Sample records for affect cell surface

  1. Nanotextured titanium surfaces stimulate spreading, migration, and growth of rat mast cells.

    PubMed

    Marcatti Amarú Maximiano, William; Marino Mazucato, Vivian; Tambasco de Oliveira, Paulo; Célia Jamur, Maria; Oliver, Constance

    2017-08-01

    Titanium is a biomaterial widely used in dental and orthopedic implants. Since tissue-implant interactions occur at the nanoscale level, nanotextured titanium surfaces may affect cellular activity and modulate the tissue response that occurs at the tissue-implant interface. Therefore, the characterization of diverse cell types in response to titanium surfaces with nanotopography is important for the rational design of implants. Mast cells are multifunctional cells of the immune system that release a range of chemical mediators involved in the inflammatory response that occurs at the tissue-implant interface. Therefore, the aim of this study was to investigate the effects of the nanotopography of titanium surfaces on the physiology of mast cells. The results show that the nanotopography of titanium surfaces promoted the spreading of mast cells, which was accompanied by the reorganization of the cytoskeleton. Also, the nanotopography of titanium surfaces enhanced cell migration and cell growth, but did not alter the number of adherent cells in first hours of culture or affect focal adhesions and mediator release. Thus, the results show that nanotopography of titanium surfaces can affect mast cell physiology, and represents an improved strategy for the rational production of surfaces that stimulate tissue integration with the titanium implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2150-2161, 2017. © 2017 Wiley Periodicals, Inc.

  2. CELL SURFACE IMMUNOGLOBULIN

    PubMed Central

    Vitetta, Ellen S.; Uhr, Jonathan W.

    1974-01-01

    A new method for the detection of cell surface immunoglobulin labeled with isotopic precursors is described. The method consists of the aggregation of surface Ig on cells with specific antibody (heterologous) and the subsequent removal of antigen-antibody complexes by the combination of high speed centrifugation and immunoprecipitation of remaining soluble complexes using antibody to the heterologous Ig. Using this method, the kinetics of appearance of cell surface Ig and its turnover were studied in murine splenocytes. The results suggest that cell surface Ig is synthesized and transported in the same manner as secretory Ig rather than being synthesized on the plasma membrane. The turnover of intracellular and cell surface Ig in lymphocytes is slow. In contrast, intracellular Ig in plasma cells is rapidly secreted and usually without a cell surface phase. Cell surface Ig was shown to be radiolabeled with [3H]glucosamine, -galactose, and -fucose. The proportion of cell surface to intracellular (nonsurface) Ig labeled with these precursors suggests the same sequence of addition of sugars to Ig destined to be on the surface of lymphocytes as with Ig which will be secreted by plasma cells. Results with this new method also confirm earlier conclusions based on experiments using cell surface iodination: 8S IgM is the predominant Ig on the surface of murine splenocytes and the molecule appears to be attached by its µ-chains. PMID:4829935

  3. Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes*

    PubMed Central

    Park, Dayoung; Brune, Kristin A.; Mitra, Anupam; Marusina, Alina I.; Maverakis, Emanual; Lebrilla, Carlito B.

    2015-01-01

    Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. PMID:26355101

  4. Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.

    PubMed

    Park, Dayoung; Brune, Kristin A; Mitra, Anupam; Marusina, Alina I; Maverakis, Emanual; Lebrilla, Carlito B

    2015-11-01

    Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Multi-scale cell/surface interaction on modified titanium aluminum vanadium surfaces

    NASA Astrophysics Data System (ADS)

    Chen, Jianbo

    This dissertation presents a series of experimental studies of the effects of multi-scale cell/surface interactions on modified Ti-6Al-4V surfaces. These include laser-grooved surfaces; porous structures and RGD-coated laser-grooved surfaces. A nano-second DPSS UV lasers with a Gaussian pulse energy profile was used to introduce the desired micro-groove geometries onto Ti-6Al-4V surfaces. This was done without inducing micro-cracks or significant changes in surface chemistry within the heat affected zones. The desired 8-12 mum groove depths and widths were achieved by the control of pulse frequency, scan speed, and the lens focal length that controls spot size. The interactions between human osteosarcoma (HOS) cells and laser-grooved Ti-6Al-4V surfaces were investigated after 48 hours of cell culture. The cell behavior, including cell spreading, alignment and adhesion, was elucidated using scanning electronic microscopy (SEM), immuno-fluorescence staining and enzymatic detachment. Contact guidance was shown to increase as grooved spacing decreased. For the range of micro-groove geometries studied, micro-grooves with groove spacings of 20 mum provided the best combination of cell orientation and adhesion. Short-term adhesion experiments (15 mins to 1 day) also revealed that there is a positive correlation between cell orientation and cell adhesion. Contact guidance on the micro-grooved surfaces is shown to be enhanced by nano- and micro-scale asperities that provide sites for the attachment of lamellopodia during cell locomotion and spreading. Contact guidance is also promoted by the geometrical confinement provided by laser grooves. An experimental study of initial cell spreading and ingrowth into Ti-6Al-4V porous structures was also carried out on porous structures with different pore sizes and geometries. A combination of SEM, the tetrazolium salt (MTT) colorimetric assay and enzymatic detachment were used to study cell spreading and adhesion. The extent of cell

  6. Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation

    PubMed Central

    Boyan, B.D.; Cheng, A.; Olivares-Navarrete, R.; Schwartz, Z.

    2016-01-01

    Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical stability of the implant, surface design ultimately contributes to osseointegration. This article reviews the surface parameters of dental implant materials that contribute to improved cell response and osseointegration. In particular, we focus on how surface design affects mesenchymal cell response and differentiation into the osteoblast lineage. Surface roughness has been largely studied at the microscale, but recent studies have highlighted the importance of hierarchical micron/submicron/nanosurface roughness, as well as surface roughness in combination with surface wettability. Integrins are transmembrane receptors that recognize changes in the surface and mediate downstream signaling pathways. Specifically, the noncanonical Wnt5a pathway has been implicated in osteoblastic differentiation of cells on titanium implant surfaces. However, much remains to be elucidated. Only recently have studies been conducted on the differences in biological response to implants based on sex, age, and clinical factors; these all point toward differences that advocate for patient-specific implant design. Finally, challenges in implant surface characterization must be addressed to optimize and compare data across studies. An understanding of both the science and the biology of the materials is crucial for developing novel dental implant materials and surface modifications for improved osseointegration. PMID:26927483

  7. Surface-modified gold nanorods for specific cell targeting

    NASA Astrophysics Data System (ADS)

    Wang, Chan-Ung; Arai, Yoshie; Kim, Insun; Jang, Wonhee; Lee, Seonghyun; Hafner, Jason H.; Jeoung, Eunhee; Jung, Deokho; Kwon, Youngeun

    2012-05-01

    Gold nanoparticles (GNPs) have unique properties that make them highly attractive materials for developing functional reagents for various biomedical applications including photothermal therapy, targeted drug delivery, and molecular imaging. For in vivo applications, GNPs need to be prepared with very little or negligible cytotoxicitiy. Most GNPs are, however, prepared using growth-directing surfactants such as cetyl trimethylammonium bromide (CTAB), which are known to have considerable cytotoxicity. In this paper, we describe an approach to remove CTAB to a non-toxic concentration. We optimized the conditions for surface modification with methoxypolyethylene glycol thiol (mPEG), which replaced CTAB and formed a protective layer on the surface of gold nanorods (GNRs). The cytotoxicities of pristine and surface-modified GNRs were measured in primary human umbilical vein endothelial cells and human cell lines derived from hepatic carcinoma cells, embryonic kidney cells, and thyroid papillary carcinoma cells. Cytotoxicity assays revealed that treating cells with GNRs did not significantly affect cell viability except for thyroid papillary carcinoma cells. Thyroid cancer cells were more susceptible to residual CTAB, so CTAB had to be further removed by dialysis in order to use GNRs for thyroid cell targeting. PEGylated GNRs are further modified to present monoclonal antibodies that recognize a specific surface marker, Na-I symporter, for thyroid cells. Antibody-conjugated GNRs specifically targeted human thyroid cells in vitro.

  8. Comparative study on morphologic changes and cell attachment of periodontitis-affected root surfaces following conditioning with CO2 and Er:YAG laser irradiations.

    PubMed

    Belal, Mahmoud Helmy; Watanabe, Hisashi

    2014-10-01

    Clinical application of lasers in periodontal therapy has continued to expand in last decades; however there are still some controversies. The present study aimed to compare the conditioning effects of the carbon dioxide (CO2) or erbium-doped: yttrium, aluminum and garnet (Er:YAG) laser on periodontally diseased root surfaces following scaling and root planing (SRP) in terms of the alteration of morphologies as well as the attachment of periodontal ligament cells. Forty-five periodontally affected root specimens were prepared and randomly assigned into three groups: I control (untreated diseased), II. SRP+CO2 laser (pulsed, noncontact mode), and III. SRP+Er:YAG laser (slight contact mode). After treatment, five specimens in each group were used for surface topographic examination. The remaining 10 specimens in each group were incubated with human periodontal ligament cell suspension. All the specimens were finally evaluated by scanning electron microscopy. The control specimens showed the lowest number of cultured cells, mostly in oval shape, with no tightly attached cells. The CO2 lased specimens showed a significant increase in the number of attached cells compared with controls, but demonstrated some major thermal alterations on the surfaces. The Er:YAG lased specimens showed the significantly highest number of attached cells, mostly in flat form, and did not show distinct thermal damage. The present study suggests that compared with the CO2 laser, the Er:YAG laser may constitute a more useful conditioning tool for enhancing periodontal cell attachment to periodontally diseased root surfaces, with fewer undesirable thermal side effects.

  9. Interdigitated Back-Surface-Contact Solar Cell Modeling Using Silvaco Atlas

    DTIC Science & Technology

    2015-06-01

    11 2. Solar Spectrum ...................................................................................13 3. PV Cell Efficiency...Figure 10. Spectrum of solar radiance, from [12]. 14 3. PV Cell Efficiency There are many factors that affect the efficiency of a solar cell. Metal...BACK-SURFACE-CONTACT SOLAR CELL MODELING USING SILVACO ATLAS by Shawn E. Green June 2015 Thesis Advisor: Sherif Michael Second Reader

  10. Surface free energy predominates in cell adhesion to hydroxyapatite through wettability.

    PubMed

    Nakamura, Miho; Hori, Naoko; Ando, Hiroshi; Namba, Saki; Toyama, Takeshi; Nishimiya, Nobuyuki; Yamashita, Kimihiro

    2016-05-01

    The initial adhesion of cells to biomaterials is critical in the regulation of subsequent cell behaviors. The purpose of this study was to investigate a mechanism through which the surface wettability of biomaterials can be improved and determine the effects of biomaterial surface characteristics on cellular behaviors. We investigated the surface characteristics of various types of hydroxyapatite after sintering in different atmospheres and examined the effects of various surface characteristics on cell adhesion to study cell-biomaterial interactions. Sintering atmosphere affects the polarization capacity of hydroxyapatite by changing hydroxide ion content and grain size. Compared with hydroxyapatite sintered in air, hydroxyapatite sintered in saturated water vapor had a higher polarization capacity that increased surface free energy and improved wettability, which in turn accelerated cell adhesion. We determined the optimal conditions of hydroxyapatite polarization for the improvement of surface wettability and acceleration of cell adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Global transcriptomic analysis of model human cell lines exposed to surface-modified gold nanoparticles: the effect of surface chemistry

    NASA Astrophysics Data System (ADS)

    Grzincic, E. M.; Yang, J. A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C. J.

    2015-01-01

    Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14 000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected.Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how

  12. Quantum Efficiency Loss after PID Stress: Wavelength Dependence on Cell Surface and Cell Edge

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Oh, Jaewon; Bowden, Stuart; TamizhMani, GovindaSamy

    2015-06-14

    It is known that the potential induced degradation (PID) stress of conventional p-base solar cells affects power, shunt resistance, junction recombination, and quantum efficiency (QE). One of the primary solutions to address the PID issue is a modification of chemical and physical properties of antireflection coating (ARC) on the cell surface. Depending on the edge isolation method used during cell processing, the ARC layer near the edges may be uniformly or non-uniformly damaged. Therefore, the pathway for sodium migration from glass to the cell junction could be either through all of the ARC surface if surface and edge ARC havemore » low quality or through the cell edge if surface ARC has high quality but edge ARC is defective due to certain edge isolation process. In this study, two PID susceptible cells from two different manufacturers have been investigated. The QE measurements of these cells before and after PID stress were performed at both surface and edge. We observed the wavelength dependent QE loss only in the first manufacturer's cell but not in the second manufacturer's cell. The first manufacturer's cell appeared to have low quality ARC whereas the second manufacturer's cell appeared to have high quality ARC with defective edge. To rapidly screen a large number of cells for PID stress testing, a new but simple test setup that does not require laminated cell coupon has been developed and is used in this investigation.« less

  13. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis

    PubMed Central

    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-01

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. PMID:25476450

  14. Acoustic sensors as a biophysical tool for probing cell attachment and cell/surface interactions.

    PubMed

    Saitakis, Michael; Gizeli, Electra

    2012-02-01

    Acoustic biosensors offer the possibility to analyse cell attachment and spreading. This is due to the offered speed of detection, the real-time non-invasive approach and their high sensitivity not only to mass coupling, but also to viscoelastic changes occurring close to the sensor surface. Quartz crystal microbalance (QCM) and surface acoustic wave (Love-wave) systems have been used to monitor the adhesion of animal cells to various surfaces and record the behaviour of cell layers under various conditions. The sensors detect cells mostly via their sensitivity in viscoelasticity and mechanical properties. Particularly, the QCM sensor detects cytoskeletal rearrangements caused by specific drugs affecting either actin microfilaments or microtubules. The Love-wave sensor directly measures cell/substrate bonds via acoustic damping and provides 2D kinetic and affinity parameters. Other studies have applied the QCM sensor as a diagnostic tool for leukaemia and, potentially, for chemotherapeutic agents. Acoustic sensors have also been used in the evaluation of the cytocompatibility of artificial surfaces and, in general, they have the potential to become powerful tools for even more diverse cellular analysis.

  15. Yeast cell surface display for lipase whole cell catalyst and its applications

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Yun; Zhang, Rui; Lian, Zhongshuai

    The cell surface display technique allows for the expression of target proteins or peptides on the microbial cell surface by fusing an appropriate protein as an anchoring motif. Yeast display systems, such as Pichia pastoris, Yarowia lipolytica and Saccharomyces cerevisiae, are ideal, alternative and extensive display systems with the advantage of simple genetic manipulation and post-translational modification of expressed heterologous proteins. Engineered yeasts show high performance characteristics and variant utilizations. Herein, we comprehensively summarize the variant factors affecting lipase whole cell catalyst activity and display efficiency, including the structure and size of target proteins, screening anchor proteins, type and chainmore » length of linkers, and the appropriate matching rules among the above-mentioned display units. Furthermore, we also address novel approaches to enhance stability and activity of recombinant lipases, such as VHb gene co-expression, multi-enzyme co-display technique, and the micro-environmental interference and self-assembly techniques. Finally, we represent the variety of applications of whole cell surface displayed lipases on yeast cells in non-aqueous phases, including synthesis of esters, PUFA enrichment, resolution of chiral drugs, organic synthesis and biofuels. We demonstrate that the lipase surface display technique is a powerful tool for functionalizing yeasts to serve as whole cell catalysts, and increasing interest is providing an impetus for broad application of this technique.« less

  16. Stem cell responses to plasma surface modified electrospun polyurethane scaffolds.

    PubMed

    Zandén, Carl; Hellström Erkenstam, Nina; Padel, Thomas; Wittgenstein, Julia; Liu, Johan; Kuhn, H Georg

    2014-07-01

    The topographical effects from functional materials on stem cell behavior are currently of interest in tissue engineering and regenerative medicine. Here we investigate the influence of argon, oxygen, and hydrogen plasma surface modification of electrospun polyurethane fibers on human embryonic stem cell (hESC) and rat postnatal neural stem cell (NSC) responses. The plasma gases were found to induce three combinations of fiber surface functionalities and roughness textures. On randomly oriented fibers, plasma treatments lead to substantially increased hESC attachment and proliferation as compared to native fibers. Argon plasma was found to induce the most optimal combination of surface functionality and roughness for cell expansion. Contact guided migration of cells and alignment of cell processes were observed on aligned fibers. Neuronal differentiation around 5% was found for all samples and was not significantly affected by the induced variations of surface functional group distribution or individual fiber topography. In this study the influence of argon, oxygen, and hydrogen plasma surface modification of electrospun polyurethane fibers on human embryonic stem cell and rat postnatal neural stem cell (NSC) responses is studied with the goal of clarifying the potential effects of functional materials on stem cell behavior, a topic of substantial interest in tissue engineering and regenerative medicine. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Effects of Different Cell-Detaching Methods on the Viability and Cell Surface Antigen Expression of Synovial Mesenchymal Stem Cells.

    PubMed

    Tsuji, Kunikazu; Ojima, Miyoko; Otabe, Koji; Horie, Masafumi; Koga, Hideyuki; Sekiya, Ichiro; Muneta, Takeshi

    2017-06-09

    Flow cytometric analysis of cell surface antigens is a powerful tool for the isolation and characterization of stem cells residing in adult tissues. In contrast to the collection of hematopoietic stem cells, the process of enzymatic digestion is usually necessary to prepare mesenchymal stem cells (MSCs) suspensions, which can influence the expression of cell surface markers. In this study, we examined the effects of various cell-detaching reagents and digestion times on the expression of stem cell-related surface antigens and MSC functions. Human MSCs were detached from dishes using four different reagents: trypsin, TrypLE, collagenase, and a nonenzymatic cell dissociation reagent (C5789; Sigma-Aldrich). Following dissociation reagent incubations ranging from 5 to 120 min, cell surface markers were analyzed by flow cytometry. Trypsin and TrypLE quickly dissociated the cells within 5 min, while collagenase and C5789 required 60 min to obtain maximum cell yields. C5789 significantly decreased cell viability at 120 min. Trypsin treatment significantly reduced CD44+, CD55+, CD73+, CD105+, CD140a+, CD140b+, and CD201+ cell numbers within 30 min. Collagenase treatment reduced CD140a expression by 30 min. In contrast, TrypLE treatment did not affect the expression of any cell surface antigens tested by 30 min. Despite the significant loss of surface antigen expression after 60 min of treatment with trypsin, adverse effects of enzymatic digestion on multipotency of MSCs were limited. Overall, our data indicated that TrypLE is advantageous over other cell dissociation reagents tested for the rapid preparation of viable MSC suspensions.

  18. Cell surface fucosylation does not affect development of colon tumors in mice with germline Smad3 mutation

    PubMed Central

    Domino, Steven E.; Karnak, David M.; Hurd, Elizabeth A.

    2006-01-01

    Background/Aims: Neoplasia-related alterations in cell surface α(1,2)fucosylated glycans have been reported in multiple tumors including colon, pancreas, endometrium, cervix, bladder, lung, and choriocarcinoma. Spontaneous colorectal tumors from mice with a germline null mutation of transforming growth factor-β signaling gene Smad3 (Madh3) were tested for α(1,2)fucosylated glycan expression. Methods: Ulex Europaeus Agglutinin-I lectin staining, fucosyltransferase gene northern blot analysis, and a cross of mutant mice with Fut2 and Smad3 germline mutations were performed. Results: Spontaneous colorectal tumors from Smad3 (-/-) homozygous null mice were found to express α(1,2)fucosylated glycans in an abnormal pattern compared to adjacent nonneoplastic colon. Northern blot analysis of α(1,2)fucosyltransferase genes Fut1 and Fut2 revealed that Fut2, but not Fut1, steady-state mRNA levels were significantly increased in tumors relative to adjacent normal colonic mucosa. Mutant mice with a Fut2-inactivating germline mutation were crossed with Smad3 targeted mice. In Smad3 (-/-)/Fut2 (-/-) double knock-out mice, UEA-I lectin staining was eliminated from colon and colon tumors, however, the number and size of tumors present by 24 weeks of age did not vary regardless of the Fut2 genotype. Conclusions: In this model of colorectal cancer, cell surface α(1,2)fucosylation does not affect development of colon tumors. PMID:17264540

  19. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

    PubMed

    Özlü, Nurhan; Qureshi, Mohammad H; Toyoda, Yusuke; Renard, Bernhard Y; Mollaoglu, Gürkan; Özkan, Nazlı E; Bulbul, Selda; Poser, Ina; Timm, Wiebke; Hyman, Anthony A; Mitchison, Timothy J; Steen, Judith A

    2015-01-13

    The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy. © 2014 The Authors.

  20. Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth

    NASA Astrophysics Data System (ADS)

    Kim, Eunhee; Yoo, Seung-Jun; Kim, Eunjung; Kwon, Tae-Hwan; Zhang, Li; Moon, Cheil; Choi, Hongsoo

    2016-04-01

    Mimicking the nanoscale surface texture of the extracellular matrix can affect the regulation of cellular behavior, including adhesion, differentiation, and neurite outgrowth. In this study, SU-8-based polymer surfaces with well-ordered nanowell arrays were fabricated using nanosphere lithography with polystyrene nanoparticles. We show that the SU-8 surface with nanowells resulted in similar neuronal development of rat pheochromocytoma (PC12) cells compared with an unpatterned poly-L-lysine (PLL)-coated SU-8 surface. Additionally, even after soaking the substrate in cell culture medium for two weeks, cells on the nanowell SU-8 surface showed long-term neurite outgrowth compared to cells on the PLL-coated SU-8 surface. The topographical surface modification of the nanowell array demonstrates potential as a replacement for cell adhesive material coatings such as PLL, for applications requiring long-term use of polymer-based implantable devices.

  1. Does the Economy or Surface Warfare Officer Career Pay Affect Surface Warfare Officer Retention?

    DTIC Science & Technology

    2014-12-01

    be a career SWO (Surface Warfare Enterprise, 2013). 6 Military pay was more important to men than women ...OR SURFACE WARFARE OFFICER CAREER PAY AFFECT SURFACE WARFARE OFFICER RETENTION? by Meagan B. Makarenko December 2014 Thesis Advisor...2014 3. REPORT TYPE AND DATES COVERED Master’s Thesis 4. TITLE AND SUBTITLE DOES THE ECONOMY OR SURFACE WARFARE OFFICER CAREER PAY AFFECT

  2. Comparison of detection methods for cell surface globotriaosylceramide.

    PubMed

    Kim, Minji; Binnington, Beth; Sakac, Darinka; Fernandes, Kimberly R; Shi, Sheryl P; Lingwood, Clifford A; Branch, Donald R

    2011-08-31

    The cell surface-expressed glycosphingolipid (GSL), globotriaosylceramide (Gb(3)), is becoming increasingly important and is widely studied in the areas of verotoxin (VT)-mediated cytotoxicity, human immunodeficiency virus (HIV) infection, immunology and cancer. However, despite its diverse roles and implications, an optimized detection method for cell surface Gb(3) has not been determined. GSLs are differentially organized in the plasma membrane which can affect their availability for protein binding. To examine various detection methods for cell surface Gb(3), we compared four reagents for use in flow cytometry analysis. A natural ligand (VT1B) and three different monoclonal antibodies (mAbs) were optimized and tested on various human cell lines for Gb(3) detection. A differential detection pattern of cell surface Gb(3) expression, which was influenced by the choice of reagent, was observed. Two mAb were found to be suboptimal. However, two other methods were found to be useful as defined by their high percentage of positivity and mean fluorescence intensity (MFI) values. Rat IgM anti-Gb(3) mAb (clone 38-13) using phycoerythrin-conjugated secondary antibody was found to be the most specific detection method while the use of VT1B conjugated to Alexa488 fluorochrome was found to be the most sensitive; showing a rare crossreactivity only when Gb(4) expression was highly elevated. The findings of this study demonstrate the variability in detection of Gb(3) depending on the reagent and cell target used and emphasize the importance of selecting an optimal methodology in studies for the detection of cell surface expression of Gb(3). Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

    PubMed

    Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

    2015-09-30

    Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

  4. Downregulation of GbpB, a Component of the VicRK Regulon, Affects Biofilm Formation and Cell Surface Characteristics of Streptococcus mutans▿ †

    PubMed Central

    Duque, Cristiane; Stipp, Rafael N.; Wang, Bing; Smith, Daniel J.; Höfling, José F.; Kuramitsu, Howard K.; Duncan, Margaret J.; Mattos-Graner, Renata O.

    2011-01-01

    The virulence of the dental caries pathogen Streptococcus mutans relies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced by S. mutans participate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressed gbpB antisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation of gbpB by the two-component system VicRK was investigated, and phenotypic analysis of a vicK mutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrose-dependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties. PMID:21078847

  5. An Inducible Endothelial Cell Surface Glycoprotein Mediates Melanoma Adhesion

    NASA Astrophysics Data System (ADS)

    Rice, G. Edgar; Bevilacqua, Michael P.

    1989-12-01

    Hematogenous metastasis requires the arrest and extravasation of blood-borne tumor cells, possibly involving direct adhesive interactions with vascular endothelium. Cytokine activation of cultured human endothelium increases adhesion of melanoma and carcinoma cell lines. An inducible 110-kD endothelial cell surface glycoprotein, designated INCAM-110, appears to mediate adhesion of melanoma cells. In addition, an inducible endothelial receptor for neutrophils, ELAM-1, supports the adhesion of a human colon carcinoma cell line. Thus, activation of vascular endothelium in vivo that results in increased expression of INCAM-110 and ELAM-1 may promote tumor cell adhesion and affect the incidence and distribution of metastases.

  6. Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

    PubMed

    Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

    2005-04-01

    Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

  7. Functional roles of cell surface peptidases in reproductive organs

    PubMed Central

    2004-01-01

    A number of biologically active peptides have been proposed to regulate function and differentiation of reproductive organs in an autocrine and/or paracrine fashion. Regulation of the local concentrations of these peptides is one of the important factors influencing their physiological effects on target cells. Membrane‐bound cell surface peptidases can activate or inactivate biologically active peptides before peptide factors access their receptors on the cell surface. Aminopeptidase A (EC 3.4.11.7), placental leucine aminopeptidase (EC 3.4.11.3), aminopeptidase‐N/CD13 (EC 3.4.11.2), dipeptidyl peptidases IV/CD26 (EC.3.4.14.5), carboxypeptidase‐M (EC 3.4.17.12), neutral endopeptidase/CD10 (EC 3.4.24.11) and endothelin converting enzyme‐1 (EC 3.4.23) are differentially expressed on the ovary, endometrium and placenta. The inhibition of enzyme activity affects steroid hormone production by granulosa and thecal cells, decidualization of endometrium and migration of extravillous trophoblasts. These findings suggest that membrane‐bound cell surface peptidases are local regulators for cellular growth and differentiation in reproductive organs by controlling extracellular concentration of peptide factors. (Reprod Med Biol 2004; 3: 165 –176) PMID:29662383

  8. Collagen Self-Assembly on Orthopedic Magnesium Biomaterials Surface and Subsequent Bone Cell Attachment

    PubMed Central

    Zhao, Nan; Zhu, Donghui

    2014-01-01

    Magnesium (Mg) biomaterials are a new generation of biodegradable materials and have promising potential for orthopedic applications. After implantation in bone tissues, these materials will directly interact with extracellular matrix (ECM) biomolecules and bone cells. Type I collagen, the major component of bone ECM, forms the architecture scaffold that provides physical support for bone cell attachment. However, it is still unknown how Mg substrate affects collagen assembly on top of it as well as subsequent cell attachment and growth. Here, we studied the effects of collagen monomer concentration, pH, assembly time, and surface roughness of two Mg materials (pure Mg and AZ31) on collagen fibril formation. Results showed that formation of fibrils would not initiate until the monomer concentration reached a certain level depending on the type of Mg material. The thickness of collagen fibril increased with the increase of assembly time. The structures of collagen fibrils formed on semi-rough surfaces of Mg materials have a high similarity to that of native bone collagen. Next, cell attachment and growth after collagen assembly were examined. Materials with rough surface showed higher collagen adsorption but compromised bone cell attachment. Interestingly, surface roughness and collagen structure did not affect cell growth on AZ31 for up to a week. Findings from this work provide some insightful information on Mg-tissue interaction at the interface and guidance for future surface modifications of Mg biomaterials. PMID:25303459

  9. Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid in vitro

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schindeler, Aaron; Little, David G.; Discipline of Paediatrics and Child Health, Faculty of Medicine, University of Sydney, Sydney

    2005-12-16

    Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption. Recent interest has centered on the effects of bisphosphonates on osteoblasts. Chronic dosing of osteoblasts with solubilized bisphosphonates has been reported to enhance osteogenesis and mineralization in vitro. However, this methodology poorly reflects the in vivo situation, where free bisphosphonate becomes rapidly bound to mineralized bone surfaces. To establish a more clinically relevant cell culture model, we cultured bone cells on calcium phosphate coated quartz discs pre-treated with the potent nitrogen-containing bisphosphonate, zoledronic acid (ZA). Binding studies utilizing [{sup 14}C]-labeled ZA confirmed that the bisphosphonate bound in a concentration-dependent manner over themore » 1-50 {mu}M dose range. When grown on ZA-treated discs, the viability of bone-marrow derived osteoclasts was greatly reduced, while the viability and mineralization of the osteoblastic MC3T3-E1 cell line were largely unaffected. This suggests that only bone resorbing cells are affected by bound bisphosphonate. However, this system does not account for transient exposure to unbound bisphosphonate in the hours following a clinical dosing. To model this event, we transiently treated osteoblasts with ZA in the absence of a calcified surface. Osteoblasts proved highly resistant to all transitory treatment regimes, even when utilizing ZA concentrations that prevented mineralization and/or induced cell death when dosed chronically. This study represents a pharmacologically more relevant approach to modeling bisphosphonate treatment on cultured bone cells and implies that bisphosphonate therapies may not directly affect osteoblasts at bone surfaces.« less

  10. CELL SURFACE IMMUNOGLOBULIN

    PubMed Central

    Vitetta, Ellen S.; Grundke-Iqbal, Inge; Holmes, Kathryn V.; Uhr, Jonathan W.

    1974-01-01

    Lymphoid cells from the spleen, lymph nodes, and thoracic duct of axenic and control mice were incubated with [3H]tyrosine and synthesis and secretion of protein and Ig studied. It was found that only IgM was synthesized by cells from axenic mice whereas cells from control mice also synthesized IgG. Splenocytes from both axenic and control mice had 8S IgM on their surface. Radiolabeled splenocytes from axenic mice were incubated to determine the kinetics of release of 125I-labeled cell surface IgM and [3H]tyrosine-labeled IgM. Cell surface IgM was shed as 8S with an initial half-life of release of 5–8 h whereas [3H]tyrosine-labeled Ig was secreted as 19S with an initial half-life of 2–3 h. These findings suggest that two independent pathways are involved. It is suggested that small lymphocytes shed 8S IgM and plasma cells secrete 19S IgM. It was observed that lymphoid cells from axenic mice synthesize a higher proportion of IgM relative to total protein. Electron microscopic examination of splenocytes from such mice revealed a markedly higher proportion of plasma cells and a paucity of lymphoblasts compared to controls. It was suggested, therefore, that axenic mice lack a population of stimulated T cells which can induce a switch from IgM to IgG synthesis and which is capable of suppressing IgM synthesis. Lymphoid cells from axenic mice synthesize and secrete less protein that coprecipitates with antigen-antibody complexes. PMID:4544585

  11. Surface engineering approaches to micropattern surfaces for cell-based assays.

    PubMed

    Falconnet, Didier; Csucs, Gabor; Grandin, H Michelle; Textor, Marcus

    2006-06-01

    The ability to produce patterns of single or multiple cells through precise surface engineering of cell culture substrates has promoted the development of cellular bioassays that provide entirely new insights into the factors that control cell adhesion to material surfaces, cell proliferation, differentiation and molecular signaling pathways. The ability to control shape and spreading of attached cells and cell-cell contacts through the form and dimension of the cell-adhesive patches with high precision is important. Commitment of stem cells to different specific lineages depends strongly on cell shape, implying that controlled microenvironments through engineered surfaces may not only be a valuable approach towards fundamental cell-biological studies, but also of great importance for the design of cell culture substrates for tissue engineering. Furthermore, cell patterning is an important tool for organizing cells on transducers for cell-based sensing and cell-based drug discovery concepts. From a material engineering standpoint, patterning approaches have greatly profited by combining microfabrication technologies, such as photolithography, with biochemical functionalization to present to the cells biological cues in spatially controlled regions where the background is rendered non-adhesive ("non-fouling") by suitable chemical modification. The focus of this review is on the surface engineering aspects of biologically motivated micropatterning of two-dimensional (flat) surfaces with the aim to provide an introductory overview and critical assessment of the many techniques described in the literature. In particular, the importance of non-fouling surface chemistries, the combination of hard and soft lithography with molecular assembly techniques as well as a number of less well known, but useful patterning approaches, including direct cell writing, are discussed.

  12. High hydrostatic pressure inactivation of Lactobacillus plantarum cells in (O/W)-emulsions is independent from cell surface hydrophobicity and lipid phase parameters

    NASA Astrophysics Data System (ADS)

    Kafka, T. A.; Reitermayer, D.; Lenz, C. A.; Vogel, R. F.

    2017-07-01

    Inactivation efficiency of high hydrostatic pressure (HHP) processing of food is strongly affected by food matrix composition. We investigated effects of fat on HHP inactivation of spoilage-associated Lactobacillus (L.) plantarum strains using defined oil-in-water (O/W)-emulsion model systems. Since fat-mediated effects on HHP inactivation could be dependent on interactions between lipid phase and microbial cells, three major factors possibly influencing such interactions were considered, that is, cell surface hydrophobicity, presence and type of surfactants, and oil droplet size. Pressure tolerance varied noticeably among L. plantarum strains and was independent of cell surface hydrophobicity. We showed that HHP inactivation of all strains tended to be more effective in presence of fat. The observation in both, surfactant-stabilized and surfactant-free (O/W)-emulsion, indicates that cell surface hydrophobicity is no intrinsic pressure resistance factor. In contrast to the presence of fat per se, surfactant type and oil droplet size did not affect inactivation efficiency.

  13. Plasma Membrane Sterol Distribution Resembles the Surface Topography of Living Cells

    PubMed Central

    2007-01-01

    Cholesterol is an important constituent of cellular membranes. It has been suggested that cholesterol segregates into sterol-rich and -poor domains in the plasma membrane, although clear evidence for this is lacking. By fluorescence imaging of the natural sterol dehydroergosterol (DHE), the lateral sterol distribution has been visualized in living cells. The spatial labeling pattern of DHE coincided with surface structures such as ruffles, microvilli, and filopodia with correlation lengths in the range of 0.8–2.5 μm. DHE staining of branched tubules and of nanotubes connecting two cells was detected. Dynamics of DHE in folded and plane membrane regions was comparable as determined by fluorescence recovery after photobleaching. DHE colocalized with fluid membrane-preferring phospholipids in surface structures and at sites of cell attachment as well as in the cleavage furrow of dividing cells, but it was not particularly enriched in those regions. Fluorescent sterol showed homogeneous staining in membrane blebs induced by F-actin disruption. Cross-linking the ganglioside GM1—a putative raft marker—did not affect the cell surface distribution of DHE. The results suggest that spatial heterogeneities of plasma membrane staining of DHE resolvable by light microscopy reflect the cell surface topography but not phase-separated sterol domains in the bilayer plane. PMID:17065557

  14. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover

    PubMed Central

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca2+-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  15. The Regulatory Protein RosR Affects Rhizobium leguminosarum bv. trifolii Protein Profiles, Cell Surface Properties, and Symbiosis with Clover.

    PubMed

    Rachwał, Kamila; Boguszewska, Aleksandra; Kopcińska, Joanna; Karaś, Magdalena; Tchórzewski, Marek; Janczarek, Monika

    2016-01-01

    Rhizobium leguminosarum bv. trifolii is capable of establishing a symbiotic relationship with plants from the genus Trifolium. Previously, a regulatory protein encoded by rosR was identified and characterized in this bacterium. RosR possesses a Cys2-His2-type zinc finger motif and belongs to Ros/MucR family of rhizobial transcriptional regulators. Transcriptome profiling of the rosR mutant revealed a role of this protein in several cellular processes, including the synthesis of cell-surface components and polysaccharides, motility, and bacterial metabolism. Here, we show that a mutation in rosR resulted in considerable changes in R. leguminosarum bv. trifolii protein profiles. Extracellular, membrane, and periplasmic protein profiles of R. leguminosarum bv. trifolii wild type and the rosR mutant were examined, and proteins with substantially different abundances between these strains were identified. Compared with the wild type, extracellular fraction of the rosR mutant contained greater amounts of several proteins, including Ca(2+)-binding cadherin-like proteins, a RTX-like protein, autoaggregation protein RapA1, and flagellins FlaA and FlaB. In contrast, several proteins involved in the uptake of various substrates were less abundant in the mutant strain (DppA, BraC, and SfuA). In addition, differences were observed in membrane proteins of the mutant and wild-type strains, which mainly concerned various transport system components. Using atomic force microscopy (AFM) imaging, we characterized the topography and surface properties of the rosR mutant and wild-type cells. We found that the mutation in rosR gene also affected surface properties of R. leguminosarum bv. trifolii. The mutant cells were significantly more hydrophobic than the wild-type cells, and their outer membrane was three times more permeable to the hydrophobic dye N-phenyl-1-naphthylamine. The mutation of rosR also caused defects in bacterial symbiotic interaction with clover plants. Compared with

  16. Construction of Hydrophobic Wood Surface and Mechanical Property of Wood Cell Wall on Nanoscale Modified by Dimethyldichlorosilane

    NASA Astrophysics Data System (ADS)

    Yang, Rui; Wang, Siqun; Zhou, Dingguo; Zhang, Jie; Lan, Ping; Jia, Chong

    2018-01-01

    Dimethyldichlorosilane was used to improve the hydrophobicity of wood surface. The water contact angle of the treated wood surface increased from 85° to 143°, which indicated increased hydrophobicity. The nanomechanical properties of the wood cell wall were evaluated using a nanoindentation test to analyse the hydrophobic mechanism on the nano scale. The elastic modulus of the cell wall was significantly affected by the concentration but the influence of treatment time is insignificant. The hardness of the cell wall for treated samples was significantly affected by both treatment time and concentration. The interaction between treatment time and concentration was extremely significant for the elastic modulus of the wood cell wall.

  17. Profound re-organization of cell surface proteome in equine retinal pigment epithelial cells in response to in vitro culturing.

    PubMed

    Szober, Christoph M; Hauck, Stefanie M; Euler, Kerstin N; Fröhlich, Kristina J H; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A

    2012-10-31

    The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

  18. Application of atomic force microscopy to microbial surfaces: from reconstituted cell surface layers to living cells.

    PubMed

    Dufrêne, Y F

    2001-02-01

    The application of atomic force microscopy (AFM) to probe the ultrastructure and physical properties of microbial cell surfaces is reviewed. The unique capabilities of AFM can be summarized as follows: imaging surface topography with (sub)nanometer lateral resolution; examining biological specimens under physiological conditions; measuring local properties and interaction forces. AFM is being used increasingly for: (i) visualizing the surface ultrastructure of microbial cell surface layers, including bacterial S-layers, purple membranes, porin OmpF crystals and fungal rodlet layers; (ii) monitoring conformational changes of individual membrane proteins; (iii) examining the morphology of bacterial biofilms, (iv) revealing the nanoscale structure of living microbial cells, including fungi, yeasts and bacteria, (v) mapping interaction forces at microbial surfaces, such as van der Waals and electrostatic forces, solvation forces, and steric/bridging forces; and (vi) probing the local mechanical properties of cell surface layers and of single cells.

  19. Cell/surface interactions on laser micro-textured titanium-coated silicon surfaces.

    PubMed

    Mwenifumbo, Steven; Li, Mingwei; Chen, Jianbo; Beye, Aboubaker; Soboyejo, Wolé

    2007-01-01

    This paper examines the effects of nano-scale titanium coatings, and micro-groove/micro-grid patterns on cell/surface interactions on silicon surfaces. The nature of the cellular attachment and adhesion to the coated/uncoated micro-textured surfaces was elucidated by the visualization of the cells and relevant cytoskeletal & focal adhesion proteins through scanning electron microscopy and immunofluorescence staining. Increased cell spreading and proliferation rates are observed on surfaces with 50 nm thick Ti coatings. The micro-groove geometries have been shown to promote contact guidance, which leads to reduced scar tissue formation. In contrast, smooth surfaces result in random cell orientations and the increased possibility of scar tissue formation. Immunofluorescence cell staining experiments also reveal that the actin stress fibers are aligned along the groove dimensions, with discrete focal adhesions occurring along the ridges, within the grooves and at the ends of the cell extensions. The implications of the observed cell/surface interactions are discussed for possible applications of silicon in implantable biomedical systems.

  20. Two DD-carboxypeptidases from Mycobacterium smegmatis affect cell surface properties through regulation of peptidoglycan cross-linking and glycopeptidolipids.

    PubMed

    Pandey, Satya Deo; Pal, Shilpa; Kumar N, Ganesh; Bansal, Ankita; Mallick, Sathi; Ghosh, Anindya S

    2018-05-07

    of Glycopeptidolipids (a significant surface lipid present in many non-tuberculous mycobacteria including Mycobacterium smegmatis ) and affect other physiological parameters like cell morphology, growth rate, biofilm formation, antibiotic susceptibility and existence within murine macrophages. Thus, unraveling the physiology of DD-CPases might help us design anti-mycobacterial therapeutics in future. Copyright © 2018 American Society for Microbiology.

  1. Improved bone marrow stromal cell adhesion on micropatterned titanium surfaces.

    PubMed

    Iskandar, Maria E; Cipriano, Aaron F; Lock, Jaclyn; Gott, Shannon C; Rao, Masaru P; Liu, Huinan

    2012-01-01

    Implant longevity is desired for all bone replacements and fixatives. Titanium (Ti) implants fail due to lack of juxtaposed bone formation, resulting in implant loosening. Implant surface modifications have shown to affect the interactions between the implant and bone. In clinical applications, it is crucial to improve osseointegration and implant fixation at the implant and bone interface. Moreover, bone marrow derived cells play a significant role for implant and tissue integration. Therefore, the objective of this study is to investigate how surface micropatterning on Ti influences its interactions with bone marrow derived cells containing mesenchymal and hematopoietic stem cells. Bone marrow derived mesenchymal stem cells (BMSC) have the capability of differentiating into osteoblasts that contribute to bone growth, and therefore implant/bone integration. Hematopoietic stem cell derivatives are precursor cells that contribute to inflammatory response. By using all three cells naturally contained within bone marrow, we mimic the physiological environment to which an implant is exposed. Primary rat bone marrow derived cells were seeded onto Ti with surfaces composed of arrays of grooves of equal width and spacing ranging from 0.5 to 50 µm, fabricated using a novel plasma-based dry etching technique. Results demonstrated enhanced total cell adhesion on smaller micrometer-scale Ti patterns compared with larger micrometer-scale Ti patterns, after 24-hr culture. Further studies are needed to determine bone marrow derived cell proliferation and osteogenic differentiation potential on micropatterned Ti, and eventually nanopatterned Ti.

  2. Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

    NASA Astrophysics Data System (ADS)

    Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

    2014-04-01

    To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

  3. Back surface reflectors for solar cells

    NASA Technical Reports Server (NTRS)

    Chai, A. T.

    1980-01-01

    Sample solar cells were fabricated to study the effects of various back surface reflectors on the device performance. They are typical 50 micrometers thick, space quality, silicon solar cells except for variations of the back contact configuration. The back surfaces of the sample cells are polished to a mirror like finish, and have either conventional full contacts or grid finger contacts. Measurements and evaluation of various metallic back surface reflectors, as well as cells with total internal reflection, are presented. Results indicate that back surface reflectors formed using a grid finger back contact are more effective reflectors than cells with full back metallization and that Au, Ag, or Cu are better back surface reflector metals than Al.

  4. Polymer microfilters with nanostructured surfaces for the culture of circulating cancer cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu

    There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and withoutmore » an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. Copyright 2016 The Authors. Published by Elsevier B.V. All rights reserved.« less

  5. Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

    PubMed Central

    Sumiyoshi, Mika; Ricciuto, Jessica; Tisdale, Ann; Gipson, Ilene K.; Mantelli, Flavio; Argüeso, Pablo

    2008-01-01

    Purpose Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal–limbal epithelial (HCLE) cells. Methods Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-α-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell–cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Results Benzyl-α-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-α-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. Conclusions These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface–associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye). PMID:18172093

  6. Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

    PubMed Central

    Cowden Dahl, Karen D.; Robertson, Sarah E.; Weaver, Valerie M.; Simon, M. Celeste

    2005-01-01

    Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin. PMID:15689487

  7. Rare TREM2 variants associated with Alzheimer's disease display reduced cell surface expression.

    PubMed

    Sirkis, Daniel W; Bonham, Luke W; Aparicio, Renan E; Geier, Ethan G; Ramos, Eliana Marisa; Wang, Qing; Karydas, Anna; Miller, Zachary A; Miller, Bruce L; Coppola, Giovanni; Yokoyama, Jennifer S

    2016-09-02

    Rare variation in TREM2 has been associated with greater risk for Alzheimer's disease (AD). TREM2 encodes a cell surface receptor expressed on microglia and related cells, and the R47H variant associated with AD appears to affect the ability of TREM2 to bind extracellular ligands. In addition, other rare TREM2 mutations causing early-onset neurodegeneration are thought to impair cell surface expression. Using a sequence kernel association (SKAT) analysis in two independent AD cohorts, we found significant enrichment of rare TREM2 variants not previously characterized at the protein level. Heterologous expression of the identified variants showed that novel variants S31F and R47C displayed significantly reduced cell surface expression. In addition, we identified rare variant R136Q in a patient with language-predominant AD that also showed impaired surface expression. The results suggest rare TREM2 variants enriched in AD may be associated with altered TREM2 function and that AD risk may be conferred, in part, from altered TREM2 surface expression.

  8. Do cell surface trafficking impairments account for variable cell surface sodium iodide symporter levels in breast cancer?

    PubMed Central

    Beyer, S.J.; Jimenez, R.E.; Shapiro, C.L.; Cho, J.Y.; Jhiang, S.M.

    2009-01-01

    The Na+/I- symporter (NIS) is a transmembrane glycoprotein that mediates iodide uptake into thyroid follicular cells and serves as the molecular basis of radioiodine imaging and therapy for thyroid cancer patients. The finding that NIS protein is present in 80-90% of breast tumors suggests that breast cancer patients may also benefit from NIS-mediated radionuclide imaging and targeted therapy. However, only 17-25% of NIS-positive breast tumors have detectable radionuclide uptake activity. The discrepancy between NIS expression and radionuclide uptake activity is most likely contributed by variable cell surface NIS protein levels. Apart from the prevalent view that NIS cell surface trafficking impairments account for the variability, our current study proposes that differential levels of NIS expression may also account for variable cell surface NIS levels among breast tumors. We address the need to confirm the identity of intracellular NIS staining to reveal the mechanisms underlying variable cell surface NIS levels. In addition, we warrant a quantitative correlation between cell surface NIS levels and radionuclide uptake activity in patients such that the cell surface NIS levels required for radionuclide imaging can be defined and the defects impairing NIS activity can be recognized. PMID:18500672

  9. Mendelian and non-mendelian mutations affecting surface antigen expression in Paramecium tetraurelia.

    PubMed Central

    Epstein, L M; Forney, J D

    1984-01-01

    A screening procedure was devised for the isolation of X-ray-induced mutations affecting the expression of the A immobilization antigen (i-antigen) in Paramecium tetraurelia. Two of the mutations isolated by this procedure proved to be in modifier genes. The two genes are unlinked to each other and unlinked to the structural A i-antigen gene. These are the first modifier genes identified in a Paramecium sp. that affect surface antigen expression. Another mutation was found to be a deletion of sequences just downstream from the A i-antigen gene. In cells carrying this mutation, the A i-antigen gene lies in close proximity to the end of a macronuclear chromosome. The expression of the A i-antigen is not affected in these cells, demonstrating that downstream sequences are not important for the regulation and expression of the A i-antigen gene. A stable cell line was also recovered which shows non-Mendelian inheritance of a macronuclear deletion of the A i-antigen gene. This mutant does not contain the gene in its macronucleus, but contains a complete copy of the gene in its micronucleus. In the cytoplasm of wild-type animals, the micronuclear gene is included in the developing macronucleus; in the cytoplasm of the mutant, the incorporation of the A i-antigen gene into the macronucleus is inhibited. This is the first evidence that a mechanism is available in ciliates to control the expression of a gene by regulating its incorporation into developing macronuclei. Images PMID:6092921

  10. Targeting Prostate Cancer Stemlike Cells through Cell Surface Expressed GRP78

    DTIC Science & Technology

    2016-12-01

    NOTES 14. ABSTRACT This study investigated a function for cell surface GRP78 in regulating prostate cancer stem -like cells . In year 1, we showed that...enrichment of cell surface GRP78+ cancer stem like cells in sphere culture. We also showed that the signaling axis activated by cell surface GRP78 is...but not the GRP78(-) cells , exhibited cancer stem -like cell behavior. Furthermore an GRP78 monoclonal antibody inhibited sphere forming ability of

  11. The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status.

    PubMed

    Horváthová, Mira; Ilavská, Silvia; Štefíková, Kornélia; Szabová, Michaela; Krivošíková, Zora; Jahnová, Eva; Tulinská, Jana; Spustová, Viera; Gajdoš, Martin

    2017-07-11

    The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells ( p < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause.

  12. The Cell Surface Markers Expression in Postmenopausal Women and Relation to Obesity and Bone Status

    PubMed Central

    Horváthová, Mira; Ilavská, Silvia; Štefíková, Kornélia; Szabová, Michaela; Krivošíková, Zora; Jahnová, Eva; Tulinská, Jana; Spustová, Viera; Gajdoš, Martin

    2017-01-01

    The age-related changes and hormonal deprivation in postmenopausal women are associated with the immune response alteration. The excessive fat accumulation, local and systemic inflammation may lead to dysregulation in immune function and relevant health problems, including obesity and osteoporosis. We analyzed the expression of cell surface markers in the venous blood specimens, stained with fluorophores-conjugated monoclonal antibodies and analysed by multicolour flow cytometry. The significant changes of cytotoxic, naive, and memory T-lymphocytes, plasmacytoid dendritic cells (DCs) were in postmenopausal women versus fertile women. Body mass index (BMI) affected markedly the cell surface expression of CD265/RANK. Osteoporosis is linked to reduced percentage of plasmacytoid DCs, and elevated natural Treg cells (p < 0.05). The confounding factors such as women age, BMI, bone mineral density (BMD), waist size and tissue fat affect the expression of RANK on myeloid DCs and CD40L on T-lymphocytes that might be the immunophenotypic modulators after menopause. PMID:28696349

  13. Identification of a regulatory T cell specific cell surface molecule that mediates suppressive signals and induces Foxp3 expression.

    PubMed

    Wang, Rui; Wan, Qi; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

    2008-07-16

    Regulatory T (T(reg)) cells control immune activation and maintain tolerance. How T(regs) mediate their suppressive function is unclear. Here we identified a cell surface molecule, called GARP, (or LRRC32), which within T cells is specifically expressed in T(regs) activated through the T cell receptor (TCR). Ectopic expression of GARP in human naïve T (T(N)) cells inhibited their proliferation and cytokine secretion upon TCR activation. Remarkably, GARP over-expression in T(N) cells induced expression of T(reg) master transcription factor Foxp3 and endowed them with a partial suppressive function. The extracellular but not the cytoplasmic region of GARP, was necessary for these functions. Silencing Foxp3 in human T(reg) cells reduced expression of GARP and attenuated their suppressive function. However, GARP function was not affected when Foxp3 was downregulated in GARP-overexpressing cells, while silencing GARP in Foxp3-overexpressing cells reduced their suppressive activity. These findings reveal a novel cell surface molecule-mediated regulatory mechanism, with implications for modulating aberrant immune responses.

  14. Biomolecular strategies for cell surface engineering

    NASA Astrophysics Data System (ADS)

    Wilson, John Tanner

    Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of ultrathin conformal coatings that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Significantly, this work provides novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond. Encapsulation of cells and tissue offers a rational approach for attenuating deleterious host responses towards transplanted cells, but a need exists to develop cell encapsulation strategies that minimize transplant volume. Towards this end, we endeavored to generate nanothin films of diverse architecture with tunable properties on the extracellular surface of individual pancreatic islets through a process of layer-by-layer (LbL) self assembly. We first describe the formation of poly(ethylene glycol) (PEG)-rich conformal coatings on islets via LbL self assembly of poly(L-lysine)-g-PEG(biotin) and streptavidin. Multilayer thin films conformed to the geometrically and chemically heterogeneous islet surface, and could be assembled without loss of islet viability or function. Significantly, coated islets performed comparably to untreated controls in a murine model of allogenic intraportal islet transplantation, and, to our knowledge, this is the first study to report in vivo survival and function of nanoencapsulated cells or cell aggregates. Based on these findings, we next postulated that structurally similar PLL-g-PEG copolymers comprised of shorter PEG grafts might be used to initiate and propagate the assembly of polyelectrolyte multilayer (PEM) films on pancreatic islets, while simultaneously preserving islet viability. Through control of PLL

  15. Cell Adhesion on Surface-Functionalized Magnesium.

    PubMed

    Wagener, Victoria; Schilling, Achim; Mainka, Astrid; Hennig, Diana; Gerum, Richard; Kelch, Marie-Luise; Keim, Simon; Fabry, Ben; Virtanen, Sannakaisa

    2016-05-18

    The biocompatibility of commercially pure magnesium-based (cp Mg) biodegradable implants is compromised of strong hydrogen evolution and surface alkalization due to high initial corrosion rates of cp Mg in the physiological environment. To mitigate this problem, the addition of corrosion-retarding alloying elements or coating of implant surfaces has been suggested. In the following work, we explored the effect of organic coatings on long-term cell growth. cp Mg was coated with aminopropyltriehtoxysilane + vitamin C (AV), carbonyldiimidazole (CDI), or stearic acid (SA). All three coatings have been previously suggested to reduce initial corrosion and to enhance protein adsorption and hence cell adhesion on magnesium surfaces. Endothelial cells (DH1+/+) and osteosarcoma cells (MG63) were cultured on coated samples for up to 20 days. To quantify Mg corrosion, electrochemical impedance spectroscopy (EIS) was measured after 1, 3, and 5 days of cell culture. We also investigated the speed of initial cell spreading after seeding using fluorescently labeled fibroblasts (NIH/3T3). Hydrogen evolution after contact with cell culture medium was markedly decreased on AV- and SA-coated Mg compared to uncoated Mg. These coatings also showed improved cell adhesion and spreading after 24 h of culture comparable to tissue-treated plastic surfaces. On AV-coated cp Mg, a confluent layer of endothelial cells formed after 5 days and remained intact for up to 20 days. Together, these data demonstrate that surface coating with AV is a viable strategy for improving long-term biocompatibility of cp Mg-based implants. EIS measurements confirmed that the presence of a confluent cell layer increased the corrosion resistance.

  16. Colloid Surface Chemistry Critically Affects Multiple Particle Tracking Measurements of Biomaterials

    PubMed Central

    Valentine, M. T.; Perlman, Z. E.; Gardel, M. L.; Shin, J. H.; Matsudaira, P.; Mitchison, T. J.; Weitz, D. A.

    2004-01-01

    Characterization of the properties of complex biomaterials using microrheological techniques has the promise of providing fundamental insights into their biomechanical functions; however, precise interpretations of such measurements are hindered by inadequate characterization of the interactions between tracers and the networks they probe. We here show that colloid surface chemistry can profoundly affect multiple particle tracking measurements of networks of fibrin, entangled F-actin solutions, and networks of cross-linked F-actin. We present a simple protocol to render the surface of colloidal probe particles protein-resistant by grafting short amine-terminated methoxy-poly(ethylene glycol) to the surface of carboxylated microspheres. We demonstrate that these poly(ethylene glycol)-coated tracers adsorb significantly less protein than particles coated with bovine serum albumin or unmodified probe particles. We establish that varying particle surface chemistry selectively tunes the sensitivity of the particles to different physical properties of their microenvironments. Specifically, particles that are weakly bound to a heterogeneous network are sensitive to changes in network stiffness, whereas protein-resistant tracers measure changes in the viscosity of the fluid and in the network microstructure. We demonstrate experimentally that two-particle microrheology analysis significantly reduces differences arising from tracer surface chemistry, indicating that modifications of network properties near the particle do not introduce large-scale heterogeneities. Our results establish that controlling colloid-protein interactions is crucial to the successful application of multiple particle tracking techniques to reconstituted protein networks, cytoplasm, and cells. PMID:15189896

  17. Inflammatory cell response to ultra-thin amorphous and crystalline hydroxyapatite surfaces.

    PubMed

    Rydén, Louise; Omar, Omar; Johansson, Anna; Jimbo, Ryo; Palmquist, Anders; Thomsen, Peter

    2017-01-01

    It has been suggested that surface modification with a thin hydroxyapatite (HA) coating enhances the osseointegration of titanium implants. However, there is insufficient information about the biological processes involved in the HA-induced response. This study aimed to investigate the inflammatory cell response to titanium implants with either amorphous or crystalline thin HA. Human mononuclear cells were cultured on titanium discs with a machined surface or with a thin, 0.1 μm, amorphous or crystalline HA coating. Cells were cultured for 24 and 96 h, with and without lipopolysaccharide (LPS) stimulation. The surfaces were characterized with respect to chemistry, phase composition, wettability and topography. Biological analyses included the percentage of implant-adherent cells and the secretion of pro-inflammatory cytokine (TNF-α) and growth factors (BMP-2 and TGF-β1). Crystalline HA revealed a smooth surface, whereas the amorphous HA displayed a porous structure, at nano-scale, and a hydrophobic surface. Higher TNF-α secretion and a higher ratio of adherent cells were demonstrated for the amorphous HA compared with the crystalline HA. TGF-β1 secretion was detected in all groups, but without any difference. No BMP-2 secretion was detected in any of the groups. The addition of LPS resulted in a significant increase in TNF-α in all groups, whereas TGF-β1 was not affected. Taken together, the results show that thin HA coatings with similar micro-roughness but a different phase composition, nano-scale roughness and wettability are associated with different monocyte responses. In the absence of strong inflammatory stimuli, crystalline hydroxyapatite elicits a lower inflammatory response compared with amorphous hydroxyapatite.

  18. F 2 excimer laser (157 nm) radiation modification and surface ablation of PHEMA hydrogels and the effects on bioactivity: Surface attachment and proliferation of human corneal epithelial cells

    NASA Astrophysics Data System (ADS)

    Zainuddin; Chirila, Traian V.; Barnard, Zeke; Watson, Gregory S.; Toh, Chiong; Blakey, Idriss; Whittaker, Andrew K.; Hill, David J. T.

    2011-02-01

    Physical and chemical changes at the surface of poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels modified by ablation with an F 2 excimer laser were investigated experimentally. An important observation was that only the outer exposed surface layers of the hydrogel were affected by the exposure to 157 nm radiation. The effect of the surface changes on the tendency of cells to adhere to the PHEMA was also investigated. A 0.5 cm 2 area of the hydrogel surfaces was exposed to laser irradiation at 157 nm to fluences of 0.8 and 4 J cm -2. The changes in surface topography were analysed by light microscopy and atomic force microscopy, while the surface chemistry was characterized by attenuated total reflection infrared and X-ray photoelectron spectroscopies. Cell-interfacial interactions were examined based on the proliferation of human corneal limbal epithelial (HLE) cells cultured on the laser-modified hydrogels, and on the unexposed hydrogels and tissue culture plastic for comparison. It was observed that the surface topography of laser-exposed hydrogels showed rippled patterns with a surface roughness increasing at the higher exposure dose. The changes in surface chemistry were affected not only by an indirect effect of hydrogen and hydroxyl radicals, formed by water photolysis, on the PHEMA, but also by the direct action of laser radiation on PHEMA if the surface layers of the gel become depleted of water. The laser treatment led to a change in the surface characteristics, with a lower concentration of ester side-chains and the formation of new oxygenated species at the surface. The surface also became more hydrophobic. Most importantly, the surface chemistry and the newly created surface topographical features were able to improve the attachment, spreading and growth of HLE cells.

  19. In vitro investigation of anodization and CaP deposited titanium surface using MG63 osteoblast-like cells

    NASA Astrophysics Data System (ADS)

    Lee, J. M.; Lee, J. I.; Lim, Y. J.

    2010-03-01

    The aim of the present study was to investigate surface characteristics in four different titanium surfaces (AN: anodized at 270 V; AN-CaP: anodic oxidation and CaP deposited; SLA: sandblasted and acid etched; MA: machined) and to evaluate biological behaviors such as cell adhesion, cell proliferation, cytoskeletal organization, and osteogenic protein expression of MG63 osteoblast-like cells at the early stage. Surface analysis was performed using scanning electron microscopy, thin-film X-ray diffractometry, and a confocal laser scanning microscope. In order to evaluate cellular responses, MG63 osteoblast-like cells were used. The cell viability was evaluated by MTT assay. Immunofluorescent analyses of actin, type I collagen, osteonectin and osteocalcin were performed. The anodized and CaP deposited specimen showed homogeneously distributed CaP particles around micropores and exhibited anatase type oxides, titanium, and HA crystalline structures. This experiment suggests that CaP particles on the anodic oxidation surface affect cellular attachment and spreading. When designing an in vitro biological study for CaP coated titanium, it must be taken into account that preincubation in medium prior to cell seeding and the cell culture medium may affect the CaP coatings. All these observations illustrate the importance of the experimental conditions and the physicochemical parameters of the CaP coating. It is considered that further evaluations such as long-term in vitro cellular assays and in vivo experiments should be necessary to figure out the effect of CaP deposition to biological responses.

  20. Cell behavior on surface modified polydimethylsiloxane (PDMS).

    PubMed

    Stanton, Morgan M; Rankenberg, Johanna M; Park, Byung-Wook; McGimpsey, W Grant; Malcuit, Christopher; Lambert, Christopher R

    2014-07-01

    Designing complex tissue culture systems requires cell alignment and directed extracellular matrix (ECM) and gene expression. Here, a micro-rough, polydimethylsiloxane (PDMS) surface, that also integrates a micro-pattern of 50 µm wide lines of fibronectin (FN) separated by 60 µm wide lines of bovine serum albumin (BSA), is developed. Human fibroblasts cultured on the rough, patterned substrate have aligned growth and a significant change in morphology when compared to cells on a flat, patterned surface. The rough PDMS topography significantly decreases cell area and induces the upregulation of several ECM related genes by two-fold when compared to cells cultured on flat PDMS. This study describes a simple surface engineering procedure for creating surface architecture for scaffolds to design and control the cell-surface interface. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Different culture media affect growth characteristics, surface marker distribution and chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells.

    PubMed

    Hagmann, Sebastien; Moradi, Babak; Frank, Sebastian; Dreher, Thomas; Kämmerer, Peer Wolfgang; Richter, Wiltrud; Gotterbarm, Tobias

    2013-07-30

    Bone marrow-derived mesenchymal stromal cells (BM-MSCs) play an important role in modern tissue engineering, while distinct variations of culture media compositions and supplements have been reported. Because MSCs are heterogeneous regarding their regenerative potential and their surface markers, these parameters were compared in four widely used culture media compositions. MSCs were isolated from bone marrow and expanded in four established cell culture media. MSC yield/1000 MNCs, passage time and growth index were observed. In P4, typical MSC surface markers were analysed by fluorescence cytometry. Additionally, chondrogenic, adipogenic and osteogenic differentiation potential were evaluated. Growth index and P0 cell yield varied importantly between the media. The different expansion media had a significant influence on the expression of CD10, CD90, CD105, CD140b CD146 and STRO-1. While no significant differences were observed regarding osteogenic and adipogenic differentiation, chondrogenic differentiation was superior in medium A as reflected by GAG/DNA content. The choice of expansion medium can have a significant influence on growth, differentiation potential and surface marker expression of mesenchymal stromal cells, which is of fundamental importance for tissue engineering procedures.

  2. Accelerated cell-sheet recovery from a surface successively grafted with polyacrylamide and poly(N-isopropylacrylamide).

    PubMed

    Akiyama, Yoshikatsu; Kikuchi, Akihiko; Yamato, Masayuki; Okano, Teruo

    2014-08-01

    A double polymeric nanolayer consisting of poly(N-isopropylacrylamide) (PIPAAm) and hydrophilic polyacrylamide (PAAm) was deposited on tissue culture polystyrene (TCPS) surfaces using electron beam irradiation to form a new temperature-responsive cell culture surface in which the basal hydrophilic PAAm component in the double polymeric layer promotes the hydration of the upper PIPAAm layer and induces rapid cell detachment compared to a conventional temperature-responsive cell culture surface, PIPAAm-grafted TCPS (PIPAAm-TCPS). Take-off angle-dependent X-ray photoelectron spectroscopy spectral analysis demonstrated that the grafted PIPAAm and PAAm components were located in the upper and basal regions of the double polymeric layer, respectively, suggesting that the double polymeric layer forms an inter-penetrating-network-like structure with PAAm at the basal portion of the PIPAAm grafted chains. The wettability of the temperature-responsive cell culture surfaces with the double polymeric layer tended to be more hydrophilic, with an increase in the basal PAAm graft density at a constant PIPAAm graft density. However, when the graft densities of the upper PIPAAm and basal PAAm were optimized, the resulting temperature-responsive cell culture surface with the double polymeric layer exhibited rapid cell detachment while maintaining cell adhesive character comparable to that of PIPAAm-TCPS. The cell adhesive character was altered from cell-adhesive to cell-repellent with increasing PAAm or PIPAAm graft density. The cell adhesive character of the temperature-responsive cell culture surfaces was relatively consistent with their contact angles. These results strongly suggest that the basal PAAm surface properties affect the degree of hydration and dehydration of the subsequently grafted PIPAAm. In addition, the roles of the hydrophilic component in accelerating cell detachment are further discussed in terms of the mobility of the grafted PIPAAm chains. Applications of

  3. The Azospirillum brasilense Che1 chemotaxis pathway controls swimming velocity, which affects transient cell-to-cell clumping.

    PubMed

    Bible, Amber; Russell, Matthew H; Alexandre, Gladys

    2012-07-01

    The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche.

  4. ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation.

    PubMed

    Hsueh, Yi-Huang; Ke, Wan-Ju; Hsieh, Chien-Te; Lin, Kuen-Song; Tzou, Dong-Ying; Chiang, Chao-Lung

    2015-01-01

    Zinc oxide nanoparticles (ZnO NPs) are an important antimicrobial additive in many industrial applications. However, mass-produced ZnO NPs are ultimately disposed of in the environment, which can threaten soil-dwelling microorganisms that play important roles in biodegradation, nutrient recycling, plant protection, and ecological balance. This study sought to understand how ZnO NPs affect Bacillus subtilis, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on B. subtilis growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that B. subtilis growth is inhibited by high concentrations of ZnO NPs (≥ 50 ppm), with cells exhibiting a prolonged lag phase and delayed medial FtsZ ring formation. RedoxSensor and Phag-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, B. subtilis reductase activity, membrane stability, and protein expression all decrease. SDS-PAGE Stains-All staining results and FT-IR data further demonstrate that ZnO NPs negatively affect exopolysaccharide production. Moreover, it was found that B. subtilis biofilm surface structures became smooth under ZnO-NP concentrations of only 5-10 ppm, with concentrations ≤ 25 ppm significantly reducing biofilm formation activity. XANES and EXAFS spectra analysis further confirmed the presence of ZnO in co-cultured B. subtilis cells, which suggests penetration of cell membranes by either ZnO NPs or toxic Zn+ ions from ionized ZnO NPs, the latter of which may be deionized to ZnO within bacterial cells. Together, these results demonstrate that ZnO NPs can affect B. subtilis viability through the inhibition of cell growth, cytosolic protein expression, and biofilm formation, and suggest that future ZnO-NP waste management strategies would do well to mitigate the potential environmental impact engendered by the disposal of these nanoparticles.

  5. ZnO Nanoparticles Affect Bacillus subtilis Cell Growth and Biofilm Formation

    PubMed Central

    Hsueh, Yi-Huang; Ke, Wan-Ju; Hsieh, Chien-Te; Lin, Kuen-Song; Tzou, Dong-Ying; Chiang, Chao-Lung

    2015-01-01

    Zinc oxide nanoparticles (ZnO NPs) are an important antimicrobial additive in many industrial applications. However, mass-produced ZnO NPs are ultimately disposed of in the environment, which can threaten soil-dwelling microorganisms that play important roles in biodegradation, nutrient recycling, plant protection, and ecological balance. This study sought to understand how ZnO NPs affect Bacillus subtilis, a plant-beneficial bacterium ubiquitously found in soil. The impact of ZnO NPs on B. subtilis growth, FtsZ ring formation, cytosolic protein activity, and biofilm formation were assessed, and our results show that B. subtilis growth is inhibited by high concentrations of ZnO NPs (≥ 50 ppm), with cells exhibiting a prolonged lag phase and delayed medial FtsZ ring formation. RedoxSensor and Phag-GFP fluorescence data further show that at ZnO-NP concentrations above 50 ppm, B. subtilis reductase activity, membrane stability, and protein expression all decrease. SDS-PAGE Stains-All staining results and FT-IR data further demonstrate that ZnO NPs negatively affect exopolysaccharide production. Moreover, it was found that B. subtilis biofilm surface structures became smooth under ZnO-NP concentrations of only 5–10 ppm, with concentrations ≤ 25 ppm significantly reducing biofilm formation activity. XANES and EXAFS spectra analysis further confirmed the presence of ZnO in co-cultured B. subtilis cells, which suggests penetration of cell membranes by either ZnO NPs or toxic Zn+ ions from ionized ZnO NPs, the latter of which may be deionized to ZnO within bacterial cells. Together, these results demonstrate that ZnO NPs can affect B. subtilis viability through the inhibition of cell growth, cytosolic protein expression, and biofilm formation, and suggest that future ZnO-NP waste management strategies would do well to mitigate the potential environmental impact engendered by the disposal of these nanoparticles. PMID:26039692

  6. Apatite formation on bioactive calcium-silicate cements for dentistry affects surface topography and human marrow stromal cells proliferation.

    PubMed

    Gandolfi, Maria Giovanna; Ciapetti, Gabriela; Taddei, Paola; Perut, Francesca; Tinti, Anna; Cardoso, Marcio Vivan; Van Meerbeek, Bart; Prati, Carlo

    2010-10-01

    The effect of ageing in phosphate-containing solution of bioactive calcium-silicate cements on the chemistry, morphology and topography of the surface, as well as on in vitro human marrow stromal cells viability and proliferation was investigated. A calcium-silicate cement (wTC) mainly based on dicalcium-silicate and tricalcium-silicate was prepared. Alpha-TCP was added to wTC to obtain wTC-TCP. Bismuth oxide was inserted in wTC to prepare a radiopaque cement (wTC-Bi). A commercial calcium-silicate cement (ProRoot MTA) was tested as control. Cement disks were aged in DPBS for 5 h ('fresh samples'), 14 and 28 days, and analyzed by ESEM/EDX, SEM/EDX, ATR-FTIR, micro-Raman techniques and scanning white-light interferometry. Proliferation, LDH release, ALP activity and collagen production of human marrow stromal cells (MSC) seeded for 1-28 days on the cements were evaluated. Fresh samples exposed a surface mainly composed of calcium-silicate hydrates CSH (from the hydration of belite and alite), calcium hydroxide, calcium carbonate, and ettringite. Apatite nano-spherulites rapidly precipitated on cement surfaces within 5 h. On wTC-TCP the Ca-P deposits appeared thicker than on the other cements. Aged cements showed an irregular porous calcium-phosphate (Ca-P) coating, formed by aggregated apatite spherulites with interspersed calcite crystals. All the experimental cements exerted no acute toxicity in the cell assay system and allowed cell growth. Using biochemical results, the scores were: fresh cements>aged cements for cell proliferation and ALP activity (except for wTC-Bi), whereas fresh cementscell proliferation than aged cements, probably favoured by the presence of Si-OH gel and the early formation of apatite nano-spherulites; (2) the alpha-TCP doped cement aged for 28 days displayed the highest bioactivity and cell proliferation; (3) the deleterious effect of bismuth on cell

  7. Basic surface properties of mononuclear cells from Didelphis marsupialis.

    PubMed

    Nacife, V P; de Meirelles, M de N; Silva Filho, F C

    1998-01-01

    The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5 degrees and 40.8 degrees, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

  8. Multijunction Solar Cell Technology for Mars Surface Applications

    NASA Technical Reports Server (NTRS)

    Stella, Paul M.; Mardesich, Nick; Ewell, Richard C.; Mueller, Robert L.; Endicter, Scott; Aiken, Daniel; Edmondson, Kenneth; Fetze, Chris

    2006-01-01

    Solar cells used for Mars surface applications have been commercial space qualified AM0 optimized devices. Due to the Martian atmosphere, these cells are not optimized for the Mars surface and as a result operate at a reduced efficiency. A multi-year program, MOST (Mars Optimized Solar Cell Technology), managed by JPL and funded by NASA Code S, was initiated in 2004, to develop tools to modify commercial AM0 cells for the Mars surface solar spectrum and to fabricate Mars optimized devices for verification. This effort required defining the surface incident spectrum, developing an appropriate laboratory solar simulator measurement capability, and to develop and test commercial cells modified for the Mars surface spectrum. This paper discusses the program, including results for the initial modified cells. Simulated Mars surface measurements of MER cells and Phoenix Lander cells (2007 launch) are provided to characterize the performance loss for those missions. In addition, the performance of the MER rover solar arrays is updated to reflect their more than two (2) year operation.

  9. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells

    PubMed Central

    Xiong, Jimin; Menicanin, Danijela; Marino, Victor

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein “spots” were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population. PMID:27579043

  10. Investigation of the Cell Surface Proteome of Human Periodontal Ligament Stem Cells.

    PubMed

    Xiong, Jimin; Menicanin, Danijela; Zilm, Peter S; Marino, Victor; Bartold, P Mark; Gronthos, Stan

    2016-01-01

    The present study examined the cell surface proteome of human periodontal ligament stem cells (PDLSC) compared to human fibroblasts. Cell surface proteins were prelabelled with CyDye before processing to extract the membrane lysates, which were separated using 2D electrophoresis. Selected differentially expressed protein "spots" were identified using Mass spectrometry. Four proteins were selected for validation: CD73, CD90, Annexin A2, and sphingosine kinase 1 previously associated with mesenchymal stem cells. Flow cytometric analysis found that CD73 and CD90 were highly expressed by human PDLSC and gingival fibroblasts but not by keratinocytes, indicating that these antigens could be used as potential markers for distinguishing between mesenchymal cells and epithelial cell populations. Annexin A2 was also found to be expressed at low copy number on the cell surface of human PDLSC and gingival fibroblasts, while human keratinocytes lacked any cell surface expression of Annexin A2. In contrast, sphingosine kinase 1 expression was detected in all the cell types examined using immunocytochemical analysis. These proteomic studies form the foundation to further define the cell surface protein expression profile of PDLSC in order to better characterise this cell population and help develop novel strategies for the purification of this stem cell population.

  11. Morphological Observations of Mesenchymal Stem Cell Adhesion to a Nanoperiodic-Structured Titanium Surface Patterned Using Femtosecond Laser Processing

    NASA Astrophysics Data System (ADS)

    Oya, Kei; Aoki, Shun; Shimomura, Kazunori; Sugita, Norihiko; Suzuki, Kenji; Nakamura, Norimasa; Fujie, Hiromichi

    2012-12-01

    It is known that the adhesive and anisotropic properties of cell-derived biomaterials are affected by micro- or nanoscale structures processed on culture surfaces. In the present study, the femtosecond laser processing technique was used to scan a laser beam at an intensity of approximately the ablation threshold level on a titanium surface for nanoscale processing. Microscopy observation revealed that the processed titanium exhibited a periodic-patterned groove structure at the surface; the width and depth of the groove were 292 ±50 and 99 ±31 nm, respectively, and the periodic pitch of the groove was 501 ±100 nm. Human synovium-derived mesenchymal stem cells were cultured on the surface at a cell density of 3.0×103 cells/cm2 after 4 cell passages. For comparison, the cells were also cultured on a nonprocessed titanium surface under the condition identical to that of the processed surface. Results revealed that the duration for cell attachment to the surface was markedly reduced on the processed titanium as compared with the nonprocessed titanium. Moreover, on the processed titanium, cell extension area significantly increased while cell orientation was aligned along the direction of the periodic grooves. These results suggest that the femtosecond laser processing improves the adhesive and anisotropic properties of cells by producing the nanoperiodic structure on titanium culture surfaces.

  12. A Cell-surface Phylome for African Trypanosomes

    PubMed Central

    Jackson, Andrew P.; Allison, Harriet C.; Barry, J. David; Field, Mark C.; Hertz-Fowler, Christiane; Berriman, Matthew

    2013-01-01

    The cell surface of Trypanosoma brucei, like many protistan blood parasites, is crucial for mediating host-parasite interactions and is instrumental to the initiation, maintenance and severity of infection. Previous comparisons with the related trypanosomatid parasites T. cruzi and Leishmania major suggest that the cell-surface proteome of T. brucei is largely taxon-specific. Here we compare genes predicted to encode cell surface proteins of T. brucei with those from two related African trypanosomes, T. congolense and T. vivax. We created a cell surface phylome (CSP) by estimating phylogenies for 79 gene families with putative surface functions to understand the more recent evolution of African trypanosome surface architecture. Our findings demonstrate that the transferrin receptor genes essential for bloodstream survival in T. brucei are conserved in T. congolense but absent from T. vivax and include an expanded gene family of insect stage-specific surface glycoproteins that includes many currently uncharacterized genes. We also identify species-specific features and innovations and confirm that these include most expression site-associated genes (ESAGs) in T. brucei, which are absent from T. congolense and T. vivax. The CSP presents the first global picture of the origins and dynamics of cell surface architecture in African trypanosomes, representing the principal differences in genomic repertoire between African trypanosome species and provides a basis from which to explore the developmental and pathological differences in surface architectures. All data can be accessed at: http://www.genedb.org/Page/trypanosoma_surface_phylome. PMID:23556014

  13. Glycobiology of the cell surface: Its debt to cell electrophoresis 1940-65.

    PubMed

    Cook, Geoffrey M W

    2016-06-01

    This Review describes how in the period 1940-1959 cell electrophoresis (in the earlier literature often referred to as 'microelectrophoresis') was used to explore the surface chemistry of cells. Using the erythrocyte as a suitable model for the study of biological membranes, the early investigators were agreed on the presence of negatively charged groups at the surface of this cell. The contemporary dogma was that these were phosphate groups associated with phospholipids. Work in the 1960s, particularly on changes in the electrokinetic properties of erythrocytes following treatment with proteolytic enzymes, lead to the realization that the negatively charged groups at the red cell surface are predominantly due to sialic acids carried on glycoproteins. It quickly became apparent from cell electrophoresis that sialic acids have a ubiquitous presence on the surface of animal cells. This finding required that any complete model of the plasma membrane must include glycosylated molecules at the cell periphery, thus laying the foundations for the field termed 'Glycobiology of the Cell Surface'. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Differential responses of osteoblast lineage cells to nanotopographically-modified, microroughened titanium-aluminum-vanadium alloy surfaces.

    PubMed

    Gittens, Rolando A; Olivares-Navarrete, Rene; McLachlan, Taylor; Cai, Ye; Hyzy, Sharon L; Schneider, Jennifer M; Schwartz, Zvi; Sandhage, Kenneth H; Boyan, Barbara D

    2012-12-01

    Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications. Copyright © 2012 Elsevier Ltd. All rights reserved.

  15. Differential Responses of Osteoblast Lineage Cells to Nanotopographically-Modified, Microroughened Titanium-Aluminum-Vanadium Alloy Surfaces

    PubMed Central

    Gittens, Rolando A.; Olivares-Navarrete, Rene; McLachlan, Taylor; Cai, Ye; Hyzy, Sharon L.; Schneider, Jennifer M.; Schwartz, Zvi; Sandhage, Kenneth H.; Boyan, Barbara D.

    2013-01-01

    Surface structural modifications at the micrometer and nanometer scales have driven improved success rates of dental and orthopaedic implants by mimicking the hierarchical structure of bone. However, how initial osteoblast-lineage cells populating an implant surface respond to different hierarchical surface topographical cues remains to be elucidated, with bone marrow mesenchymal stem cells (MSCs) or immature osteoblasts as possible initial colonizers. Here we show that in the absence of any exogenous soluble factors, osteoblastic maturation of primary human osteoblasts (HOBs) but not osteoblastic differentiation of MSCs is strongly influenced by nanostructures superimposed onto a microrough Ti6Al4V (TiAlV) alloy. The sensitivity of osteoblasts to both surface microroughness and nanostructures led to a synergistic effect on maturation and local factor production. Osteoblastic differentiation of MSCs was sensitive to TiAlV surface microroughness with respect to production of differentiation markers, but no further enhancement was found when cultured on micro/nanostructured surfaces. Superposition of nanostructures to microroughened surfaces affected final MSC numbers and enhanced production of vascular endothelial growth factor (VEGF) but the magnitude of the response was lower than for HOB cultures. Our results suggest that the differentiation state of osteoblast-lineage cells determines the recognition of surface nanostructures and subsequent cell response, which has implications for clinical evaluation of new implant surface nanomodifications. PMID:22989383

  16. Surface cell immobilization within perfluoroalkoxy microchannels

    NASA Astrophysics Data System (ADS)

    Stojkovič, Gorazd; Krivec, Matic; Vesel, Alenka; Marinšek, Marjan; Žnidaršič-Plazl, Polona

    2014-11-01

    Perfluoroalkoxy (PFA) is one of the most promising materials for the fabrication of cheap, solvent resistant and reusable microfluidic chips, which have been recently recognized as effective tools for biocatalytic process development. The application of biocatalysts significantly depends on efficient immobilization of enzymes or cells within the reactor enabling long-term biocatalyst use. Functionalization of PFA microchannels by 3-aminopropyltriethoxysilane (ATPES) and glutaraldehyde was used for rapid preparation of microbioreactors with surface-immobilized cells. X-ray photoelectron spectroscopy and scanning electron microscopy were used to accurately monitor individual treatment steps and to select conditions for cell immobilization. The optimized protocol for Saccharomyces cerevisiae immobilization on PFA microchannel walls comprised ethanol surface pretreatment, 4 h contacting with 10% APTES aqueous solution, 10 min treatment with 1% glutaraldehyde and 20 min contacting with cells in deionized water. The same protocol enabled also immobilization of Escherichia coli, Pseudomonas putida and Bacillus subtilis cells on PFA surface in high densities. Furthermore, the developed procedure has been proved to be very efficient also for surface immobilization of tested cells on other materials that are used for microreactor fabrication, including glass, polystyrene, poly (methyl methacrylate), polycarbonate, and two olefin-based polymers, namely Zeonor® and Topas®.

  17. Aggregate formation affects ultrasonic disruption of microalgal cells.

    PubMed

    Wang, Wei; Lee, Duu-Jong; Lai, Juin-Yih

    2015-12-01

    Ultrasonication is a cell disruption process of low energy efficiency. This study dosed K(+), Ca(2+) and Al(3+) to Chlorella vulgaris cultured in Bold's Basal Medium at 25°C and measured the degree of cell disruption under ultrasonication. Adding these metal ions yielded less negatively charged surfaces of cells, while with the latter two ions large and compact cell aggregates were formed. The degree of cell disruption followed: control=K(+)>Ca(2+)>Al(3+) samples. Surface charges of cells and microbubbles have minimal effects on the microbubble number in the proximity of the microalgal cells. Conversely, cell aggregates with large size and compact interior resist cell disruption under ultrasonication. Staining tests revealed high diffusional resistance of stains over the aggregate interior. Microbubbles may not be effective generated and collapsed inside the compact aggregates, hence leading to low cell disruption efficiencies. Effective coagulation/flocculation in cell harvesting may lead to adverse effect on subsequent cell disruption efficiency. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. A Molecular Smart Surface for Spatio-Temporal Studies of Cell Mobility

    PubMed Central

    Lee, Eun-ju; Luo, Wei; Chan, Eugene W. L.; Yousaf, Muhammad N.

    2015-01-01

    Active migration in both healthy and malignant cells requires the integration of information derived from soluble signaling molecules with positional information gained from interactions with the extracellular matrix and with other cells. How a cell responds and moves involves complex signaling cascades that guide the directional functions of the cytoskeleton as well as the synthesis and release of proteases that facilitate movement through tissues. The biochemical events of the signaling cascades occur in a spatially and temporally coordinated manner then dynamically shape the cytoskeleton in specific subcellular regions. Therefore, cell migration and invasion involve a precise but constantly changing subcellular nano-architecture. A multidisciplinary effort that combines new surface chemistry and cell biological tools is required to understand the reorganization of cytoskeleton triggered by complex signaling during migration. Here we generate a class of model substrates that modulate the dynamic environment for a variety of cell adhesion and migration experiments. In particular, we use these dynamic substrates to probe in real-time how the interplay between the population of cells, the initial pattern geometry, ligand density, ligand affinity and integrin composition affects cell migration and growth. Whole genome microarray analysis indicates that several classes of genes ranging from signal transduction to cytoskeletal reorganization are differentially regulated depending on the nature of the surface conditions. PMID:26030281

  19. Iron oxide nanoparticles surface coating and cell uptake affect biocompatibility and inflammatory responses of endothelial cells and macrophages

    NASA Astrophysics Data System (ADS)

    Orlando, Antonina; Colombo, Miriam; Prosperi, Davide; Gregori, Maria; Panariti, Alice; Rivolta, Ilaria; Masserini, Massimo; Cazzaniga, Emanuela

    2015-09-01

    Engineered iron oxide nanoparticles (IONP) offer the possibility of a wide range of medical uses, from clinical imaging to magnetically based hyperthermia for tumor treatment. These applications require their systemic administration in vivo. An important property of nanoparticles is their stability in biological media. For this purpose, a multicomponent nanoconstruct combining high colloidal stability and improved physical properties was synthesized and characterized. IONP were coated with an amphiphilic polymer (PMA), which confers colloidal stability, and were pegylated in order to obtain the nanoconstruct PEG-IONP-PMA. The aim of this study was to utilize cultured human endothelial cells (HUVEC) and murine macrophages, taken as model of cells exposed to NP after systemic administration, to assess the biocompatibility of PEG-IONP-PMA (23.1 ± 1.4 nm) or IONP-PMA (15.6 ± 3.4 nm). PEG-IONP-PMA, tested at different concentrations as high as 20 μg mL-1, exhibited no cytotoxicity or inflammatory responses. By contrast, IONP-PMA showed a concentration-dependent increase of cytotoxicity and of TNF-α production by macrophages and NO production by HUVECs. Cell uptake analysis suggested that after PEGylation, IONP were less internalized either by macrophages or by HUVEC. These results suggest that the choice of the polymer and the chemistry of surface functionalization are a crucial feature to confer to IONP biocompatibility.

  20. Surface micro- and nano-texturing of stainless steel by femtosecond laser for the control of cell migration.

    PubMed

    Martínez-Calderon, M; Manso-Silván, M; Rodríguez, A; Gómez-Aranzadi, M; García-Ruiz, J P; Olaizola, S M; Martín-Palma, R J

    2016-11-02

    The precise control over the interaction between cells and the surface of materials plays a crucial role in optimizing the integration of implanted biomaterials. In this regard, material surface with controlled topographic features at the micro- and nano-scales has been proved to affect the overall cell behavior and therefore the final osseointegration of implants. Within this context, femtosecond (fs) laser micro/nano machining technology was used in this work to modify the surface structure of stainless steel aiming at controlling cell adhesion and migration. The experimental results show that cells tend to attach and preferentially align to the laser-induced nanopatterns oriented in a specific direction. Accordingly, the laser-based fabrication method here described constitutes a simple, clean, and scalable technique which allows a precise control of the surface nano-patterning process and, subsequently, enables the control of cell adhesion, migration, and polarization. Moreover, since our surface-patterning approach does not involve any chemical treatments and is performed in a single step process, it could in principle be applied to most metallic materials.

  1. Surface micro- and nano-texturing of stainless steel by femtosecond laser for the control of cell migration

    PubMed Central

    Martínez-Calderon, M.; Manso-Silván, M.; Rodríguez, A.; Gómez-Aranzadi, M.; García-Ruiz, J. P.; Olaizola, S. M.; Martín-Palma, R. J.

    2016-01-01

    The precise control over the interaction between cells and the surface of materials plays a crucial role in optimizing the integration of implanted biomaterials. In this regard, material surface with controlled topographic features at the micro- and nano-scales has been proved to affect the overall cell behavior and therefore the final osseointegration of implants. Within this context, femtosecond (fs) laser micro/nano machining technology was used in this work to modify the surface structure of stainless steel aiming at controlling cell adhesion and migration. The experimental results show that cells tend to attach and preferentially align to the laser-induced nanopatterns oriented in a specific direction. Accordingly, the laser-based fabrication method here described constitutes a simple, clean, and scalable technique which allows a precise control of the surface nano-patterning process and, subsequently, enables the control of cell adhesion, migration, and polarization. Moreover, since our surface-patterning approach does not involve any chemical treatments and is performed in a single step process, it could in principle be applied to most metallic materials. PMID:27805063

  2. Clostridium perfringens Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry

    PubMed Central

    Stiles, Bradley G.; Hale, Martha L.; Marvaud, Jean-Christophe; Popoff, Michel R.

    2000-01-01

    The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane. PMID:10816501

  3. Clostridium perfringens iota toxin: binding studies and characterization of cell surface receptor by fluorescence-activated cytometry.

    PubMed

    Stiles, B G; Hale, M L; Marvaud, J C; Popoff, M R

    2000-06-01

    The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37 degrees C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37 degrees C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane.

  4. Direct Peptide Interaction with Surface Glycosaminoglycans Contributes to the Cell Penetration of Maurocalcine*

    PubMed Central

    Ram, Narendra; Aroui, Sonia; Jaumain, Emilie; Bichraoui, Hicham; Mabrouk, Kamel; Ronjat, Michel; Lortat-Jacob, Hugues; De Waard, Michel

    2008-01-01

    Maurocalcine (MCa), initially identified from a tunisian scorpion venom, defines a new member of the family of cell penetrating peptides by its ability to efficiently cross the plasma membrane. The initiating mechanistic step required for the cell translocation of a cell penetrating peptide implicates its binding onto cell surface components such as membrane lipids and/or heparan sulfate proteoglycans. Here we characterized the interaction of wild-type MCa and MCa K20A, a mutant analogue with reduced cell-penetration efficiency, with heparin (HP) and heparan sulfates (HS) through surface plasma resonance. HP and HS bind both to MCa, indicating that heparan sulfate proteoglycans may represent an important entry route of the peptide. This is confirmed by the fact that (i) both compounds bind with reduced affinity to MCa K20A and (ii) the cell penetration of wild-type or mutant MCa coupled to fluorescent streptavidin is reduced by about 50% in mutant Chinese hamster ovary cell lines lacking either all glycosaminoglycans (GAGs) or just HS. Incubating MCa with soluble HS, HP, or chondroitin sulfates also inhibits the cell penetration of MCa-streptavidin complexes. Analyses of the cell distributions of MCa/streptavidin in several Chinese hamster ovary cell lines show that the distribution of the complex coincides with the endosomal marker Lyso-Tracker red and is not affected by the absence of GAGs. The distribution of MCa/streptavidin is not coincident with that of transferrin receptors nor affected by a dominant-negative dynamin 2 K44A mutant, an inhibitor of clathrin-mediated endocytosis. However, entry of the complex is greatly diminished by amiloride, indicating the importance of macropinocytosis in MCa/streptavidin entry. It is concluded that (i) interaction of MCa with GAGs quantitatively improves the cell penetration of MCa, and (ii) GAG-dependent and -independent MCa penetration rely similarly on the macropinocytosis pathway. PMID:18603532

  5. Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

    Betz, N. A.; Fattaey, H. K.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1994-01-01

    While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

  6. The Azospirillum brasilense Che1 Chemotaxis Pathway Controls Swimming Velocity, Which Affects Transient Cell-to-Cell Clumping

    PubMed Central

    Bible, Amber; Russell, Matthew H.

    2012-01-01

    The Che1 chemotaxis-like pathway of Azospirillum brasilense contributes to chemotaxis and aerotaxis, and it has also been found to contribute to regulating changes in cell surface adhesive properties that affect the propensity of cells to clump and to flocculate. The exact contribution of Che1 to the control of chemotaxis and flocculation in A. brasilense remains poorly understood. Here, we show that Che1 affects reversible cell-to-cell clumping, a cellular behavior in which motile cells transiently interact by adhering to one another at their nonflagellated poles before swimming apart. Clumping precedes and is required for flocculation, and both processes appear to be independently regulated. The phenotypes of a ΔaerC receptor mutant and of mutant strains lacking cheA1, cheY1, cheB1, or cheR1 (alone or in combination) or with che1 deleted show that Che1 directly mediates changes in the flagellar swimming velocity and that this behavior directly modulates the transient nature of clumping. Our results also suggest that an additional receptor(s) and signaling pathway(s) are implicated in mediating other Che1-independent changes in clumping identified in the present study. Transient clumping precedes the transition to stable clump formation, which involves the production of specific extracellular polysaccharides (EPS); however, production of these clumping-specific EPS is not directly controlled by Che1 activity. Che1-dependent clumping may antagonize motility and prevent chemotaxis, thereby maintaining cells in a metabolically favorable niche. PMID:22522896

  7. Characterizing deformability and surface friction of cancer cells

    PubMed Central

    Byun, Sangwon; Son, Sungmin; Amodei, Dario; Cermak, Nathan; Shaw, Josephine; Kang, Joon Ho; Hecht, Vivian C.; Winslow, Monte M.; Jacks, Tyler; Mallick, Parag; Manalis, Scott R.

    2013-01-01

    Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell’s passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces. PMID:23610435

  8. Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chang, Eun-Ju; Kim, Hong-Hee; Huh, Jung-Eun

    2005-02-01

    The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growthmore » factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.« less

  9. DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells

    PubMed Central

    Mulrooney, Tiernan J.; Posch, Phillip E.; Hurley, Carolyn Katovich

    2013-01-01

    KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules. PMID:23715743

  10. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against alpha-Enolase.

    PubMed

    López-Alemany, Roser; Longstaff, Colin; Hawley, Stephen; Mirshahi, Massoud; Fábregas, Pere; Jardí, Merce; Merton, Elizabeth; Miles, Lindsey A; Félez, Jordi

    2003-04-01

    Localization of plasmin activity on leukocyte surfaces plays a critical role in fibrinolysis as well as in pathological and physiological processes in which cells must degrade the extracellular matrix in order to migrate. The binding of plasminogen to leukocytic cell lines induces a 30- to 80-fold increase in the rate of plasminogen activation by tissue-type (tPA) and urokinase-type (uPA) plasminogen activators. In the present study we have examined the role of alpha-enolase in plasminogen activation on the cell surface. We produced and characterized a monoclonal antibody (MAb) 11G1 against purified alpha-enolase, which abrogated about 90% of cell-dependent plasminogen activation by either uPA or tPA on leukocytoid cell lines of different lineages: B-lymphocytic, T-lymphocytic, granulocytic, and monocytic cells. In addition, MAb 11G1 also blocked enhancement of plasmin formation by peripheral blood neutrophils and monocytes. In contrast, MAb 11G1 did not affect plasmin generation in the presence of fibrin, indicating that this antibody did not interact with fibrinolytic components in the absence of cells. These data suggest that, although leukocytic cells display several molecules that bind plasminogen, alpha-enolase is responsible for the majority of the promotion of plasminogen activation on the surfaces of leukocytic cells. Copyright 2003 Wiley-Liss, Inc.

  11. Redesigning of Microbial Cell Surface and Its Application to Whole-Cell Biocatalysis and Biosensors.

    PubMed

    Han, Lei; Zhao, Yukun; Cui, Shan; Liang, Bo

    2018-06-01

    Microbial cell surface display technology can redesign cell surfaces with functional proteins and peptides to endow cells some unique features. Foreign peptides or proteins are transported out of cells and immobilized on cell surface by fusing with anchoring proteins, which is an effective solution to avoid substance transfer limitation, enzyme purification, and enzyme instability. As the most frequently used prokaryotic and eukaryotic protein surface display system, bacterial and yeast surface display systems have been widely applied in vaccine, biocatalysis, biosensor, bioadsorption, and polypeptide library screening. In this review of bacterial and yeast surface display systems, different cell surface display mechanisms and their applications in biocatalysis as well as biosensors are described with their strengths and shortcomings. In addition to single enzyme display systems, multi-enzyme co-display systems are presented here. Finally, future developments based on our and other previous reports are discussed.

  12. Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells.

    PubMed

    Khor, Hwei Ling; Kuan, Yujun; Kukula, Hildegard; Tamada, Kaoru; Knoll, Wolfgang; Moeller, Martin; Hutmacher, Dietmar W

    2007-05-01

    Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.

  13. Biological Behavior of Osteoblast Cell and Apatite Forming Ability of the Surface Modified Ti Alloys.

    PubMed

    Zhao, Jingming; Hwang, K H; Choi, W S; Shin, S J; Lee, J K

    2016-02-01

    Titanium as one kind of biomaterials comes in direct contact with the body, making evaluation of biocompatibility an important aspect to biomaterials development. Surface chemistry of titanium plays an important role in osseointegration. Different surface modification alters the surface chemistry and result in different biological response. In this study, three kinds of mixed acid solutions were used to treat Ti specimens to induce Ca-P formation. Following a strong mixed acid activation process, Ca-P coating successfully formed on the Ti surfaces in simulated body fluid. Strong mixed acid increased the roughness of the metal surface, because the porous and rough surface allows better adhesion between Ca-P coatings and substrates. After modification of titanium surface by mixed acidic solution and subsequently H2O2/HCL treatment evaluation of biocompatibility was conducted from hydroxyapatite formation by biomimetic process and cell viability on modified titanium surface. Nano-scale modification of titanium surfaces can alter cellular and tissue responses, which may benefit osseointegration and dental implant therapy. Results from this study indicated that surface treatment methods affect the surface morphology, type of TiO2 layer formed and subsequent apatite deposition and biological responses. The thermo scientific alamarblue cell viability assay reagent is used to quantitatively measure the viability of mammalian cell lines, bacteria and fungi by incorporating a rapid, sensitive and reliable fluorometric/colorimetric growth indicator, without any toxic and side effect to cell line. In addition, mixed acid treatment uses a lower temperature and shorter time period than widely used alkali treatment.

  14. Proton exchange membrane and electrode surface areas as factors that affect power generation in microbial fuel cells.

    PubMed

    Oh, Sang-Eun; Logan, Bruce E

    2006-03-01

    Power generation in microbial fuel cells (MFCs) is a function of the surface areas of the proton exchange membrane (PEM) and the cathode relative to that of the anode. To demonstrate this, the sizes of the anode and cathode were varied in two-chambered MFCs having PEMs with three different surface areas (A (PEM)=3.5, 6.2, or 30.6 cm(2)). For a fixed anode and cathode surface area (A (An)=A (Cat)=22.5 cm(2)), the power density normalized to the anode surface area increased with the PEM size in the order 45 mW/m(2) (A (PEM)=3.5 cm(2)), 68 mW/m(2) (A (PEM)=6.2 cm(2)), and 190 mW/m(2) (A (PEM)=30.6 cm(2)). PEM surface area was shown to limit power output when the surface area of the PEM was smaller than that of the electrodes due to an increase in internal resistance. When the relative cross sections of the PEM, anode, and cathode were scaled according to 2A (Cat)=A(PEM)=2A (An), the maximum power densities of the three different MFCs, based on the surface area of the PEM (A (PEM)=3.5, 6.2, or 30.6 cm(2)), were the same (168+/-4.53 mW/m(2)). Increasing the ionic strength and using ferricyanide at the cathode also increased power output.

  15. Laser surface treatment of polyamide and NiTi alloy and the effects on mesenchymal stem cell response

    NASA Astrophysics Data System (ADS)

    Waugh, D. G.; Lawrence, J.; Shukla, P.; Chan, C.; Hussain, I.; Man, H. C.; Smith, G. C.

    2015-07-01

    Mesenchymal stem cells (MSCs) are known to play important roles in development, post-natal growth, repair, and regeneration of mesenchymal tissues. What is more, surface treatments are widely reported to affect the biomimetic nature of materials. This paper will detail, discuss and compare laser surface treatment of polyamide (Polyamide 6,6), using a 60 W CO2 laser, and NiTi alloy, using a 100 W fiber laser, and the effects of these treatments on mesenchymal stem cell response. The surface morphology and composition of the polyamide and NiTi alloy were studied by scanning electron microscopy (SEM) and X-ray photoemission spectroscopy (XPS), respectively. MSC cell morphology cell counting and viability measurements were done by employing a haemocytometer and MTT colorimetric assay. The success of enhanced adhesion and spreading of the MSCs on each of the laser surface treated samples, when compared to as-received samples, is evidenced in this work.

  16. Surface complexation modeling of proton and Cd adsorption onto an algal cell wall.

    PubMed

    Kaulbach, Emily S; Szymanowski, Jennifer E S; Fein, Jeremy B

    2005-06-01

    This study quantifies Cd adsorption onto the cell wall of the algal species Pseudokirchneriella subcapitata by applying a surface complexation approach to model the observed adsorption behavior. We use potentiometric titrations to determine deprotonation constants and site concentrations for the functional groups on the algal cell wall. Adsorption and desorption kinetics experiments illustrate that adsorption of Cd onto the cell wall is rapid and reversible, except under low pH conditions. Adsorption experiments conducted as a function of pH and total Cd concentration yield the stoichiometry and site-specific stability constants for the important Cd-algal surface complexes. We model the acid/base properties of the algal cell wall by invoking four discrete surface functional group types, with pKa values of 3.9 +/- 0.3, 5.4 +/- 0.1, 7.6 +/- 0.3, and 9.6 +/- 0.4. The results of the Cd adsorption experiments indicate that the first, third, and fourth sites contribute to Cd adsorption under the experimental conditions, with calculated log stability constant values of 4.1 +/- 0.5, 5.4 +/- 0.5, and 6.1 +/- 0.4, respectively. Our results suggest that the stabilities of the Cd-surface complexes are high enough for algal adsorption to affect the fate and transport of Cd under some conditions and that on a per gram basis, algae and bacteria exhibit broadly similar extents of Cd adsorption.

  17. Optimizing surface characteristics for cell adhesion and proliferation on titanium plasma spray coatings on polyetheretherketone.

    PubMed

    Yoon, Byung Jo Victor; Xavier, Fred; Walker, Brendon R; Grinberg, Samuel; Cammisa, Frank P; Abjornson, Celeste

    2016-10-01

    Titanium plasma spray coating on polyetheretherketone (PEEK) is a recent innovation to interbody spacer technology. The inherent hydrophobic properties of standard, uncoated PEEK implants can hamper cell attachment and bone healing during fusion. The addition of titanium coating not only offers initial stability due to increased surface roughness but also long-term stability due to bony ongrowth created from osteoconductive microenvironment on the device surface. The previously established hydrophilic and osteophilic properties of commercially pure titanium (CPTi) can potentially provide an ideal environment promoting cell attachment and bony ongrowth when applied at the end plate level of the fusion site. Because the surface material composition and topography is what seems to directly affect cell adhesion, it is important to determine the ideal titanium coating for the highest effectiveness. The purpose of the study is to determine whether there is an optimal surface roughness for the titanium coatings and whether different polishing methods have a greater effect than roughness or topography in mediating cell adhesion to the surface. The study was divided into two phases. In Phase 1, the effects of varying surface roughnesses on identical polishing method were compared. In Phase 2, the effect of varying polishing methods was compared on identical surface roughnesses. Coating thickness, porosity, and surface roughness were characterized using an optical microscope as per ASTM F 1854 standards. For both phases, PEEK coupons with plasma-sprayed CPTi were used, and human mesenchymal stem cells (hMSCs) at an initial density of 25,000 cells/cm 2 were seeded and cultured for 24 hours before fixation in 10% formalin. The cultured hMSCs were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining, a fluorescent stain that binds to the DNA of living cells. Samples were imaged using an environmental scanning electron microscope (eSEM) (Carl Zeiss Microscopy, Thornwood

  18. Red cell surface changes in cold agglutination

    PubMed Central

    Salsbury, A. J.; Clarke, J. A.; Shand, W. S.

    1968-01-01

    Surface changes in red blood cells undergoing cold agglutination have been investigated using the Cambridge Stereoscan electron microscope. On incubation of red cells with a cold agglutinin of anti-I specificity at 4°C, circular shadows on the red cell membrane developed within 2 min. At the same time the membrane showed a granularity and processes began to develop on the surface. These processes increased in length, the processes of contiguous cells became interlinked and agglutination was complete after incubation of 1 hr. On warming an agglutinated specimen, the process was reversed with separation of red cells and retraction of the finger-like processes to yield discrete red cells of normal appearance. The addition of heparin in vivo prevented agglutination but did not inhibit surface changes completely. Complement appeared to play no part in the production of cold agglutination due to these antibodies or in the reversal of agglutination by warming. The significance of the surface changes described in relation to previous information on the mechanism of agglutination, has been discussed. ImagesFig. 1Fig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11 PMID:5655472

  19. Combined use of atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry for cell surface analysis.

    PubMed

    Dague, Etienne; Delcorte, Arnaud; Latgé, Jean-Paul; Dufrêne, Yves F

    2008-04-01

    Understanding the surface properties of microbial cells is a major challenge of current microbiological research and a key to efficiently exploit them in biotechnology. Here, we used three advanced surface analysis techniques with different sensitivity, probing depth, and lateral resolution, that is, in situ atomic force microscopy, X-ray photoelectron spectroscopy, and secondary ion mass spectrometry, to gain insight into the surface properties of the conidia of the human fungal pathogen Aspergillus fumigatus. We show that the native ultrastructure, surface protein and polysaccharide concentrations, and amino acid composition of three mutants affected in hydrophobin production are markedly different from those of the wild-type, thereby providing novel insight into the cell wall architecture of A. fumigatus. The results demonstrate the power of using multiple complementary techniques for probing microbial cell surfaces.

  20. Isolation by cell-column chromatography of immunoglobulins specific for cell surface carbohydrates

    PubMed Central

    1977-01-01

    A new method of affinity chromatography using glutaraldehyde-fixed cells immobilized on Sephadex beads has been used to isolate immunoglobulins (Ig's) specific for cell surface glycoproteins. Ig's that specifically bound and agglutinated the same cells as those originally fixed on the columns were isolated from nonimmune sera of various species. Periodate treatment of the cell-columns and the free cells destroyed their ability to bind the Ig's, and the binding of the Ig's to untreated cells was inhibited by monosaccharides such as D- galactose and sialic acid. The binding of antibodies directed against cell surfaces obtained by immunizing animals with the same mouse tumor cell lines used on the columns (P388 and EL4) was not inhibited by various saccharides. Surface glycoproteins obtained from the mouse tumor cells by immunoprecipitation with the column-isolated Ig's yielded specific electrophoretic patterns that differed from those obtained using Ig's from the sera of rabbits immunized with the tumor cells. The data suggest that the Ig's isolated by cell-column chromatography were directed against carbohydrates, probably those in terminal positions of the polysaccharide portions of the tumor cell surface glycoproteins. Column-isolated Ig's specific for carbohydrates were also useful in studies of cell interactions in nonmammalian systems including Dictyostelium discoideum and Saccharomyces cerevisiae. The cell-column method appears to be adaptable to the isolation of a variety of molecules in addition to antibodies. PMID:833547

  1. Modeling electrochemical resistance with coal surface properties in a direct carbon fuel cell based on molten carbonate

    NASA Astrophysics Data System (ADS)

    Eom, Seongyong; Ahn, Seongyool; Kang, Kijoong; Choi, Gyungmin

    2017-12-01

    In this study, a numerical model of activation and ohmic polarization is modified, taking into account the correlation function between surface properties and inner resistance. To investigate the correlation function, the surface properties of coal are changed by acid treatment, and the correlations between the inner resistance measured by half-cell tests and the surface characteristics are analyzed. A comparison between the model and experimental results demonstrates that the absolute average deviations for each fuel are less than 10%. The numerical results show that the sensitivities of the coal surface properties affecting polarization losses change depending on the operating temperature. The surface oxygen concentrations affect the activation polarization and the sensitivity decreased with increasing temperature. The surface ash of coal is an additional index to be considered along with ohmic polarization and it has the greatest effect on the surface properties at 973 K.

  2. Micropattern array with gradient size (µPAGS) plastic surfaces fabricated by PDMS (polydimethylsiloxane) mold-based hot embossing technique for investigation of cell-surface interaction.

    PubMed

    Choi, Min Jin; Park, Ju Young; Cha, Kyoung Je; Rhie, Jong-Won; Cho, Dong-Woo; Kim, Dong Sung

    2012-12-01

    Recently, it was found that the variations of physical environment significantly affect cell behaviors including cell proliferation, migration and differentiation. Through a plastic surface with controlled mechanical properties such as stiffness, one can change the orientation and migration of cells in a particular direction, thereby determining cell behaviors. In this study, we demonstrate a polydimethylsiloxane (PDMS) mold-based hot embossing technique for rapid, simple and low-cost replication of polystyrene (PS) surfaces having micropatterns. The PDMS mold was fabricated by UV-photolithography followed by PDMS casting; the elastomeric properties of PDMS enabled us to obtain conformal contact of the PDMS mold to a PS surface and to create high transcription quality of micropatterns on the PS surface. Two different types of circular micropillar and microwell arrays were successfully replicated on the PS surfaces based on the suggested technique. The micropatterns were designed to have various diameters (2-150 µm), spacings (2-160 µm) and heights (1.4, 2.4, 8.2 and 14.9 µm), so as to generate the gradient of physical properties on the surface. Experimental parametric studies indicated that (1) the embossing temperature became a critical processing parameter as the aspect ratio of micropattern increased and (2) the PDMS mold-based hot embossing could successfully replicate micropatterns, even having an aspect ratio of 2.7 for micropattern diameter of 6 µm, with an optimal processing condition (embossing pressure and temperature of 0.4 MPa and 130 °C, respectively) in this study. We carried out cell experiments with adipose-derived stem cells on the replicated PS surface with the height of 1.4 µm to investigate cellular behaviors in response to the micropattern array with gradient size. Cellular experiment results showed that the micropillar-arrayed surface improved cell proliferation as compared with the microwell-arrayed surface. We could also estimate the

  3. The cell surface environment for pathogen recognition and entry.

    PubMed

    Stow, Jennifer L; Condon, Nicholas D

    2016-04-01

    The surface of mammalian cells offers an interface between the cell interior and its surrounding milieu. As part of the innate immune system, macrophages have cell surface features optimised for probing and sampling as they patrol our tissues for pathogens, debris or dead cells. Their highly dynamic and constantly moving cell surface has extensions such as lamellipodia, filopodia and dorsal ruffles that help detect pathogens. Dorsal ruffles give rise to macropinosomes for rapid, high volume non-selective fluid sampling, receptor internalisation and plasma membrane turnover. Ruffles can also generate phagocytic cups for the receptor-mediated uptake of pathogens or particles. The membrane lipids, actin cytoskeleton, receptors and signalling proteins that constitute these cell surface domains are discussed. Although the cell surface is designed to counteract pathogens, many bacteria, viruses and other pathogens have evolved to circumvent or hijack these cell structures and their underlying machinery for entry and survival. Nevertheless, these features offer important potential for developing vaccines, drugs and preventative measures to help fight infection.

  4. New Monoclonal Antibodies to Defined Cell Surface Proteins on Human Pluripotent Stem Cells.

    PubMed

    O'Brien, Carmel M; Chy, Hun S; Zhou, Qi; Blumenfeld, Shiri; Lambshead, Jack W; Liu, Xiaodong; Kie, Joshua; Capaldo, Bianca D; Chung, Tung-Liang; Adams, Timothy E; Phan, Tram; Bentley, John D; McKinstry, William J; Oliva, Karen; McMurrick, Paul J; Wang, Yu-Chieh; Rossello, Fernando J; Lindeman, Geoffrey J; Chen, Di; Jarde, Thierry; Clark, Amander T; Abud, Helen E; Visvader, Jane E; Nefzger, Christian M; Polo, Jose M; Loring, Jeanne F; Laslett, Andrew L

    2017-03-01

    The study and application of human pluripotent stem cells (hPSCs) will be enhanced by the availability of well-characterized monoclonal antibodies (mAbs) detecting cell-surface epitopes. Here, we report generation of seven new mAbs that detect cell surface proteins present on live and fixed human ES cells (hESCs) and human iPS cells (hiPSCs), confirming our previous prediction that these proteins were present on the cell surface of hPSCs. The mAbs all show a high correlation with POU5F1 (OCT4) expression and other hPSC surface markers (TRA-160 and SSEA-4) in hPSC cultures and detect rare OCT4 positive cells in differentiated cell cultures. These mAbs are immunoreactive to cell surface protein epitopes on both primed and naive state hPSCs, providing useful research tools to investigate the cellular mechanisms underlying human pluripotency and states of cellular reprogramming. In addition, we report that subsets of the seven new mAbs are also immunoreactive to human bone marrow-derived mesenchymal stem cells (MSCs), normal human breast subsets and both normal and tumorigenic colorectal cell populations. The mAbs reported here should accelerate the investigation of the nature of pluripotency, and enable development of robust cell separation and tracing technologies to enrich or deplete for hPSCs and other human stem and somatic cell types. Stem Cells 2017;35:626-640. © 2016 The Authors Stem Cells published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  5. Engineering of global regulators and cell surface properties toward enhancing stress tolerance in Saccharomyces cerevisiae.

    PubMed

    Kuroda, Kouichi; Ueda, Mitsuyoshi

    2017-12-01

    Microbial cell factories are subject to various stresses, leading to the reductions of metabolic activity and bioproduction efficiency. Therefore, the development of stress-tolerant microorganisms is important for improving bio-production efficiency. Recently, modifications of cell surface properties and master regulators have been shown to be effective approaches for enhancing stress tolerance. The cell surface is an attractive target owing to its interactions with the environment and its role in transmitting environmental information. Cell surface engineering in yeast has enabled the convenient modification of cell surface properties. Displaying random peptide libraries and subsequent screening can successfully improve stress tolerance. Furthermore, master regulators including transcription factors are also promising target to be engineered because stress tolerance is determined by many cooperative factors and modification of master regulators can simultaneously affect the expression of multiple downstream genes. The key single amino acid mutations in transcription factors have been identified by analyzing tolerant yeasts that were isolated by adaptive evolution under stress conditions. This enabled the reconstruction of stress-tolerant yeast without burdening cells by introducing the identified mutations. Therefore, for the construction of stress-tolerant yeast from any strains, these two approaches are promising alternatives to conventional overexpression and deletion of stress-related genes. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  6. Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration.

    PubMed

    Cai, Zhenyu; Zhang, Anling; Choksi, Swati; Li, Weihua; Li, Tao; Zhang, Xue-Min; Liu, Zheng-Gang

    2016-08-01

    Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. TNF-induced necroptosis is mediated by receptor-interacting protein kinases, RIP1 and RIP3, and the mixed lineage kinase domain-like (MLKL). After being phosphorylated by RIP3, MLKL is translocated to the plasma membrane and mediates necroptosis. However, the execution of necroptosis and its role in inflammation and other cellular responses remain largely elusive. In this study, we report that MLKL-mediated activation of cell-surface proteases of the a disintegrin and metalloprotease (ADAM) family promotes necroptosis, inflammation and cell migration. ADAMs are specifically activated at the early stage of necroptosis when MLKL is phosphorylated and translocated to the cell plasma membrane. Activation of ADAMs induces ectodomain shedding of diverse cell-surface proteins including adhesion molecules, receptors, growth factors and cytokines. Importantly, the shedding of cell-surface proteins disrupts cell adhesion and accelerates necroptosis, while the soluble fragments of the cleaved proteins trigger the inflammatory responses. We also demonstrate that the shedding of E-cadherin ectodomain from necroptotic cells promotes cell migration. Thus, our study provides a novel mechanism of necroptosis-induced inflammation and new insights into the physiological and pathological functions of this unique form of cell death.

  7. Activation of cell-surface proteases promotes necroptosis, inflammation and cell migration

    PubMed Central

    Cai, Zhenyu; Zhang, Anling; Choksi, Swati; Li, Weihua; Li, Tao; Zhang, Xue-Min; Liu, Zheng-Gang

    2016-01-01

    Necroptosis is a programmed, caspase-independent cell death that is morphologically similar to necrosis. TNF-induced necroptosis is mediated by receptor-interacting protein kinases, RIP1 and RIP3, and the mixed lineage kinase domain-like (MLKL). After being phosphorylated by RIP3, MLKL is translocated to the plasma membrane and mediates necroptosis. However, the execution of necroptosis and its role in inflammation and other cellular responses remain largely elusive. In this study, we report that MLKL-mediated activation of cell-surface proteases of the a disintegrin and metalloprotease (ADAM) family promotes necroptosis, inflammation and cell migration. ADAMs are specifically activated at the early stage of necroptosis when MLKL is phosphorylated and translocated to the cell plasma membrane. Activation of ADAMs induces ectodomain shedding of diverse cell-surface proteins including adhesion molecules, receptors, growth factors and cytokines. Importantly, the shedding of cell-surface proteins disrupts cell adhesion and accelerates necroptosis, while the soluble fragments of the cleaved proteins trigger the inflammatory responses. We also demonstrate that the shedding of E-cadherin ectodomain from necroptotic cells promotes cell migration. Thus, our study provides a novel mechanism of necroptosis-induced inflammation and new insights into the physiological and pathological functions of this unique form of cell death. PMID:27444869

  8. Living Toroids - Cells on Toroidal Surfaces

    NASA Astrophysics Data System (ADS)

    Chang, Ya-Wen; Angelini, Thomas; Marquez, Samantha; Kim, Harold; Fernandez-Nieves, Alberto

    2014-03-01

    Cellular environment influences a multitude of cellular functions by providing chemical and physical signals that modulate cell behavior, dynamics, development, and eventually survival. Substrate mechanics has been recognized as one of the important physical cues that governs cell behavior at single cell level as well as in collective cell motion. Past research has suggested several contact-guided behaviors to be the result of surface curvature. However, studies on the effect of curvature are relatively scarce likely due to the difficulty in generating substrates with well-defined curvature. Here we describe the generation of toroidal droplets, which unlike spherical droplets, have regions of both positive and negative Gaussian curvature. Additionally, the range of curvatures can be controlled by varying the size and aspect ratio of the torus. Cells are either encapsulated inside toroidal droplets or located on toroidal hydrogel surfaces. Preliminary studies use B. Subtilis to study the organization of bacteria biofilms. When confined in droplets surrounded by yield-stress fluid, bacteria self-organize into heterogeneous biofilm at fluid- substrate interface. It is found that the surface curvature in the sub-millimeter scale has little effect on biofilm architecture.

  9. Differential effect of rebamipide on transmembrane mucin biosynthesis in stratified ocular surface epithelial cells.

    PubMed

    Uchino, Yuichi; Woodward, Ashley M; Argüeso, Pablo

    2016-12-01

    Mucins are a group of highly glycosylated glycoproteins responsible for the protection of wet-surfaced epithelia. Recent data indicate that transmembrane mucins differ in their contribution to the protective function of the ocular surface, with MUC16 being the most effective barrier on the apical surface glycocalyx. Here, we investigated the role of the mucoprotective drug rebamipide in the regulation of transmembrane mucin biosynthesis using stratified cultures of human corneal and conjunctival epithelial cells. We find that the addition of rebamipide to corneal, but not conjunctival, epithelial cells increased MUC16 protein biosynthesis. Rebamipide did not affect the levels of MUC1, 4 and 20 compared to control. In these experiments, rebamipide had no effect on the expression levels of Notch intracellular domains, suggesting that the rebamipide-induced increase in MUC16 biosynthesis in differentiated corneal cultures is not regulated by Notch signaling. Overall these findings indicate that rebamipide induces the differential upregulation of MUC16 in stratified cultures of human corneal epithelial cells, which may have implications to the proper restoration of barrier function in ocular surface disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Cell surface acid-base properties of the cyanobacterium Synechococcus: Influences of nitrogen source, growth phase and N:P ratios

    NASA Astrophysics Data System (ADS)

    Liu, Yuxia; Alessi, D. S.; Owttrim, G. W.; Kenney, J. P. L.; Zhou, Qixing; Lalonde, S. V.; Konhauser, K. O.

    2016-08-01

    The distribution of many trace metals in the oceans is controlled by biological uptake. Recently, Liu et al. (2015) demonstrated the propensity for a marine cyanobacterium to adsorb cadmium from seawater, suggesting that cell surface reactivity might also play an important role in the cycling of metals in the oceans. However, it remains unclear how variations in cyanobacterial growth rates and nutrient supply might affect the chemical properties of their cellular surfaces. In this study we used potentiometric titrations and Fourier Transform Infrared (FT-IR) spectrometry to profile the key metabolic changes and surface chemical responses of a Synechococcus strain, PCC 7002, during different growth regimes. This included testing various nitrogen (N) to phosphorous (P) ratios (both nitrogen and phosphorous dependent), nitrogen sources (nitrate, ammonium and urea) and growth stages (exponential, stationary, and death phase). FT-IR spectroscopy showed that varying the growth substrates on which Synechococcus cells were cultured resulted in differences in either the type or abundance of cellular exudates produced or a change in the cell wall components. Potentiometric titration data were modeled using three distinct proton binding sites, with resulting pKa values for cells of the various growth conditions in the ranges of 4.96-5.51 (pKa1), 6.67-7.42 (pKa2) and 8.13-9.95 (pKa3). According to previous spectroscopic studies, these pKa ranges are consistent with carboxyl, phosphoryl, and amine groups, respectively. Comparisons between the titration data (for the cell surface) and FT-IR spectra (for the average cellular changes) generally indicate (1) that the nitrogen source is a greater determinant of ligand concentration than growth phase, and (2) that phosphorus limitation has a greater impact on Synechococcus cellular and extracellular properties than does nitrogen limitation. Taken together, these techniques indicate that nutritional quality during cell growth can

  11. Functional dynamics of cell surface membrane proteins

    NASA Astrophysics Data System (ADS)

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules.

  12. B-cell acquisition of antigen: Sensing the surface.

    PubMed

    Knight, Andrew M

    2015-06-01

    B-cell antigen receptor (BCR) recognition and acquisition of antigen by B cells is the essential first step in the generation of effective antibody responses. As B-cell-mediated antigen presentation is also believed to play a significant role in the activation of CD4(+) Th-cell responses, considerable effort has focused on clarifying the nature of antigen/BCR interactions. Following earlier descriptions of interactions of soluble antigens with the BCR, it is now clear that B cells also recognize, physically extract and present antigens that are tethered to, or integral components of, the surfaces or extracellular matrix of other cells. In this issue of the European Journal of Immunology, Zeng et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] examine how the physical property or "stiffness" of the surface displaying antigens to B cells influences the B-cell response. This commentary reports that antigen tethered on "less stiff" surfaces induces increased B-cell activation and antibody responses. I then infer how "sensing the surface" by B cells may represent a new component of the immune system's ability to detect "damage," and how this understanding may influence approaches to clinical therapies where immune activity is either unwanted or desired. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Estimating intercellular surface tension by laser-induced cell fusion.

    PubMed

    Fujita, Masashi; Onami, Shuichi

    2011-12-01

    Intercellular surface tension is a key variable in understanding cellular mechanics. However, conventional methods are not well suited for measuring the absolute magnitude of intercellular surface tension because these methods require determination of the effective viscosity of the whole cell, a quantity that is difficult to measure. In this study, we present a novel method for estimating the intercellular surface tension at single-cell resolution. This method exploits the cytoplasmic flow that accompanies laser-induced cell fusion when the pressure difference between cells is large. Because the cytoplasmic viscosity can be measured using well-established technology, this method can be used to estimate the absolute magnitudes of tension. We applied this method to two-cell-stage embryos of the nematode Caenorhabditis elegans and estimated the intercellular surface tension to be in the 30-90 µN m(-1) range. Our estimate was in close agreement with cell-medium surface tensions measured at single-cell resolution.

  14. Myotube formation is affected by adipogenic lineage cells in a cell-to-cell contact-independent manner

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Takegahara, Yuki; Yamanouchi, Keitaro, E-mail: akeita@mail.ecc.u-tokyo.ac.jp; Nakamura, Katsuyuki

    2014-05-15

    Intramuscular adipose tissue (IMAT) formation is observed in some pathological conditions such as Duchenne muscular dystrophy (DMD) and sarcopenia. Several studies have suggested that IMAT formation is not only negatively correlated with skeletal muscle mass but also causes decreased muscle contraction in sarcopenia. In the present study, we examined w hether adipocytes affect myogenesis. For this purpose, skeletal muscle progenitor cells were transfected with siRNA of PPARγ (siPPARγ) in an attempt to inhibit adipogenesis. Myosin heavy chain (MHC)-positive myotube formation was promoted in cells transfected with siPPARγ compared to that of cells transfected with control siRNA. To determine whether directmore » cell-to-cell contact between adipocytes and myoblasts is a prerequisite for adipocytes to affect myogenesis, skeletal muscle progenitor cells were cocultured with pre- or mature adipocytes in a Transwell coculture system. MHC-positive myotube formation was inhibited when skeletal muscle progenitor cells were cocultured with mature adipocytes, but was promoted when they were cocultured with preadipocytes. Similar effects were observed when pre- or mature adipocyte-conditioned medium was used. These results indicate that preadipocytes play an important role in maintaining skeletal muscle mass by promoting myogenesis; once differentiated, the resulting mature adipocytes negatively affect myogenesis, leading to the muscle deterioration observed in skeletal muscle pathologies. - Highlights: • We examined the effects of pre- and mature adipocytes on myogenesis in vitro. • Preadipocytes and mature adipocytes affect myoblast fusion. • Preadipocytes play an important role in maintaining skeletal muscle mass. • Mature adipocytes lead to muscle deterioration observed in skeletal muscle pathologies.« less

  15. Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

    PubMed

    Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

    2016-10-01

    High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH 2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Cell density-dependent differential proliferation of neural stem cells on omnidirectional nanopore-arrayed surface.

    PubMed

    Cha, Kyoung Je; Kong, Sun-Young; Lee, Ji Soo; Kim, Hyung Woo; Shin, Jae-Yeon; La, Moonwoo; Han, Byung Woo; Kim, Dong Sung; Kim, Hyun-Jung

    2017-10-12

    Recently, the importance of surface nanotopography in the determination of stem cell fate and behavior has been revealed. In the current study, we generated polystyrene cell-culture dishes with an omnidirectional nanopore arrayed surface (ONAS) (diameter: 200 nm, depth: 500 nm, center-to-center distance: 500 nm) and investigated the effects of nanotopography on rat neural stem cells (NSCs). NSCs cultured on ONAS proliferated better than those on the flat surface when cell density was low and showed less spontaneous differentiation during proliferation in the presence of mitogens. Interestingly, NSCs cultured on ONAS at clonal density demonstrated a propensity to generate neurospheres, whereas those on the flat surface migrated out, proliferated as individuals, and spread out to attach to the surface. However, the differential patterns of proliferation were cell density-dependent since the distinct phenomena were lost when cell density was increased. ONAS modulated cytoskeletal reorganization and inhibited formation of focal adhesion, which is generally observed in NSCs grown on flat surfaces. ONAS appeared to reinforce NSC-NSC interaction, restricted individual cell migration and prohibited NSC attachment to the nanopore surface. These data demonstrate that ONAS maintains NSCs as undifferentiated while retaining multipotency and is a better topography for culturing low density NSCs.

  17. Functional dynamics of cell surface membrane proteins.

    PubMed

    Nishida, Noritaka; Osawa, Masanori; Takeuchi, Koh; Imai, Shunsuke; Stampoulis, Pavlos; Kofuku, Yutaka; Ueda, Takumi; Shimada, Ichio

    2014-04-01

    Cell surface receptors are integral membrane proteins that receive external stimuli, and transmit signals across plasma membranes. In the conventional view of receptor activation, ligand binding to the extracellular side of the receptor induces conformational changes, which convert the structure of the receptor into an active conformation. However, recent NMR studies of cell surface membrane proteins have revealed that their structures are more dynamic than previously envisioned, and they fluctuate between multiple conformations in an equilibrium on various timescales. In addition, NMR analyses, along with biochemical and cell biological experiments indicated that such dynamical properties are critical for the proper functions of the receptors. In this review, we will describe several NMR studies that revealed direct linkage between the structural dynamics and the functions of the cell surface membrane proteins, such as G-protein coupled receptors (GPCRs), ion channels, membrane transporters, and cell adhesion molecules. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Biological response of human bone marrow mesenchymal stem cells to fluoride-modified titanium surfaces.

    PubMed

    Guida, Luigi; Annunziata, Marco; Rocci, Antonio; Contaldo, Maria; Rullo, Rosario; Oliva, Adriana

    2010-11-01

    The aim of the present study was to examine the behaviour of human bone marrow-derived mesenchymal stem cells (BM-MSC) to fluoride-modified grit-blasted (F-TiO) titanium surfaces compared with grit-blasted ones (TiO). Implant surfaces were analysed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). BM-MSC were isolated from healthy donors and grown on the implant surfaces. Cell adhesion and proliferation, type I collagen (Col I) synthesis, osteoblastic differentiation (in terms of alkaline phosphatase activity, osteocalcin synthesis and extracellular matrix mineralization) were assessed. Furthermore, the ability to affect the osteoblastic/osteoclastic balance in terms of osteoprotegerin (OPG) and activator of nuclear factor κ B ligand (RANKL) ratio was investigated. F-TiO surface showed higher S(a) values (P<0.05) and the presence of nano-scale structures at the AFM and SEM analysis. Comparable cell morphology and similar adhesion values on both surfaces were detected at early time, whereas higher proliferation values on F-TiO samples were observed at 7 and 10 days. Increased Col I and OPG levels for cells grown on F-TiO were found, whereas RANKL was not detectable in any of the conditioned media. BM-MSC showed a similar expression of early and late osteogenic markers on both TiO and F-TiO surfaces. The results of the present study show that the chemical and micro/nano-scale modifications induced by fluoride treatment of TiO-grit blasted surfaces stimulate the proliferation and the extracellular matrix synthesis by BM-MSC, as well as the increase of OPG synthesis, thus preventing osteoclast activation and differentiation. © 2010 John Wiley & Sons A/S.

  19. CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

    PubMed Central

    Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

    2009-01-01

    The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

  20. Stem cell behavior on tailored porous oxide surface coatings.

    PubMed

    Lavenus, Sandrine; Poxson, David J; Ogievetsky, Nika; Dordick, Jonathan S; Siegel, Richard W

    2015-07-01

    Nanoscale surface topographies are known to have a profound influence on cell behavior, including cell guidance, migration, morphology, proliferation, and differentiation. In this study, we have observed the behavior of human mesenchymal stem cells cultured on a range of tailored porous SiO2 and TiO2 nanostructured surface coatings fabricated via glancing angle electron-beam deposition. By controlling the physical vapor deposition angle during fabrication, we could control systematically the deposited coating porosity, along with associated topographic features. Immunocytochemistry and image analysis quantitatively revealed the number of adherent cells, as well as their basic cellular morphology, on these surfaces. Signaling pathway studies showed that even with subtle changes in nanoscale surface structures, the behavior of mesenchymal stem cells was strongly influenced by the precise surface structures of these porous coatings. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier*

    PubMed Central

    Argüeso, Pablo; Guzman-Aranguez, Ana; Mantelli, Flavio; Cao, Zhiyi; Ricciuto, Jessica; Panjwani, Noorjahan

    2009-01-01

    Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins. PMID:19556244

  2. Surface Molecules Released by Trypanosoma cruzi Metacyclic Forms Downregulate Host Cell Invasion

    PubMed Central

    Clemente, Tatiana Mordente; Cortez, Cristian; Novaes, Antônio da Silva; Yoshida, Nobuko

    2016-01-01

    Background The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. Methodology/Principal Findings Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the presence of G-CM or CL-CM. Parasite invasion was significantly inhibited by G-CM, but not by CL-CM. As G strain MT invasion rate in D10 is very low, assays with this strain were performed in PBS++, which induces invasion-promoting lysosome-spreading. G-CM, but not CL-CM, significantly inhibited G strain internalization, effect that was counteracted by preincubating G-CM with an anti-gp90 monoclonal antibody or anti-gp82 polyclonal antibody that do not recognize live MT. G strain CM generated in PBS++ contained much lower amounts of gp90 and gp82 as compared to CM produced in D10, and exhibited lower inhibitory effect on host cell invasion. Conclusion/Significance Our data suggest that the surface molecules spontaneously released by MT impair parasite-host cell interaction, gp82 presumably competing with the molecule expressed on MT surface for the host cell receptor, and gp90 further contributing to down modulate invasion. PMID:27483135

  3. Cystatin F Affects Natural Killer Cell Cytotoxicity

    PubMed Central

    Perišić Nanut, Milica; Sabotič, Jerica; Švajger, Urban; Jewett, Anahid; Kos, Janko

    2017-01-01

    Cystatin F is a cysteine peptidase inhibitor which, unlike other cystatin family members, is targeted to endosomal/lysosomal compartments. It is synthesized as an inactive disulfide-linked dimer which is then converted to an active monomer by proteolytic cleavage of 15 N-terminal residues. Cystatin F has been suggested to regulate the cytotoxicity of natural killer (NK) cells by inhibiting the major granzyme convertases, cathepsins C and H. To test this hypothesis, we prepared variants of cystatin F and analyzed their uptake, subcellular trafficking, and peptidase inhibition, as well as their impact on the cytotoxicity of NK-92 cells and primary NK cells. The N-glycosylation pattern is responsible for the secretion, uptake, and subcellular sorting of cystatin F in HeLa and Hek293 cells, whereas the legumain binding site had no effect on these processes. Active, N-terminally truncated, monomeric cystatin F can also be internalized by recipient cells and targeted to endo/lysosomes, affecting also cells lacking the activating peptidase. Cystatin F mutants capable of cell internalization and trafficking through the endo/lysosomal pathway significantly decreased cathepsin C and H activities, both in situ, following transfection and in trans, using conditioned media. Further, incubation of IL-2 stimulated NK-92 and primary NK cells with full-length and N-terminally truncated cystatin F mutants led to suppression of their granule-mediated cytotoxicity. This effect was most significant with the N-terminally truncated mutants. These results suggest that cystatin F can be an important mediator within tumor microenvironment affecting the cytotoxicity of NK cells and consequently antitumor immune response. PMID:29180998

  4. Lactoperoxidase catalyzed radioiodination of cell surface immunoglobulin: incorporated radioactivity may not reflect relative cell surface Ig density. [/sup 125/I

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wilder, R.L.; Yuen, C.C.; Mage, R.G.

    1979-02-01

    Rabbit and mouse splenic lymphocytes were radioiodinated by the lactoperoxidase technique, extracted with non-ionic detergent, immunoprecipitated with high titered rabbit anti-kappa antisera, and compared by SDS-PAGE. Mouse sIg peaks were reproducibly larger in size than rabbit sIg peaks (often greater than 10 times). Neither differences in incorporation of label into the rabbit cell surface, nor differences in average sIg density explain this result. Total TCA-precipitable radioactivity was similar in each species. Estimation of the relative amounts of sIg in the mouse and rabbit showed similar average sIg densities. Differences in detergent solubility, proteolytic lability, or antisera used also do notmore » adequately account for this difference. Thus, these data indicate that radioactivity incorporated after lactoperoxidase catalyzed cell surface radioiodination may not reflect cell surface Ig density. Conclusions about cell surface density based upon relative incorporation of radioactivity should be confirmed by other approaches.« less

  5. The Cell-Surface N-Glycome of Human Embryonic Stem Cells and Differentiated Hepatic Cells thereof.

    PubMed

    Montacir, Houda; Freyer, Nora; Knöspel, Fanny; Urbaniak, Thomas; Dedova, Tereza; Berger, Markus; Damm, Georg; Tauber, Rudolf; Zeilinger, Katrin; Blanchard, Véronique

    2017-07-04

    Human embryonic stem cells (hESCs) are pluripotent stem cells that offer a wide range of applications in regenerative medicine. In addition, they have been proposed as an appropriate alternative source of hepatocytes. In this work, hESCs were differentiated into definitive endodermal cells (DECs), followed by maturation into hepatocyte-like cells (HLCs). Their cell-surface N-glycome was profiled and also compared with that of primary human hepatocytes (PHHs). Undifferentiated hESCs contained large amounts of high-mannose N-glycans. In contrast, complex-type N-glycans such as asialylated or monosialylated biantennary and triantennary N-glycans were dominant in HLCs, and fully galactosylated structures were significantly more abundant than in undifferentiated hESCs. The cell-surface N-glycosylation of PHHs was more biologically processed than that of HLCs, with bisialylated biantennary and trisialylated triantennary structures predominant. This is the first report of the cell surface N-glycome of PHHs and of HLCs being directly generated from hESCs without embryoid body formation. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Maintaining the pluripotency of mouse embryonic stem cells on gold nanoparticle layers with nanoscale but not microscale surface roughness

    NASA Astrophysics Data System (ADS)

    Lyu, Zhonglin; Wang, Hongwei; Wang, Yanyun; Ding, Kaiguo; Liu, Huan; Yuan, Lin; Shi, Xiujuan; Wang, Mengmeng; Wang, Yanwei; Chen, Hong

    2014-05-01

    Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold

  7. Surface deformation during an action potential in pearled cells

    NASA Astrophysics Data System (ADS)

    Mussel, Matan; Fillafer, Christian; Ben-Porath, Gal; Schneider, Matthias F.

    2017-11-01

    Electric pulses in biological cells (action potentials) have been reported to be accompanied by a propagating cell-surface deformation with a nanoscale amplitude. Typically, this cell surface is covered by external layers of polymer material (extracellular matrix, cell wall material, etc.). It was recently demonstrated in excitable plant cells (Chara braunii) that the rigid external layer (cell wall) hinders the underlying deformation. When the cell membrane was separated from the cell wall by osmosis, a mechanical deformation, in the micrometer range, was observed upon excitation of the cell. The underlying mechanism of this mechanical pulse has, to date, remained elusive. Herein we report that Chara cells can undergo a pearling instability, and when the pearled fragments were excited even larger and more regular cell shape changes were observed (˜10 -100 μ m in amplitude). These transient cellular deformations were captured by a curvature model that is based on three parameters: surface tension, bending rigidity, and pressure difference across the surface. In this paper these parameters are extracted by curve-fitting to the experimental cellular shapes at rest and during excitation. This is a necessary step to identify the mechanical parameters that change during an action potential.

  8. Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Whitson, P. A.

    1993-01-01

    Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.

  9. Driving Cell Seeding Using Vibration Induced Surface Waves

    NASA Astrophysics Data System (ADS)

    Li, Haiyan; Friend, James; Yeo, Leslie

    2007-11-01

    The ability to load cells into scaffold matrices is an important step in in-vitro cell culturing. Efficient and rapid cell seeding is however difficult and has traditionally been carried out using a static method by allowing gravity to drive the perfusion of the cell suspension into the porous scaffold. Nevertheless, due to the large capillary pressures associated with the small scaffold pore dimensions, the static cell seeding method is both slow and inefficient; the majority of cells are distributed close to the surface of the scaffold due to the inability of the fluid to penetrate deep into the scaffold. By driving the liquid into the scaffold using small amplitude surface vibrations on a piezoelectric substrate, we demonstrate that the cells can be infused much quicker (approximately 10 seconds) than if allowed to perfuse by gravity alone, which requires seeding times in excess of 30 minutes. Greater penetration of the fluid and hence the cells into the scaffold is also achieved with the vibration forcing, thus giving rise to a more uniform cell distribution within the scaffold. Moreover, we have verified that 80% of the yeast cells seeded by the surface waves remained viable.

  10. Mapping cell surface adhesion by rotation tracking and adhesion footprinting

    NASA Astrophysics Data System (ADS)

    Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

    2017-03-01

    Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

  11. The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.

    PubMed

    Lu, Ting; Lin, Zongwei; Ren, Jianwei; Yao, Peng; Wang, Xiaowei; Wang, Zhe; Zhang, Qunye

    2016-01-01

    MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs. Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it. These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.

  12. Cell surface engineering of industrial microorganisms for biorefining applications.

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-11-15

    In order to decrease carbon emissions and negative environmental impacts of various pollutants, biofuel/biochemical production should be promoted for replacing fossil-based industrial processes. Utilization of abundant lignocellulosic biomass as a feedstock has recently become an attractive option. In this review, we focus on recent efforts of cell surface display using industrial microorganisms such as Escherichia coli and yeast. Cell surface display is used primarily for endowing cellulolytic activity on the host cells, and enables direct fermentation to generate useful fuels and chemicals from lignocellulosic biomass. Cell surface display systems are systematically summarized, and the drawbacks/perspectives as well as successful application of surface display for industrial biotechnology are discussed. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Surface acoustic wave actuated cell sorting (SAWACS).

    PubMed

    Franke, T; Braunmüller, S; Schmid, L; Wixforth, A; Weitz, D A

    2010-03-21

    We describe a novel microfluidic cell sorter which operates in continuous flow at high sorting rates. The device is based on a surface acoustic wave cell-sorting scheme and combines many advantages of fluorescence activated cell sorting (FACS) and fluorescence activated droplet sorting (FADS) in microfluidic channels. It is fully integrated on a PDMS device, and allows fast electronic control of cell diversion. We direct cells by acoustic streaming excited by a surface acoustic wave which deflects the fluid independently of the contrast in material properties of deflected objects and the continuous phase; thus the device underlying principle works without additional enhancement of the sorting by prior labelling of the cells with responsive markers such as magnetic or polarizable beads. Single cells are sorted directly from bulk media at rates as fast as several kHz without prior encapsulation into liquid droplet compartments as in traditional FACS. We have successfully directed HaCaT cells (human keratinocytes), fibroblasts from mice and MV3 melanoma cells. The low shear forces of this sorting method ensure that cells survive after sorting.

  14. Cell-surface marker discovery for lung cancer

    PubMed Central

    Cohen, Allison S.; Khalil, Farah K.; Welsh, Eric A.; Schabath, Matthew B.; Enkemann, Steven A.; Davis, Andrea; Zhou, Jun-Min; Boulware, David C.; Kim, Jongphil; Haura, Eric B.; Morse, David L.

    2017-01-01

    Lung cancer is the leading cause of cancer deaths in the United States. Novel lung cancer targeted therapeutic and molecular imaging agents are needed to improve outcomes and enable personalized care. Since these agents typically cannot cross the plasma membrane while carrying cytotoxic payload or imaging contrast, discovery of cell-surface targets is a necessary initial step. Herein, we report the discovery and characterization of lung cancer cell-surface markers for use in development of targeted agents. To identify putative cell-surface markers, existing microarray gene expression data from patient specimens were analyzed to select markers with differential expression in lung cancer compared to normal lung. Greater than 200 putative cell-surface markers were identified as being overexpressed in lung cancers. Ten cell-surface markers (CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 and TMPRSS4) were selected based on differential mRNA expression in lung tumors vs. non-neoplastic lung samples and other normal tissues, and other considerations involving known biology and targeting moieties. Protein expression was confirmed by immunohistochemistry (IHC) staining and scoring of patient tumor and normal tissue samples. As further validation, marker expression was determined in lung cancer cell lines using microarray data and Kaplan–Meier survival analyses were performed for each of the markers using patient clinical data. High expression for six of the markers (CA9, CA12, CXorf61, GPR87, LYPD3, and SLC7A11) was significantly associated with worse survival. These markers should be useful for the development of novel targeted imaging probes or therapeutics for use in personalized care of lung cancer patients. PMID:29371917

  15. Surface etching technologies for monocrystalline silicon wafer solar cells

    NASA Astrophysics Data System (ADS)

    Tang, Muzhi

    With more than 200 GW of accumulated installations in 2015, photovoltaics (PV) has become an important green energy harvesting method. The PV market is dominated by solar cells made from crystalline silicon wafers. The engineering of the wafer surfaces is critical to the solar cell cost reduction and performance enhancement. Therefore, this thesis focuses on the development of surface etching technologies for monocrystalline silicon wafer solar cells. It aims to develop a more efficient alkaline texturing method and more effective surface cleaning processes. Firstly, a rapid, isopropanol alcohol free texturing method is successfully demonstrated to shorten the process time and reduce the consumption of chemicals. This method utilizes the special chemical properties of triethylamine, which can form Si-N bonds with wafer surface atoms. Secondly, a room-temperature anisotropic emitter etch-back process is developed to improve the n+ emitter passivation. Using this method, 19.0% efficient screen-printed aluminium back surface field solar cells are developed that show an efficiency gain of 0.15% (absolute) compared with conventionally made solar cells. Finally, state-of-the-art silicon surface passivation results are achieved using hydrogen plasma etching as a dry alternative to the classical hydrofluoric acid wet-chemical process. The effective native oxide removal and the hydrogenation of the silicon surface are shown to be the reasons for the excellent level of surface passivation achieved with this novel method.

  16. Engineering Novel and Improved Biocatalysts by Cell Surface Display

    PubMed Central

    Smith, Mason R.; Khera, Eshita; Wen, Fei

    2017-01-01

    Biocatalysts, especially enzymes, have the ability to catalyze reactions with high product selectivity, utilize a broad range of substrates, and maintain activity at low temperature and pressure. Therefore, they represent a renewable, environmentally friendly alternative to conventional catalysts. Most current industrial-scale chemical production processes using biocatalysts employ soluble enzymes or whole cells expressing intracellular enzymes. Cell surface display systems differ by presenting heterologous enzymes extracellularly, overcoming some of the limitations associated with enzyme purification and substrate transport. Additionally, coupled with directed evolution, cell surface display is a powerful platform for engineering enzymes with enhanced properties. In this review, we will introduce the molecular and cellular principles of cell surface display and discuss how it has been applied to engineer enzymes with improved properties as well as to develop surface-engineered microbes as whole-cell biocatalysts. PMID:29056821

  17. Rear surface effects in high efficiency silicon solar cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wenham, S.R.; Robinson, S.J.; Dai, X.

    1994-12-31

    Rear surface effects in PERL solar cells can lead not only to degradation in the short circuit current and open circuit voltage, but also fill factor. Three mechanisms capable of changing the effective rear surface recombination velocity with injection level are identified, two associated with oxidized p-type surfaces, and the third with two dimensional effects associated with a rear floating junction. Each of these will degrade the fill factor if the range of junction biases corresponding to the rear surface transition, coincides with the maximum power point. Despite the identified non idealities, PERL cells with rear floating junctions (PERF cells)more » have achieved record open circuit voltages for silicon solar cells, while simultaneously achieving fill factor improvements relative to standard PERL solar cells. Without optimization, a record efficiency of 22% has been demonstrated for a cell with a rear floating junction. The results of both theoretical and experimental studies are provided.« less

  18. Surface topography of hairy cell leukemia cells compared to other leukemias as seen by scanning electron microscopy.

    PubMed

    Polliack, Aaron; Tadmor, Tamar

    2011-06-01

    This short review deals with the ultrastructural surface architecture of hairy cell leukemia (HCL) compared to other leukemic cells, as seen by scanning electron microscopy (SEM). The development of improved techniques for preparing blood cells for SEM in the 1970s readily enabled these features to be visualized more accurately. This review returns us to the earlier history of SEM, when the surface topography of normal and neoplastic cells was visualized and reported for the first time, in an era before the emergence and use of monoclonal antibodies and flow cytometry, now used routinely to define cells by their immunophenotype. Surface microvilli are characteristic for normal and leukemic lymphoid cells, myelo-monocytic cells lack microvilli and show surface ruffles, while leukemic plasma and myeloma cells and megakaryocytes display large surface blebs. HCL cell surfaces are complex and typically 'hybrid' in nature, displaying both lymphoid and monocytic features with florid ruffles of varying sizes interspersed with clumps of short microvilli cytoplasm. The surface features of other leukemic cells and photomicrographs of immuno-SEM labeling of cells employing antibodies and colloidal gold, reported more than 20 years ago, are shown.

  19. Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

    PubMed

    Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

    2014-07-04

    Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Investigation of back surface fields effect on bifacial solar cells

    NASA Astrophysics Data System (ADS)

    Sepeai, Suhaila; Sulaiman, M. Y.; Sopian, Kamaruzzaman; Zaidi, Saleem H.

    2012-11-01

    A bifacial solar cell, in contrast with a conventional monofacial solar cell, produces photo-generated current from both front and back sides. Bifacial solar cell is an attractive candidate for enhancing photovoltaic (PV) market competitiveness as well as supporting the current efforts to increase efficiency and lower material costs. This paper reports on the fabrication of bifacial solar cells using phosphorus-oxytrichloride (POCl3) emitter formation on p-type, nanotextured silicon (Si) wafer. Backside surface field was formed through Al-diffusion using conventional screen-printing process. Bifacial solar cells with a structure of n+pp+ with and without back surface field (BSF) were fabricated in which silicon nitride (SiN) anti reflection and passivation films were coated on both sides, followed by screen printing of Argentum (Ag) and Argentum/Aluminum (Ag/Al) on front and back contacts, respectively. Bifacial solar cells without BSF exhibited open circuit voltage (VOC) of 535 mV for front and 480 mV for back surface. With Al-alloyed BSF bifacial solar cells, the VOC improved to 580 mV for the front surface and 560 mV for the back surface. Simulation of bifacial solar cells using PC1D and AFORS software demonstrated good agreement with experimental results. Simulations showed that best bifacial solar cells are achieved through a combination of high lifetime wafer, low recombination back surface field, reduced contact resistance, and superior surface passivation.

  1. Keratinocyte Motility Is Affected by UVA Radiation-A Comparison between Normal and Dysplastic Cells.

    PubMed

    Niculiţe, Cristina M; Nechifor, Marina T; Urs, Andreea O; Olariu, Laura; Ceafalan, Laura C; Leabu, Mircea

    2018-06-07

    UVA radiation induces multiple and complex changes in the skin, affecting epidermal cell behavior. This study reports the effects of UVA exposure on normal (HaCaT) and dysplastic (DOK) keratinocytes. The adherence, spreading and proliferation were investigated by time-lapse measurement of cell layer impedance on different matrix proteins. Prior to UVA exposure, the time required for adherence and spreading did not differ significantly for HaCaT and DOK cells, while spreading areas were larger for HaCaT cells. Under UVA exposure, HaCaT and DOK cells behavior differed in terms of movement and proliferation. The cells' ability to cover the denuded surface and individual cell trajectories were recorded by time-lapse videomicroscopy, during wound healing experiments. Dysplastic keratinocytes showed more sensitivity to UVA, exhibiting transient deficiencies in directionality of movement and a delay in re-coating the denuded area. The actin cytoskeleton displayed a cortical organization immediately after irradiation, in both cell lines, similar to mock-irradiated cells. Post-irradiation, DOK cells displayed a better organization of stress fibers, persistent filopodia, and new, stronger focal contacts. In conclusion, after UVA exposure HaCaT and DOK cells showed a different behavior in terms of adherence, spreading, motility, proliferation, and actin cytoskeleton dynamics, with the dyplastic keratinocytes being more sensitive.

  2. Display of fungal hydrophobin on the Pichia pastoris cell surface and its influence on Candida antarctica lipase B

    PubMed Central

    Wang, Pan; He, Jie; Sun, Yufei; Reynolds, Matthew; Zhang, Li; Han, Shuangyan; Liang, Shuli; Sui, Haixin; Lin, Ying

    2016-01-01

    To modify the Pichia pastoris cell surface, two classes of hydrophobins, SC3 from Schizophyllum commune and HFBI from Trichoderma reesei, were separately displayed on the cell wall. There was an observable increase in the hydrophobicity of recombinant strains. Candida antarctica lipase B (CALB) was then co-displayed on the modified cells, generating strains GS115/SC3-61/CALB-51 and GS115/HFBI-61/CALB-51. Interestingly, the hydrolytic and synthetic activities of strain GS115/HFBI-61/CALB-51 increased by 37% and 109%, respectively, but decreased by 26% and 43%, respectively, in strain GS115/SC3-61/CALB-51 compared with the hydrophobin-minus recombinant strain GS115/CALB-GCW51. The amount of glycerol by-product from the transesterification reaction adsorbed on the cell surface was significantly decreased following hydrophobin modification, removing the glycerol barrier and allowing substrates to access the active sites of lipases. Electron micrographs indicated that the cell wall structures of both recombinant strains appeared altered, including changes to the inner glucan layer and outer mannan layer. These results suggest that the display of hydrophobins can change the surface structure and hydrophobic properties of P. pastoris, and affect the catalytic activities of CALB displayed on the surface of P. pastoris cells. PMID:26969039

  3. Enhanced Imaging of Specific Cell-Surface Glycosylation Based on Multi-FRET.

    PubMed

    Yuan, Baoyin; Chen, Yuanyuan; Sun, Yuqiong; Guo, Qiuping; Huang, Jin; Liu, Jianbo; Meng, Xiangxian; Yang, Xiaohai; Wen, Xiaohong; Li, Zenghui; Li, Lie; Wang, Kemin

    2018-05-15

    Cell-surface glycosylation contains abundant biological information that reflects cell physiological state, and it is of great value to image cell-surface glycosylation to elucidate its functions. Here we present a hybridization chain reaction (HCR)-based multifluorescence resonance energy transfer (multi-FRET) method for specific imaging of cell-surface glycosylation. By installing donors through metabolic glycan labeling and acceptors through aptamer-tethered nanoassemblies on the same glycoconjugate, intramolecular multi-FRET occurs due to near donor-acceptor distance. Benefiting from amplified effect and spatial flexibility of the HCR nanoassemblies, enhanced multi-FRET imaging of specific cell-surface glycosylation can be obtained. With this HCR-based multi-FRET method, we achieved obvious contrast in imaging of protein-specific GalNAcylation on 7211 cell surfaces. In addition, we demonstrated the general applicability of this method by visualizing the protein-specific sialylation on CEM cell surfaces. Furthermore, the expression changes of CEM cell-surface protein-specific sialylation under drug treatment was accurately monitored. This developed imaging method may provide a powerful tool in researching glycosylation functions, discovering biomarkers, and screening drugs.

  4. Effects of titanium nanotubes on the osseointegration, cell differentiation, mineralisation and antibacterial properties of orthopaedic implant surfaces.

    PubMed

    Su, E P; Justin, D F; Pratt, C R; Sarin, V K; Nguyen, V S; Oh, S; Jin, S

    2018-01-01

    The development and pre-clinical evaluation of nano-texturised, biomimetic, surfaces of titanium (Ti) implants treated with titanium dioxide (TiO 2 ) nanotube arrays is reviewed. In vitro and in vivo evaluations show that TiO 2 nanotubes on Ti surfaces positively affect the osseointegration, cell differentiation, mineralisation, and anti-microbial properties. This surface treatment can be superimposed onto existing macro and micro porous Ti implants creating a surface texture that also interacts with cells at the nano level. Histology and mechanical pull-out testing of specimens in rabbits indicate that TiO 2 nanotubes improves bone bonding nine-fold (p = 0.008). The rate of mineralisation associated with TiO 2 nanotube surfaces is about three times that of non-treated Ti surfaces. In addition to improved osseointegration properties, TiO 2 nanotubes reduce the initial adhesion and colonisation of Staphylococcus epidermidis Collectively, the properties of Ti implant surfaces enhanced with TiO 2 nanotubes show great promise. Cite this article: Bone Joint J 2018;100-B(1 Supple A):9-16. ©2018 The British Editorial Society of Bone & Joint Surgery.

  5. Modulation of human multipotent and pluripotent stem cells using surface nanotopographies and surface-immobilised bioactive signals: A review.

    PubMed

    Wang, Peng-Yuan; Thissen, Helmut; Kingshott, Peter

    2016-11-01

    The ability to control the interactions of stem cells with synthetic surfaces is proving to be effective and essential for the quality of passaged stem cells and ultimately the success of regenerative medicine. The stem cell niche is crucial for stem cell self-renewal and differentiation. Thus, mimicking the stem cell niche, and here in particular the extracellular matrix (ECM), in vitro is an important goal for the expansion of stem cells and their applications. Here, surface nanotopographies and surface-immobilised biosignals have been identified as major factors that control stem cell responses. The development of tailored surfaces having an optimum nanotopography and displaying suitable biosignals is proposed to be essential for future stem cell culture, cell therapy and regenerative medicine applications. While early research in the field has been restricted by the limited availability of micro- and nanofabrication techniques, new approaches involving the use of advanced fabrication and surface immobilisation methods are starting to emerge. In addition, new cell types such as induced pluripotent stem cells (iPSCs) have become available in the last decade, but have not been fully understood. This review summarises significant advances in the area and focuses on the approaches that are aimed at controlling the behavior of human stem cells including maintenance of their self-renewal ability and improvement of their lineage commitment using nanotopographies and biosignals. More specifically, we discuss developments in biointerface science that are an important driving force for new biomedical materials and advances in bioengineering aiming at improving stem cell culture protocols and 3D scaffolds for clinical applications. Cellular responses revolve around the interplay between the surface properties of the cell culture substrate and the biomolecular composition of the cell culture medium. Determination of the precise role played by each factor, as well as the

  6. Large granulation cells on the surface of the giant star π1 Gruis

    NASA Astrophysics Data System (ADS)

    Paladini, C.; Baron, F.; Jorissen, A.; Le Bouquin, J.-B.; Freytag, B.; van Eck, S.; Wittkowski, M.; Hron, J.; Chiavassa, A.; Berger, J.-P.; Siopis, C.; Mayer, A.; Sadowski, G.; Kravchenko, K.; Shetye, S.; Kerschbaum, F.; Kluska, J.; Ramstedt, S.

    2018-01-01

    Convection plays a major part in many astrophysical processes, including energy transport, pulsation, dynamos and winds on evolved stars, in dust clouds and on brown dwarfs. Most of our knowledge about stellar convection has come from studying the Sun: about two million convective cells with typical sizes of around 2,000 kilometres across are present on the surface of the Sun—a phenomenon known as granulation. But on the surfaces of giant and supergiant stars there should be only a few large (several tens of thousands of times larger than those on the Sun) convective cells, owing to low surface gravity. Deriving the characteristic properties of convection (such as granule size and contrast) for the most evolved giant and supergiant stars is challenging because their photospheres are obscured by dust, which partially masks the convective patterns. These properties can be inferred from geometric model fitting, but this indirect method does not provide information about the physical origin of the convective cells. Here we report interferometric images of the surface of the evolved giant star π1 Gruis, of spectral type S5,7. Our images show a nearly circular, dust-free atmosphere, which is very compact and only weakly affected by molecular opacity. We find that the stellar surface has a complex convective pattern with an average intensity contrast of 12 per cent, which increases towards shorter wavelengths. We derive a characteristic horizontal granule size of about 1.2 × 1011 metres, which corresponds to 27 per cent of the diameter of the star. Our measurements fall along the scaling relations between granule size, effective temperature and surface gravity that are predicted by simulations of stellar surface convection.

  7. Large granulation cells on the surface of the giant star π1 Gruis.

    PubMed

    Paladini, C; Baron, F; Jorissen, A; Le Bouquin, J-B; Freytag, B; Van Eck, S; Wittkowski, M; Hron, J; Chiavassa, A; Berger, J-P; Siopis, C; Mayer, A; Sadowski, G; Kravchenko, K; Shetye, S; Kerschbaum, F; Kluska, J; Ramstedt, S

    2018-01-18

    Convection plays a major part in many astrophysical processes, including energy transport, pulsation, dynamos and winds on evolved stars, in dust clouds and on brown dwarfs. Most of our knowledge about stellar convection has come from studying the Sun: about two million convective cells with typical sizes of around 2,000 kilometres across are present on the surface of the Sun-a phenomenon known as granulation. But on the surfaces of giant and supergiant stars there should be only a few large (several tens of thousands of times larger than those on the Sun) convective cells, owing to low surface gravity. Deriving the characteristic properties of convection (such as granule size and contrast) for the most evolved giant and supergiant stars is challenging because their photospheres are obscured by dust, which partially masks the convective patterns. These properties can be inferred from geometric model fitting, but this indirect method does not provide information about the physical origin of the convective cells. Here we report interferometric images of the surface of the evolved giant star π 1 Gruis, of spectral type S5,7. Our images show a nearly circular, dust-free atmosphere, which is very compact and only weakly affected by molecular opacity. We find that the stellar surface has a complex convective pattern with an average intensity contrast of 12 per cent, which increases towards shorter wavelengths. We derive a characteristic horizontal granule size of about 1.2 × 10 11 metres, which corresponds to 27 per cent of the diameter of the star. Our measurements fall along the scaling relations between granule size, effective temperature and surface gravity that are predicted by simulations of stellar surface convection.

  8. Surface Mechanoengineering of a Zr-based Bulk Metallic Glass via Ar-Nanobubble Doping to Probe Cell Sensitivity to Rigid Materials

    DOE PAGES

    Huang, Lu; Tian, Mengkun; Wu, Dong; ...

    2017-11-24

    In this paper, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-Al- Ni-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues,more » accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties.« less

  9. Surface Mechanoengineering of a Zr-based Bulk Metallic Glass via Ar-Nanobubble Doping to Probe Cell Sensitivity to Rigid Materials

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Huang, Lu; Tian, Mengkun; Wu, Dong

    In this paper, a new materials platform, utilizing the amorphous microstructure of bulk metallic glasses (BMGs) and the versatility of ion implantation, was developed for the fundamental investigation of cell responses to substrate-rigidity variations in the gigapascal modulus range, which was previously unattainable with polymeric materials. The surface rigidity of a Zr-Al- Ni-Cu-Y BMG was modulated with low-energy Ar-ion implantation owing to the impartment of Ar nanobubbles into the amorphous matrix. Surface softening was achieved due to the formation of nanobubble-doped transitional zones in the Zrbased BMG substrate. Bone-forming cell studies on this newly designed platform demonstrated that mechanical cues,more » accompanied with the potential effects of other surface properties (i.e. roughness, morphology, and chemistry), contributed to modulating cell behaviors. Cell adhesion and actin filaments were found to be less established on less stiff surfaces, especially on the surface with an elastic modulus of 51 GPa. Cell growth appeared to be affected by surface mechanical properties. A lower stiffness was generally related to a higher growth rate. Findings in this study broadened our fundamental understanding concerning the mechanosensing of bone cells on stiff substrates. It also suggests that surface mechano-engineering of metallic materials could be a potential strategy to promote osseointegration of such materials for bone-implant applications. Further investigations are proposed to fine tune the ion implantation variables in order to further distinguish the surface-mechanical effect on bone-forming cell activities from the contributions of other surface properties.« less

  10. Cell-cell contact area affects Notch signaling and Notch-dependent patterning

    PubMed Central

    Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A.; Goodyear, Richard J.; Richardson, Guy P.; Chen, Christopher S.; Sprinzak, David

    2017-01-01

    Summary During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from microns to tens of microns. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes. PMID:28292428

  11. Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning.

    PubMed

    Shaya, Oren; Binshtok, Udi; Hersch, Micha; Rivkin, Dmitri; Weinreb, Sheila; Amir-Zilberstein, Liat; Khamaisi, Bassma; Oppenheim, Olya; Desai, Ravi A; Goodyear, Richard J; Richardson, Guy P; Chen, Christopher S; Sprinzak, David

    2017-03-13

    During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from micrometers to tens of micrometers. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Surface receptors on human haematopoietic cell lines.

    PubMed Central

    Huber, C; Sundström, C; Nilsson, K; Wigzell, H

    1976-01-01

    The expression of complement receptors, of Fc receptors, of SRBC receptors and of S-Ig was investigated on human haematopoietic cell lines of proved malignant derivation. According to their origin and to a panel of phenotypic markers these lines have been classified into lymphoma lines, myeloma lines and leukemia lines. Results were compared with those obtained on non-malignant EBV carrying lymphoblastoid cell lines (LCL). Among the lymphoid cell lines the LCL showed a pattern of B-lymphocyte surface markers, i.e. surface immunoglobulins, C3 receptors but low density of Fc receptors. The non-Burkitt lymphoma lines bore in varying degree these B-lymphocyte markers. The lines U-698 M and DG-75 were exceptional in having only surface immunoglobulin. The Burkitt lymphoma lines had all B-lymphocyte markers. The myeloma lines differed from the lymphoid lines in lacking C3 and Fc receptors and showed only trace amounts of surface immunoglobulins. In contrast to lymphoid and myeloma lines, the leukaemia lines were completely lacking surface immunoglobulins, but showed C3 and Fc receptors in variable densities. On line, the ALL derived line MOLT-3 showed the capacity to spontaneous rosette formation with SRBC. The findings that LCL presented a homogeneous pattern of B-lymphocyte surface markers may be of value in order to discriminate between these lines and lines derived from haematopoietic malignancies other than Burkitt lymphomas. PMID:963908

  13. Nano hydroxyapatite-blasted titanium surface affects pre-osteoblast morphology by modulating critical intracellular pathways.

    PubMed

    Bezerra, Fábio; Ferreira, Marcel R; Fontes, Giselle N; da Costa Fernandes, Célio Jr; Andia, Denise C; Cruz, Nilson C; da Silva, Rodrigo A; Zambuzzi, Willian F

    2017-08-01

    Although, intracellular signaling pathways are proposed to predict the quality of cell-surface relationship, this study addressed pre-osteoblast behavior in response to nano hydroxyapatite (HA)-blasted titanium (Ti) surface by exploring critical intracellular pathways and pre-osteoblast morphological change. Physicochemical properties were evaluated by atomic force microscopy (AFM) and wettability considering water contact angle of three differently texturized Ti surfaces: Machined (Mac), Dual acid-etching (DAE), and nano hydroxyapatite-blasted (nHA). The results revealed critical differences in surface topography, impacting the water contact angle and later the osteoblast performance. In order to evaluate the effect of those topographical characteristics on biological responses, we have seeded pre-osteoblast cells on the Ti discs for up to 4 h and subjected the cultures to biological analysis. First, we have observed pre-osteoblasts morphological changes resulting from the interaction with the Ti texturized surfaces whereas the cells cultured on nHA presented a more advanced spreading process when compared with the cells cultured on the other surfaces. These results argued us for analyzing the molecular machinery and thus, we have shown that nHA promoted a lower Bax/Bcl2 ratio, suggesting an interesting anti-apoptotic effect, maybe explained by the fact that HA is a natural element present in bone composition. Thereafter, we investigated the potential effect of those surfaces on promoting pre-osteoblast adhesion and survival signaling by performing crystal violet and immunoblotting approaches, respectively. Our results showed that nHA promoted a higher pre-osteoblast adhesion supported by up-modulating FAK and Src activations, both signaling transducers involved during eukaryotic cell adhesion. Also, we have shown Ras-Erk stimulation by the all evaluated surfaces. Finally, we showed that all Ti-texturing surfaces were able to promote osteoblast differentiation

  14. Antifouling property of highly oleophobic substrates for solar cell surfaces

    NASA Astrophysics Data System (ADS)

    Fukada, Kenta; Nishizawa, Shingo; Shiratori, Seimei

    2014-03-01

    Reduction of solar cell conversion efficiency by bird spoor or oil smoke is a common issue. Maintaining the surface of solar cells clean to retain the incident light is of utmost importance. In this respect, there has been growing interest in the area of superhydrophobicity for developing water repelling and self-cleaning surfaces. This effect is inspired by lotus leaves that have micro papillae covered with hydrophobic wax nanostructures. Superhydrophobic surfaces on transparent substrates have been developed for removing contaminants from solar cell surfaces. However, oil cannot be removed by superhydrophobic effect. In contrast, to prevent bird spoor, a highly oleophobic surface is required. In a previous study, we reported transparent-type fabrics comprising nanoparticles with a nano/micro hierarchical structure that ensured both oleophobicity and transparency. In the current study, we developed new highly oleophobic stripes that were constructed into semi-transparent oleophobic surfaces for solar cells. Solar cell performance was successfully maintained; the total transmittance was a key factor for determining conversion efficiency.

  15. Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers.

    PubMed

    González-Robles, Arturo; Castañón, Guadalupe; Cristóbal-Ramos, Ana Ruth; Hernández-Ramírez, Verónica Ivonne; Omaña-Molina, Maritza; Martínez-Palomo, Adolfo

    2007-12-01

    Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.

  16. Theory of back-surface-field solar cells

    NASA Technical Reports Server (NTRS)

    Vonroos, O.

    1979-01-01

    Report describes simple concise theory of back-surface-field (BSF) solar cells (npp + junctions) based on Shockley's depletion-layer approximation and cites superiority of two-junction devices over conventional unijunction cells.

  17. Concise Review: Cell Surface N-Linked Glycoproteins as Potential Stem Cell Markers and Drug Targets.

    PubMed

    Boheler, Kenneth R; Gundry, Rebekah L

    2017-01-01

    Stem cells and their derivatives hold great promise to advance regenerative medicine. Critical to the progression of this field is the identification and utilization of antibody-accessible cell-surface proteins for immunophenotyping and cell sorting-techniques essential for assessment and isolation of defined cell populations with known functional and therapeutic properties. Beyond their utility for cell identification and selection, cell-surface proteins are also major targets for pharmacological intervention. Although comprehensive cell-surface protein maps are highly valuable, they have been difficult to define until recently. In this review, we discuss the application of a contemporary targeted chemoproteomic-based technique for defining the cell-surface proteomes of stem and progenitor cells. In applying this approach to pluripotent stem cells (PSCs), these studies have improved the biological understanding of these cells, led to the enhanced use and development of antibodies suitable for immunophenotyping and sorting, and contributed to the repurposing of existing drugs without the need for high-throughput screening. The utility of this latter approach was first demonstrated with human PSCs (hPSCs) through the identification of small molecules that are selectively toxic to hPSCs and have the potential for eliminating confounding and tumorigenic cells in hPSC-derived progeny destined for research and transplantation. Overall, the cutting-edge technologies reviewed here will accelerate the development of novel cell-surface protein targets for immunophenotyping, new reagents to improve the isolation of therapeutically qualified cells, and pharmacological studies to advance the treatment of intractable diseases amenable to cell-replacement therapies. Stem Cells Translational Medicine 2017;6:131-138. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  18. Cell-material interactions revealed via material techniques of surface patterning.

    PubMed

    Yao, Xiang; Peng, Rong; Ding, Jiandong

    2013-10-04

    Cell-material interactions constitute a key fundamental topic in biomaterials study. Various cell cues and matrix cues as well as soluble factors regulate cell behaviors on materials. These factors are coupled with each other as usual, and thus it is very difficult to unambiguously elucidate the role of each regulator. The recently developed material techniques of surface patterning afford unique ways to reveal the underlying science. This paper reviews the pertinent material techniques to fabricate patterns of microscale and nanoscale resolutions, and corresponding cell studies. Some issues are emphasized, such as cell localization on patterned surfaces of chemical contrast, and effects of cell shape, cell size, cell-cell contact, and seeding density on differentiation of stem cells. Material cues to regulate cell adhesion, cell differentiation and other cell events are further summed up. Effects of some physical properties, such as surface topography and matrix stiffness, on cell behaviors are also discussed; nanoscaled features of substrate surfaces to regulate cell fate are summarized as well. The pertinent work sheds new insight into the cell-material interactions, and is stimulating for biomaterial design in regenerative medicine, tissue engineering, and high-throughput detection, diagnosis, and drug screening. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering.

    PubMed

    Bi, Xiaobao; Yin, Juan; Chen Guanbang, Ashley; Liu, Chuan-Fa

    2018-06-07

    The cell surface serves important functions such as the regulation of cell-cell and cell-environment interactions. The understanding and manipulation of the cell surface is important for a wide range of fundamental studies of cellular behavior and for biotechnological and medical applications. With the rapid advance of biology, chemistry and materials science, many strategies have been developed for the functionalization of bacterial and mammalian cell surfaces. Here, we review the recent development of chemical and enzymatic approaches to cell surface engineering with particular emphasis on discussing the advantages and limitations of each of these strategies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Regulated internalization of NMDA receptors drives PKD1-mediated suppression of the activity of residual cell-surface NMDA receptors.

    PubMed

    Fang, Xiao-Qian; Qiao, Haifa; Groveman, Bradley R; Feng, Shuang; Pflueger, Melissa; Xin, Wen-Kuan; Ali, Mohammad K; Lin, Shuang-Xiu; Xu, Jindong; Duclot, Florian; Kabbaj, Mohamed; Wang, Wei; Ding, Xin-Sheng; Santiago-Sim, Teresa; Jiang, Xing-Hong; Salter, Michael W; Yu, Xian-Min

    2015-11-19

    Constitutive and regulated internalization of cell surface proteins has been extensively investigated. The regulated internalization has been characterized as a principal mechanism for removing cell-surface receptors from the plasma membrane, and signaling to downstream targets of receptors. However, so far it is still not known whether the functional properties of remaining (non-internalized) receptor/channels may be regulated by internalization of the same class of receptor/channels. The N-methyl-D-aspartate receptor (NMDAR) is a principal subtype of glutamate-gated ion channel and plays key roles in neuronal plasticity and memory functions. NMDARs are well-known to undergo two types of regulated internalization - homologous and heterologous, which can be induced by high NMDA/glycine and DHPG, respectively. In the present work, we investigated effects of regulated NMDAR internalization on the activity of residual cell-surface NMDARs and neuronal functions. In electrophysiological experiments we discovered that the regulated internalization of NMDARs not only reduced the number of cell surface NMDARs but also caused an inhibition of the activity of remaining (non-internalized) surface NMDARs. In biochemical experiments we identified that this functional inhibition of remaining surface NMDARs was mediated by increased serine phosphorylation of surface NMDARs, resulting from the activation of protein kinase D1 (PKD1). Knockdown of PKD1 did not affect NMDAR internalization but prevented the phosphorylation and inhibition of remaining surface NMDARs and NMDAR-mediated synaptic functions. These data demonstrate a novel concept that regulated internalization of cell surface NMDARs not only reduces the number of NMDARs on the cell surface but also causes an inhibition of the activity of remaining surface NMDARs through intracellular signaling pathway(s). Furthermore, modulating the activity of remaining surface receptors may be an effective approach for treating receptor

  1. Biosynthetic maturation of an ascites tumor cell surface sialomucin. Evidence for O-glycosylation of cell surface glycoprotein by the addition of new oligosaccharides during recycling.

    PubMed

    Hull, S R; Sugarman, E D; Spielman, J; Carraway, K L

    1991-07-25

    Previous biosynthetic studies of the ascites 13762 rat mammary adenocarcinoma cell surface sialomucin ASGP-1 (ascites sialoglycoprotein-1) showed that it is synthesized initially as a poorly glycosylated immature form, which is converted to a larger premature form (t1/2 30 min) and more slowly to the mature glycoprotein (t1/2 greater than 4 h). In the present study O-glycosylation of ASGP-1 polypeptide is shown to occur in two phases: an early phase complete in less than 30 min, which corresponds to the synthesis of the premature form, and a later phase that continues for hours and corresponds to the synthesis of the mature form. Pulse-chase labeling studies indicate that 95% of the ASGP-1 has moved to the cell surface in 2 h. Since transit to the cell surface is faster than the slow phase of addition of new oligosaccharides, some new oligosaccharides must be added after ASGP-1 has reached the cell surface. Initiation of new oligosaccharides on cell surface ASGP-1 was demonstrated directly using a biotinylation procedure to identify cell surface molecules. Glucosamine labeling of biotinylated ASGP-1 was shown to occur on galactosamine residues, which are linked to the polypeptide, establishing the addition of new oligosaccharides to the cell surface molecules. Finally, resialylation studies indicate that ASGP-1 rapidly recycles through a sialylating compartment. From these results we propose that ASGP-1 reaches the cell surface in an incompletely glycosylated state and that additional oligosaccharides are added to the glycoprotein in a second process involving recycling.

  2. Functional mapping of cell surface proteins with localized stimulation of single cells

    NASA Astrophysics Data System (ADS)

    Sun, Bingyun; Chiu, Daniel T.

    2003-11-01

    This paper describes the development of using individual micro and nano meter-sized vesicles as delivery vessels to functionally map the distribution of cell surface proteins at the level of single cells. The formation of different sizes of vesicles from tens of nanometers to a few micrometers in diameter that contain the desired molecules is addressed. An optical trap is used to manipulate the loaded vesicle to specific cell morphology of interest, and a pulsed UV laser is used to photo-release the stimuli onto the cell membrane. Carbachol activated cellular calcium flux, upon binding to muscarinic acetylcholine receptors, is studied by this method, and the potential of using this method for the functional mapping of localized proteins on the cell surface membrane is discussed.

  3. The role of surface implant treatments on the biological behavior of SaOS-2 osteoblast-like cells. An in vitro comparative study.

    PubMed

    Conserva, Enrico; Menini, Maria; Ravera, Giambattista; Pera, Paolo

    2013-08-01

    The aim of this study was an in vitro comparison of osteoblast adhesion, proliferation and differentiation related to six dental implants with different surface characteristics, and to determine if the interaction between cells and implant is influenced by surface structure and chemical composition. Six types of implants were tested, presenting four different surface treatments: turned, sandblasted, acid-etched, anodized. The implant macro- and microstructure were analyzed using SEM, and the surface chemical composition was investigated using energy-dispersive X-ray analysis. SaOS-2 osteoblasts were used for the evaluation of cell adhesion and proliferation by SEM, and cell viability in contact with the various surfaces was determined using cytotoxicity MTT assays. Alkaline phosphatase (ALP) enzymatic activity in contact with the six surfaces was evaluated. Data relative to MTT assay and ALP activity were statistically analyzed using Kruskal-Wallis not parametric test and Nemenyi-Damico-Wolfe-Dunn post hoc test. All the implants tested supported cell adhesion, proliferation and differentiation, revealing neither organic contaminants nor cytotoxicity effects. The industrial treatments investigated changed the implant surface microscopic aspect and SaOS-2 cell morphology appeared to be influenced by the type of surface treatment at 6, 24, and 72 h of growth. SaOS-2 cells spread more rapidly on sandblasted surfaces. Turned surfaces showed the lowest cell proliferation at SEM observation. Sandblasted surfaces showed the greatest ALP activity values per cell, followed by turned surfaces (P < 0.05). On the base of this in vitro investigation, differently surfaced implants affected osteoblast morphology, adhesion, proliferation, and differentiation. Sandblasted surfaces promoted the most suitable osteoblast behavior. © 2012 John Wiley & Sons A/S.

  4. Maintaining the pluripotency of mouse embryonic stem cells on gold nanoparticle layers with nanoscale but not microscale surface roughness.

    PubMed

    Lyu, Zhonglin; Wang, Hongwei; Wang, Yanyun; Ding, Kaiguo; Liu, Huan; Yuan, Lin; Shi, Xiujuan; Wang, Mengmeng; Wang, Yanwei; Chen, Hong

    2014-06-21

    Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.

  5. Modelling cell motility and chemotaxis with evolving surface finite elements

    PubMed Central

    Elliott, Charles M.; Stinner, Björn; Venkataraman, Chandrasekhar

    2012-01-01

    We present a mathematical and a computational framework for the modelling of cell motility. The cell membrane is represented by an evolving surface, with the movement of the cell determined by the interaction of various forces that act normal to the surface. We consider external forces such as those that may arise owing to inhomogeneities in the medium and a pressure that constrains the enclosed volume, as well as internal forces that arise from the reaction of the cells' surface to stretching and bending. We also consider a protrusive force associated with a reaction–diffusion system (RDS) posed on the cell membrane, with cell polarization modelled by this surface RDS. The computational method is based on an evolving surface finite-element method. The general method can account for the large deformations that arise in cell motility and allows the simulation of cell migration in three dimensions. We illustrate applications of the proposed modelling framework and numerical method by reporting on numerical simulations of a model for eukaryotic chemotaxis and a model for the persistent movement of keratocytes in two and three space dimensions. Movies of the simulated cells can be obtained from http://homepages.warwick.ac.uk/∼maskae/CV_Warwick/Chemotaxis.html. PMID:22675164

  6. Dramatically reduced surface expression of NK cell receptor KIR2DS3 is attributed to multiple residues throughout the molecule.

    PubMed

    VandenBussche, C J; Mulrooney, T J; Frazier, W R; Dakshanamurthy, S; Hurley, C K

    2009-03-01

    Using flow cytometry, fluorescent microscopy and examination of receptor glycosylation status, we demonstrate that an entire killer cell immunoglobulin-like receptor (KIR) locus (KIR2DS3)--assumed earlier to be surface expressed--appears to have little appreciable surface expression in transfected cells. This phenotype was noted for receptors encoded by three allelic variants including the common KIR2DS3*001 allele. Comparing the surface expression of KIR2DS3 with that of the better-studied KIR2DS1 molecule in two different cell lines, mutational analysis identified multiple polymorphic amino-acid residues that significantly alter the proportion of molecules present on the cell surface. A simultaneous substitution of five residues localized to the leader peptide (residues -18 and -7), second domain (residues 123 and 150) and transmembrane region (residue 234) was required to restore KIR2DS3 to the expression level of KIR2DS1. Corresponding simultaneous substitutions of KIR2DS1 to the KIR2DS3 residues resulted in a dramatically decreased surface expression. Molecular modeling was used to predict how these substitutions contribute to this phenotype. Alterations in receptor surface expression are likely to affect the balance of immune cell signaling impacting the characteristics of the response to pathogens or malignancy.

  7. Mouse Elberfeld (ME) virus determines the cell surface alterations when mixedly infecting poliovirus-infected cells.

    PubMed

    Zeichhardt, H; Schlehofer, J R; Wetz, K; Hampl, H; Habermehl, K O

    1982-02-01

    The surface alterations of HEp-2 cells induced by mixed infection with two different picornaviruses (poliovirus and ME virus) were compared by scanning electron microscopic and transmission electron microscopic studies and by 51Cr-release assay. The contribution of each of the viruses to the resulting surface changes was discernible, as investigations on the chronology of the cytopathic alterations demonstrated that the changes were distinct for either virus. The surface of ME virus-infected cells was characterized by large membranous structures ('sheets' and blebs) representing huge vacuoles. These sheets were not seen in poliovirus-infected cells. Poliovirus induced more prominent cell pycnosis, elongation of filopodia and condensation of collapsed microvilli on the cell surface than ME virus. Mixed infection with these two viruses led to surface alterations typical for ME virus. These ME virus-specific changes occurred irrespective of poliovirus reproduction or its inhibition by guanidine. ME virus-specific alterations also predominated in cytolytic membrane damage as expressed by 51Cr-release from infected cells. 51Cr-release was more pronounced from ME virus than from poliovirus-infected cells, even when ME virus reproduction was suppressed by interfering poliovirus. However, alteration of the internal structures of the infected cells was only dominated by ME virus when the reproduction of poliovirus was suppressed.

  8. Cell culture surfaces with immobilized gold nanostars: a new approach for laser-induced plasmonic cell optoporation

    NASA Astrophysics Data System (ADS)

    Vanzha, Ekaterina; Pylaev, Timofey; Prilepskii, Artur; Golubev, Alexander; Khlebtsov, Boris; Bogatyrev, Vladimir; Khlebtsov, Nikolai

    2017-03-01

    The application of gold nanoparticles (GNPs) for laser-induced cell transfection has been studied intensively during the past decade as efficient and gentle alternative to well-established molecule delivery methods like lipid-based transfection or electroporation. The method is based on temporal increase of membrane permeability induced by laser irradiation of GNPs attached to cell membranes. Although this approach is attractive due to high throughput and easy usability, it is not free from serious drawbacks related to random adsorption of GNPs during preincubation of cells with GNPs. This stage can affect the optoporation results because of potential nanoparticle toxicity, thus leading to decreased delivery efficiency and to low reproducibility of independent optoporation runs. Herein, we suggest a novel GNP-mediated laser transfection technique based on immobilized gold nanostars (GNSs) that are adsorbed on microplate wells and act as a plasmonic surface. The HeLa cells are grown directly on the monolayer of immobilized GNSs followed by CW NIR laser irradiation. We used the propidium iodide (PI) as a model transfecting agent to monitor simultaneously the delivery of PI into HeLa cells and their viability. These proof-of-the-concept experiments demonstrated enhanced penetration of PI into irradiated cells as compared to untreated ones.

  9. The multilevel analysis of surface acting and mental health: A moderation of positive group affective tone

    NASA Astrophysics Data System (ADS)

    Lee, Meng-Shiu; Huang, Jui-Chan; Wu, Tzu-Jung

    2017-06-01

    The purpose of this study is to investigate the relationship among surface acting, mental health, and positive group affective tone. According to the prior theory, this study attempts to establish a comprehensive research framework among these variables, and furthermore tests the moderating effect of positive group affective tone. Data were collected from 435 employees in 52 service industrial companies by questionnaire, and this study conducted multilevel analysis. The results showed that surface acting will negatively affect the mental health. In addition, the positive group affective tone have significant moderating effect on the relationship among surface acting and mental health. Finally, this study discusses managerial implications and highlights future research suggestions.

  10. Surface shape affects the three-dimensional exploratory movements of nocturnal arboreal snakes.

    PubMed

    Jayne, Bruce C; Olberding, Jeffrey P; Athreya, Dilip; Riley, Michael A

    2012-12-01

    Movement and searching behaviors at diverse spatial scales are important for understanding how animals interact with their environment. Although the shapes of branches and the voids in arboreal habitats seem likely to affect searching behaviors, their influence is poorly understood. To gain insights into how both environmental structure and the attributes of an animal may affect movement and searching, we compared the three-dimensional exploratory movements of snakes in the dark on two simulated arboreal surfaces (disc and horizontal cylinder). Most of the exploratory movements of snakes in the dark were a small fraction of the distances they could reach while bridging gaps in the light. The snakes extended farther away from the edge of the supporting surface at the ends of the cylinder than from the sides of the cylinder or from any direction from the surface of the disc. The exploratory movements were not random, and the surface shape and three-dimensional directions had significant interactive effects on how the movements were structured in time. Thus, the physical capacity for reaching did not limit the area that was explored, but the shape of the supporting surface and the orientation relative to gravity did create biased searching patterns.

  11. A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

    PubMed

    Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

    2017-05-01

    Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment.

    PubMed

    Kobayashi, Jun; Hayashi, Masaki; Ohno, Takahiro; Nishi, Masanori; Arisaka, Yoshinori; Matsubara, Yoshinori; Kakidachi, Hiroshi; Akiyama, Yoshikatsu; Yamato, Masayuki; Horii, Akihiro; Okano, Teruo

    2014-11-01

    Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces. © 2013 Wiley Periodicals, Inc.

  13. A unique cell-surface protein phenotype distinguishes human small-cell from non-small-cell lung cancer

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Baylin, S.B.; Gazdar, A.F.; Minna, J.D.

    1982-08-01

    Radioiodination (/sup 125/I) and two-dimensional polyacrylamide gel electrophoresis was used to determine that small-(oat) cell lung carcinoma (SCC)-a tumor with neuroedocrine features-possesses a surface protein pattern distinct from the other types of lung cancer cells (squamous, adeno-, and large-cell undifferentiated carcinoma). Twelve distinguishing proteins, 40 to 70 kilodaltons (kDal), characterized four separate lines of SCC; three of these, designated E (60 kDal; pI = 7.3), S (30 kDal; pI = 6.0), and U 57 kDal; pI = 5.6), may be unique SCC gene products and were identified only in (/sup 35/S)methionine labeling of SCC and not in non-SCC or humanmore » fibroblasts. Two lines of adeno-, one of squamous, and one of undifferentiated large-cell lung carcinoma exhibited similar surface protein patterns to one another. Nine distinguishing proteins (40 to 100 kDal) and at least five large proteins (>100 kDal) were unique to these lines. The surface protein phenotypes for SCC and non-SCC were distinct from those for human lymphoblastoid cells and fibroblasts. However, the neuroendocrine features of SCC were further substantiated because 6 of the 12 distinguishing SCC surface proteins, including E and U, were identified on human neuroblastoma cells. The proteins identified should (i) help define differentiation steps for normal and neoplastic bronchial epithelial cells, (ii) prove useful in better classifying lung cancers, and (iii) be instrumental in tracing formation of neuroendocrine cells.« less

  14. Interleukin-3 Does Not Affect the Differentiation of Mast Cells Derived from Human Bone Marrow Progenitors

    PubMed Central

    Shimizu, Yuji; Matsumoto, Kenji; Okayama, Yoshimichi; Kentaro, Sakai; Maeno, Toshitaka; Suga, Tatsuo; Miura, Toru; Takai, Shinji; Kurabayashi, Masahiko; Saito, Hirohisa

    2008-01-01

    Although IL-3 is commonly used for culture of human progenitor-derived mast cells together with Stem cell factor (SCF) and IL-6, the effect of IL-3 on human mast cell differentiation has not been well elucidated. Human bone marrow CD34+ progenitors were cultured for up to 12 weeks in the presence of rhSCF and rhIL-6 either with rhIL-3 (IL-3 (+)) or without rhIL-3 (IL-3 (−)) for the initial 1-week of culture. Total cell number increased at 2 weeks in IL-3 (+), as compared to IL-3 (−), but changes in the appearance of mast cells were delayed. When IL-3 was present for the initial 1-week culture, granules looked more mature with IL-3 than without IL-3. However, tryptase and chymase contents, and surface antigen expression (CD18, CD51, CD54, and CD117) were not altered by IL-3. Surface expression and mRNA level of FcεRIα and histamine release by crosslinking of FcεRIα did not differ from one preparation to the next. GeneChip analysis revealed that no significant differences were observed between IL-3 (+) and IL-3 (−) cells either when inactivated or activated by aggregation of FcεRIα. These findings indicate that initial incubation of human bone marrow CD34+ progenitors with IL-3 does not affect the differentiation of mast cells. PMID:18214796

  15. Influence of surfaces modified with biomimetic extracellular matrices on adhesion and proliferation of mesenchymal stem cells and osteosarcoma cells.

    PubMed

    Cai, Rong; Kawazoe, Naoki; Chen, Guoping

    2015-02-01

    Preparation of surfaces modified with biomimetic extracellular matrices (ECMs) is important for investigation of the interaction between ECMs and cells. In the present study, surfaces modified with ECMs from normal somatic cells, stem cells and tumor cells were prepared by cell culture method. The ECMs derived from bone marrow-derived mesenchymal stem cells (MSCs), dermal fibroblasts (FBs), osteoblasts (OBs) and MG63 osteosarcoma cells were deposited on the surfaces of cell-culture polystyrene plates (TCPS). The ECMs from different cell types had different compositions. The effects of the ECM-deposited surfaces on the adhesion, spreading and proliferation of MSCs and MG63 human osteosarcoma cells were dependent on the type of both ECMs and cells. The surfaces deposited with ECMs from MSCs, FBs and OBs promoted cell adhesion more strongly than surfaces deposited with ECMs from MG63 cells and TCPS. Compared to TCPS, the ECM-deposited surfaces promoted proliferation of MSCs while they inhibited the proliferation of MG63 cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Atomic force microscopy – looking at mechanosensors on the cell surface

    PubMed Central

    Heinisch, Jürgen J.; Lipke, Peter N.; Beaussart, Audrey; El Kirat Chatel, Sofiane; Dupres, Vincent; Alsteens, David; Dufrêne, Yves F.

    2012-01-01

    Summary Living cells use cell surface proteins, such as mechanosensors, to constantly sense and respond to their environment. However, the way in which these proteins respond to mechanical stimuli and assemble into large complexes remains poorly understood at the molecular level. In the past years, atomic force microscopy (AFM) has revolutionized the way in which biologists analyze cell surface proteins to molecular resolution. In this Commentary, we discuss how the powerful set of advanced AFM techniques (e.g. live-cell imaging and single-molecule manipulation) can be integrated with the modern tools of molecular genetics (i.e. protein design) to study the localization and molecular elasticity of individual mechanosensors on the surface of living cells. Although we emphasize recent studies on cell surface proteins from yeasts, the techniques described are applicable to surface proteins from virtually all organisms, from bacteria to human cells. PMID:23077172

  17. Differential Expression of Osteo-Modulatory Molecules in Periodontal Ligament Stem Cells in Response to Modified Titanium Surfaces

    PubMed Central

    Kim, So Yeon; Yoo, Ji-Yeon; Ohe, Joo-Young; Lee, Jung-Woo; Moon, Ji-Hoi; Kwon, Yong-Dae; Heo, Jung Sun

    2014-01-01

    This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT), and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs. PMID:25057487

  18. Differential expression of osteo-modulatory molecules in periodontal ligament stem cells in response to modified titanium surfaces.

    PubMed

    Kim, So Yeon; Yoo, Ji-Yeon; Ohe, Joo-Young; Lee, Jung-Woo; Moon, Ji-Hoi; Kwon, Yong-Dae; Heo, Jung Sun

    2014-01-01

    This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT), and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs.

  19. Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity.

    PubMed

    Faille, Christine; Jullien, Celine; Fontaine, Francoise; Bellon-Fontaine, Marie-Noelle; Slomianny, Christian; Benezech, Thierry

    2002-08-01

    The ability of bacterial spores and vegetative cells to adhere to inert surfaces was investigated by means of the number of adherent spores (Bacillus cereus and Bacillus subtilis spores) and Escherichia coli cells and their resistance to cleaning or rinsing procedures (adhesion strength). Six materials (glass, stainless steel, polyethylene high density (PEHD), polyamide-6, polyvinyl chloride, and Teflon) were tested. Slight differences in the number of adherent spores (less than 1 log unit) were observed between materials, but a higher number of adherent E. coli cells was found on the hydrophobic materials PEHD and Teflon. Conversely, the resistance of both B. cereus and B. subtilis spores to a cleaning procedure was significantly affected by the material. Hydrophobic materials were harder to clean. The topography parameter derived from the Abbott-Firestone curve, RVK, and, to a lesser extent, the widely used roughness parameters RA (average roughness) and Rz (maximal roughness), were related to the number of adherent cells. Lastly, the soiling level as well as the adhesion strength were shown to depend largely on the microorganism. The number of adhering B. cereus hydrophobic spores and their resistance to a cleaning procedure were found to be 10 times greater than those of the B. subtilis hydrophilic spores. Escherichia coli was loosely bound to all the materials tested, even after 24 h biofilm formation.

  20. Amine-Rich Organic Thin Films for Cell Culture: Possible Electrostatic Effects in Cell-Surface Interactions

    NASA Astrophysics Data System (ADS)

    Wertheimer, Michael R.; St-Georges-Robillard, Amélie; Lerouge, Sophie; Mwale, Fackson; Elkin, Bentsian; Oehr, Christian; Wirges, Werner; Gerhard, Reimund

    2012-11-01

    In recent communications from these laboratories, we observed that amine-rich thin organic layers are very efficient surfaces for the adhesion of mammalian cells. We prepare such deposits by plasma polymerization at low pressure, atmospheric pressure, or by vacuum-ultraviolet photo-polymerization. More recently, we have also investigated a commercially available material, Parylene diX AM. In this article we first briefly introduce literature relating to electrostatic interactions between cells, proteins, and charged surfaces. We then present certain selected cell-response results that pertain to applications in orthopedic and cardiovascular medicine: we discuss the influence of surface properties on the observed behaviors of two particular cell lines, human U937 monocytes, and Chinese hamster ovary cells. Particular emphasis is placed on possible electrostatic attractive forces due to positively charged R-NH3+ groups and negatively charged proteins and cells, respectively. Experiments carried out with electrets, polymers with high positive or negative surface potentials are added for comparison.

  1. Specialized cell surface structures in cellulolytic bacteria.

    PubMed Central

    Lamed, R; Naimark, J; Morgenstern, E; Bayer, E A

    1987-01-01

    The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin. Characteristic protuberant structures were observed on cells of all cellulolytic strains. These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E. A. Bayer and R. Lamed, J. Bacteriol. 167:828-836, 1986). Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria. The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose. Images PMID:3301817

  2. Remote Control of Tissue Interactions via Engineered Photo-switchable Cell Surfaces

    NASA Astrophysics Data System (ADS)

    Luo, Wei; Pulsipher, Abigail; Dutta, Debjit; Lamb, Brian M.; Yousaf, Muhammad N.

    2014-09-01

    We report a general cell surface molecular engineering strategy via liposome fusion delivery to create a dual photo-active and bio-orthogonal cell surface for remote controlled spatial and temporal manipulation of microtissue assembly and disassembly. Cell surface tailoring of chemoselective functional groups was achieved by a liposome fusion delivery method and quantified by flow cytometry and characterized by a new cell surface lipid pull down mass spectrometry strategy. Dynamic co-culture spheroid tissue assembly in solution and co-culture tissue multilayer assembly on materials was demonstrated by an intercellular photo-oxime ligation that could be remotely cleaved and disassembled on demand. Spatial and temporal control of microtissue structures containing multiple cell types was demonstrated by the generation of patterned multilayers for controlling stem cell differentiation. Remote control of cell interactions via cell surface engineering that allows for real-time manipulation of tissue dynamics may provide tools with the scope to answer fundamental questions of cell communication and initiate new biotechnologies ranging from imaging probes to drug delivery vehicles to regenerative medicine, inexpensive bioreactor technology and tissue engineering therapies.

  3. Yeast surface display of dehydrogenases in microbial fuel-cells.

    PubMed

    Gal, Idan; Schlesinger, Orr; Amir, Liron; Alfonta, Lital

    2016-12-01

    Two dehydrogenases, cellobiose dehydrogenase from Corynascus thermophilus and pyranose dehydrogenase from Agaricus meleagris, were displayed for the first time on the surface of Saccharomyces cerevisiae using the yeast surface display system. Surface displayed dehydrogenases were used in a microbial fuel cell and generated high power outputs. Surface displayed cellobiose dehydrogenase has demonstrated a midpoint potential of -28mV (vs. Ag/AgCl) at pH=6.5 and was used in a mediator-less anode compartment of a microbial fuel cell producing a power output of 3.3μWcm(-2) using lactose as fuel. Surface-displayed pyranose dehydrogenase was used in a microbial fuel cell and generated high power outputs using different substrates, the highest power output that was achieved was 3.9μWcm(-2) using d-xylose. These results demonstrate that surface displayed cellobiose dehydrogenase and pyranose dehydrogenase may successfully be used in microbial bioelectrochemical systems. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Factors affecting reconstitution of the T cell compartment in allogeneic haematopoietic cell transplant recipients.

    PubMed

    Fallen, P R; McGreavey, L; Madrigal, J A; Potter, M; Ethell, M; Prentice, H G; Guimarães, A; Travers, P J

    2003-11-01

    The factors affecting T cell reconstitution post haematopoietic cell transplantation (HCT) are not well characterised. We carried out a longitudinal analysis of T cell reconstitution in 32 HCT recipients during the first 12 months post transplant. We analysed reconstitution of naïve, memory and effector T cells, their diversity and monitored thymic output using TCR rearrangement excision circles (TRECs). Thymic-independent pathways were responsible for the rapid reconstitution of memory and effector T cells less than 6 months post HCT. Thymic-dependent pathways were activated between 6 and 12 months in the majority of patients with naïve T cell numbers increasing in parallel with TREC levels. Increasing patient age, chronic GVHD and T cell depletion (with or without pretransplant Campath-1H) predicted low TREC levels and slow naïve T cell recovery. Furthermore, increasing patient age also predicted high memory and effector T cell numbers. The effects of post HCT immunosuppression, total body irradiation, donor leucocyte infusions, T cell dose and post HCT infections on T cell recovery were also analysed. However, no effects of these single variables across a variety of different age, GVHD and T cell depletion groups were apparent. This study suggests that future analysis of the factors affecting T cell reconstitution and studies aimed at reactivating the thymus through therapeutic intervention should be analysed in age-, GVHD- and TCD-matched patient groups.

  5. Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide

    NASA Technical Reports Server (NTRS)

    Betz, N. A.; Westhoff, B. A.; Johnson, T. C.; Spooner, B. S. (Principal Investigator)

    1995-01-01

    Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

  6. Functional cell-surface display of a lipase-specific chaperone.

    PubMed

    Wilhelm, Susanne; Rosenau, Frank; Becker, Stefan; Buest, Sebastian; Hausmann, Sascha; Kolmar, Harald; Jaeger, Karl-Erich

    2007-01-02

    Lipases are important enzymes in biotechnology. Extracellular bacterial lipases from Pseudomonads and related species require the assistance of specific chaperones, designated "Lif" proteins (lipase specific foldases). Lifs, a unique family of steric chaperones, are anchored to the periplasmic side of the inner membrane where they convert lipases into their active conformation. We have previously shown that the autotransporter protein EstA from P. aeruginosa can be used to direct a variety of proteins to the cell surface of Escherichia coli. Here we demonstrate for the first time the functional cell-surface display of the Lif chaperone and FACS (fluorescence-activated cell sorting)-based analysis of bacterial cells that carried foldase-lipase complexes. The model Lif protein, LipH from P. aeruginosa, was displayed at the surface of E. coli cells. Surface exposed LipH was functional and efficiently refolded chemically denatured lipase. The foldase autodisplay system reported here can be used for a variety of applications including the ultrahigh-throughput screening of large libraries of foldase variants generated by directed evolution.

  7. Surface modification of PLGA nanoparticles by carbopol to enhance mucoadhesion and cell internalization.

    PubMed

    Surassmo, Suvimol; Saengkrit, Nattika; Ruktanonchai, Uracha Rungsardthong; Suktham, Kunat; Woramongkolchai, Noppawan; Wutikhun, Tuksadon; Puttipipatkhachorn, Satit

    2015-06-01

    Mucoadhesive poly (lactic-co-glycolic acid) (PLGA) nanoparticles having a modified shell-matrix derived from polyvinyl alcohol (PVA) and Carbopol (CP), a biodegradable polymer coating, to improve the adhesion and cell transfection properties were developed. The optimum formulations utilized a CP concentration in the range of 0.05-0.2%w/v, and were formed using modified emulsion-solvent evaporation technique. The resulting CP-PLGA nanoparticles were characterized in terms of their physical and chemical properties. The absorbed CP on the PLGA shell-matrix was found to affect the particle size and surface charge, with 0.05% CP giving rise to smooth spherical particles (0.05CP-PLGA) with the smallest size (285.90 nm), and strong negative surface charge (-25.70 mV). The introduction of CP results in an enhancement of the mucoadhesion between CP-PLGA nanoparticles and mucin particles. In vitro cell internalization studies highlighted the potential of 0.05CP-PLGA nanoparticles for transfection into SiHa cells, with uptake being time dependent. Additionally, cytotoxicity studies of CP-PLGA nanoparticles against SiHa cancer cells indicated that low concentrations of the nanoparticles were non-toxic to cells (cell viability >80%). From the various formulations studied, 0.05CP-PLGA nanoparticles proved to be the optimum model carrier having the required mucoadhesive profile and could be an alternative therapeutic efficacy carrier for targeted mucosal drug delivery systems with biodegradable polymer. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Endothelial cell behaviour on gas-plasma-treated PLA surfaces: the roles of surface chemistry and roughness.

    PubMed

    Shah, Amita; Shah, Sarita; Mani, Gopinath; Wenke, Joseph; Agrawal, Mauli

    2011-04-01

    Glow-discharge gas-plasma (GP) treatment has been shown to induce surface modifications such that cell adhesion and growth are enhanced. However, it is not known which gas used in GP treatment is optimal for endothelial cell function. Polylactic acid (PLA) films treated oxygen, argon, or nitrogen GP were characterized using contact angles, scanning electron microscopy, atomic force microscopy, optical profilometry, and x-ray photoelectron spectroscopy. All three GP treatments decreased the carbon atomic concentration and surface roughness and increased the oxygen atomic concentration. Human umbilical vein endothelial cells were cultured on the PLA films for up to 7 days. Based on proliferation and live/dead assays, surface chemistry was shown to have the greatest effect on the attachment, proliferation, and viability of these cells, while roughness did not have a significant influence. Of the different gases, endothelial cell viability, attachment and proliferation were most significantly increased on PLA surfaces treated with oxygen and argon gas plasma. Copyright © 2010 John Wiley & Sons, Ltd.

  9. The Escherichia coli Subtilase Cytotoxin A Subunit Specifically Cleaves Cell-surface GRP78 Protein and Abolishes COOH-terminal-dependent Signaling

    PubMed Central

    Ray, Rupa; de Ridder, Gustaaf G.; Eu, Jerry P.; Paton, Adrienne W.; Paton, James C.; Pizzo, Salvatore V.

    2012-01-01

    GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH2-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78low) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH2-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78. PMID:22851173

  10. The Escherichia coli subtilase cytotoxin A subunit specifically cleaves cell-surface GRP78 protein and abolishes COOH-terminal-dependent signaling.

    PubMed

    Ray, Rupa; de Ridder, Gustaaf G; Eu, Jerry P; Paton, Adrienne W; Paton, James C; Pizzo, Salvatore V

    2012-09-21

    GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH(2)-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78(low)) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH(2)-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.

  11. Metformin selectively affects human glioblastoma tumor-initiating cell viability

    PubMed Central

    Würth, Roberto; Pattarozzi, Alessandra; Gatti, Monica; Bajetto, Adirana; Corsaro, Alessandro; Parodi, Alessia; Sirito, Rodolfo; Massollo, Michela; Marini, Cecilia; Zona, Gianluigi; Fenoglio, Daniela; Sambuceti, Gianmario; Filaci, Gilberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

    2013-01-01

    Cancer stem cell theory postulates that a small population of tumor-initiating cells is responsible for the development, progression and recurrence of several malignancies, including glioblastoma. In this perspective, tumor-initiating cells represent the most relevant target to obtain effective cancer treatment. Metformin, a first-line drug for type II diabetes, was reported to possess anticancer properties affecting the survival of cancer stem cells in breast cancer models. We report that metformin treatment reduced the proliferation rate of tumor-initiating cell-enriched cultures isolated from four human glioblastomas. Metformin also impairs tumor-initiating cell spherogenesis, indicating a direct effect on self-renewal mechanisms. Interestingly, analyzing by FACS the antiproliferative effects of metformin on CD133-expressing subpopulation, a component of glioblastoma cancer stem cells, a higher reduction of proliferation was observed as compared with CD133-negative cells, suggesting a certain degree of cancer stem cell selectivity in its effects. In fact, glioblastoma cell differentiation strongly reduced sensitivity to metformin treatment. Metformin effects in tumor-initiating cell-enriched cultures were associated with a powerful inhibition of Akt-dependent cell survival pathway, while this pathway was not affected in differentiated cells. The specificity of metformin antiproliferative effects toward glioblastoma tumor-initiating cells was confirmed by the lack of significant inhibition of normal human stem cells (umbilical cord-derived mesenchymal stem cells) in vitro proliferation after metformin exposure. Altogether, these data clearly suggest that metformin exerts antiproliferative activity on glioblastoma cells, showing a higher specificity toward tumor-initiating cells, and that the inhibition of Akt pathway may represent a possible intracellular target of this effect. PMID:23255107

  12. Osteoblastlike cell adhesion on titanium surfaces modified by plasma nitriding.

    PubMed

    da Silva, Jose Sandro Pereira; Amico, Sandro Campos; Rodrigues, Almir Olegario Neves; Barboza, Carlos Augusto Galvao; Alves, Clodomiro; Croci, Alberto Tesconi

    2011-01-01

    The aim of this study was to evaluate the characteristics of various titanium surfaces modified by cold plasma nitriding in terms of adhesion and proliferation of rat osteoblastlike cells. Samples of grade 2 titanium were subjected to three different surface modification processes: polishing, nitriding by plasma direct current, and nitriding by cathodic cage discharge. To evaluate the effect of the surface treatment on the cellular response, the adhesion and proliferation of osteoblastlike cells (MC3T3) were quantified and the results were analyzed by Kruskal-Wallis and Friedman statistical tests. Cellular morphology was observed by scanning electron microscopy. There was more MC3T3 cell attachment on the rougher surfaces produced by cathodic cage discharge compared with polished samples (P < .05). Plasma nitriding improves titanium surface roughness and wettability, leading to osteoblastlike cell adhesion.

  13. Probes for anionic cell surface detection

    DOEpatents

    Smith, Bradley D.

    2013-03-05

    Embodiments of the present invention are generally directed to compositions comprising a class of molecular probes for detecting the presence of anionic cell surfaces. Embodiments include compositions that are enriched for these compositions and preparations, particularly preparations suitable for use as laboratory/clinical reagents and diagnostic indicators, either alone or as part of a kit. An embodiment of the invention provides for a highly selective agent useful in the discernment and identification of dead or dying cells, such as apoptotic cells, in a relatively calcium-free environment. An embodiment of the invention provides a selective agent for the identification of bacteria in a mixed population of bacterial cells and nonbacterial cells.

  14. Nanometer polymer surface features: the influence on surface energy, protein adsorption and endothelial cell adhesion

    NASA Astrophysics Data System (ADS)

    Carpenter, Joseph; Khang, Dongwoo; Webster, Thomas J.

    2008-12-01

    Current small diameter (<5 mm) synthetic vascular graft materials exhibit poor long-term patency due to thrombosis and intimal hyperplasia. Tissue engineered solutions have yielded functional vascular tissue, but some require an eight-week in vitro culture period prior to implantation—too long for immediate clinical bedside applications. Previous in vitro studies have shown that nanostructured poly(lactic-co-glycolic acid) (PLGA) surfaces elevated endothelial cell adhesion, proliferation, and extracellular matrix synthesis when compared to nanosmooth surfaces. Nonetheless, these studies failed to address the importance of lateral and vertical surface feature dimensionality coupled with surface free energy; nor did such studies elicit an optimum specific surface feature size for promoting endothelial cell adhesion. In this study, a series of highly ordered nanometer to submicron structured PLGA surfaces of identical chemistry were created using a technique employing polystyrene nanobeads and poly(dimethylsiloxane) (PDMS) molds. Results demonstrated increased endothelial cell adhesion on PLGA surfaces with vertical surface features of size less than 18.87 nm but greater than 0 nm due to increased surface energy and subsequently protein (fibronectin and collagen type IV) adsorption. Furthermore, this study provided evidence that the vertical dimension of nanometer surface features, rather than the lateral dimension, is largely responsible for these increases. In this manner, this study provides key design parameters that may promote vascular graft efficacy.

  15. Engineered microtopographies and surface chemistries direct cell attachment and function

    NASA Astrophysics Data System (ADS)

    Magin, Chelsea Marie

    Harrison, in 1914, first recognized that cells respond to physicochemical cues such as substratum topography when he observed that fibroblasts elongated while cultured on spider silk. Recently, techniques developed in the micro-electronics industry have been used to create molds for producing microscaled topographies with various shapes and spatial arrangements. Although these patterning techniques are well-established, very little is known about the mechanisms underlying cell sensing and response to microtopographies. In this work cellular micro-environments with varying surface topographies and chemistries were evaluated with marine organisms and mammalian cells to investigate cellular sensing and response. Biofouling---the accumulation of micro-organisms, plants, and animals on submerged surfaces---is an environmental and economic concern. Engineered topographies, replicated in polydimethylsiloxane elastomer (PDMSe) and functionalized poly(ethylene glycol)-dimethacrylate (PEGDMA) hydrogels, were evaluated for inhibition of marine fouling organism attachment. Microtopographies replicated in PDMSe inhibited attachment of the marine bacterium, Cobetia marina up to 99% versus smooth. The average normalized attachment densities of cells of C. marina and zoospores of the green algae Ulva on PDMSe topographies scaled inversely with the Engineered Roughness Index (ERIII), a representation of surface energy. Attachment densities of Ulva from four assays and C. marina from two growth phases to PDMSe surfaces scaled inversely with one equation: ERI II multiplied by the Reynolds number of the organism (Re) (R 2 = 0.77). The same microtopographies created in PDMSe reduced the initial attachment density and attachment strength of cells of the diatoms Navicula incerta and Seminavis robusta compared to smooth PDMSe. The average normalized attachment density of Navicula after exposure to shear stress (48 Pa) was correlated with the contact area between the diatom and a

  16. Development of living cell force sensors for the interrogation of cell surface interactions

    NASA Astrophysics Data System (ADS)

    Brown, Scott Chang

    The measurement of cell surface interactions, or cell interaction forces, are critical for the early diagnosis and prevention of disease, the design of targeted drug and gene delivery vehicles, the development of next-generation implant materials, and much more. However, the technologies and devices that are currently available are highly limited with respect to the dynamic force range over which they can measure cell-cell or cell-substratum interactions, and with their ability to adequately mimic biologically relevant systems. Consequently, research efforts that involve cell surface interactions have been limited. In this dissertation, existing tools for research at the nanoscale (i.e., atomic force microscopy microcantilevers) are modified to develop living cell force sensors that allow for the highly sensitive measurement of cell-mediated interactions over the entire range of forces expected in biotechnology (and nano-biotechnology) research (from a single to millions of receptor-ligand bonds). Several force sensor motifs have been developed that can be used to measure interactions using single adherent cells, single suspension culture cell, and cell monolayers (tissues) over a wide range of interaction conditions (e.g., approach velocity, shear rate, contact time) using a conventional atomic force microscope. This new tool has been applied to study the pathogenesis of spontaneous pneumothorax and the interaction of cells with 14 man-made interfaces. Consequently, a new hypothesis of the interactions that manifest spontaneous pneumothorax has been developed. Additionally, these findings have the potential to lead to the development of tools for data mining materials and surfaces for unique cell interactions that could have an immense societal impact.

  17. Effect of Stratification on Surface Properties of Corneal Epithelial Cells

    PubMed Central

    Yáñez-Soto, Bernardo; Leonard, Brian C.; Raghunathan, Vijay Krishna; Abbott, Nicholas L.; Murphy, Christopher J.

    2015-01-01

    Purpose The purpose of this study was to determine the influence of mucin expression in an immortalized human corneal epithelial cell line (hTCEpi) on the surface properties of cells, such as wettability, contact angle, and surface heterogeneity. Methods hTCEpi cells were cultured to confluence in serum-free medium. The medium was then replaced by stratification medium to induce mucin biosynthesis. The mucin expression profile was analyzed using quantitative PCR and Western blotting. Contact angles were measured using a two-immiscible liquid method, and contact angle hysteresis was evaluated by tilting the apparatus and recording advancing and receding contact angles. The spatial distribution of mucins was evaluated with fluorescently labeled lectin. Results hTCEpi cells expressed the three main ocular mucins (MUC1, MUC4, and MUC16) with a maximum between days 1 and 3 of the stratification process. Upon stratification, cells caused a very significant increase in contact angle hysteresis, suggesting the development of spatially discrete and heterogeneously distributed surface features, defined by topography and/or chemical functionality. Although atomic force microscopy measurements showed no formation of appreciable topographic features on the surface of the cells, we observed a significant increase in surface chemical heterogeneity. Conclusions The surface chemical heterogeneity of the corneal epithelium may influence the dynamic behavior of tear film by “pinning” the contact line between the cellular surface and aqueous tear film. Engineering the surface properties of corneal epithelium could potentially lead to novel treatments in dry eye disease. PMID:26747762

  18. Autonomous molecular cascades for evaluation of cell surfaces

    NASA Astrophysics Data System (ADS)

    Rudchenko, Maria; Taylor, Steven; Pallavi, Payal; Dechkovskaia, Alesia; Khan, Safana; Butler, Vincent P., Jr.; Rudchenko, Sergei; Stojanovic, Milan N.

    2013-08-01

    Molecular automata are mixtures of molecules that undergo precisely defined structural changes in response to sequential interactions with inputs. Previously studied nucleic acid-based automata include game-playing molecular devices (MAYA automata) and finite-state automata for the analysis of nucleic acids, with the latter inspiring circuits for the analysis of RNA species inside cells. Here, we describe automata based on strand-displacement cascades directed by antibodies that can analyse cells by using their surface markers as inputs. The final output of a molecular automaton that successfully completes its analysis is the presence of a unique molecular tag on the cell surface of a specific subpopulation of lymphocytes within human blood cells.

  19. Imaging and reconstruction of cell cortex structures near the cell surface

    NASA Astrophysics Data System (ADS)

    Jin, Luhong; Zhou, Xiaoxu; Xiu, Peng; Luo, Wei; Huang, Yujia; Yu, Feng; Kuang, Cuifang; Sun, Yonghong; Liu, Xu; Xu, Yingke

    2017-11-01

    Total internal reflection fluorescence microscopy (TIRFM) provides high optical sectioning capability and superb signal-to-noise ratio for imaging of cell cortex structures. The development of multi-angle (MA)-TIRFM permits high axial resolution imaging and reconstruction of cellular structures near the cell surface. Cytoskeleton is composed of a network of filaments, which are important for maintenance of cell function. The high-resolution imaging and quantitative analysis of filament organization would contribute to our understanding of cytoskeleton regulation in cell. Here, we used a custom-developed MA-TIRFM setup, together with stochastic photobleaching and single molecule localization method, to enhance the lateral resolution of TIRFM imaging to about 100 nm. In addition, we proposed novel methods to perform filament segmentation and 3D reconstruction from MA-TIRFM images. Furthermore, we applied these methods to study the 3D localization of cortical actin and microtubule structures in U373 cancer cells. Our results showed that cortical actins localize ∼ 27 nm closer to the plasma membrane when compared with microtubules. We found that treatment of cells with chemotherapy drugs nocodazole and cytochalasin B disassembles cytoskeletal network and induces the reorganization of filaments towards the cell periphery. In summary, this study provides feasible approaches for 3D imaging and analyzing cell surface distribution of cytoskeletal network. Our established microscopy platform and image analysis toolkits would facilitate the study of cytoskeletal network in cells.

  20. Targeting RAS-driven human cancer cells with antibodies to upregulated and essential cell-surface proteins.

    PubMed

    Martinko, Alexander J; Truillet, Charles; Julien, Olivier; Diaz, Juan E; Horlbeck, Max A; Whiteley, Gordon; Blonder, Josip; Weissman, Jonathan S; Bandyopadhyay, Sourav; Evans, Michael J; Wells, James A

    2018-01-23

    While there have been tremendous efforts to target oncogenic RAS signaling from inside the cell, little effort has focused on the cell-surface. Here, we used quantitative surface proteomics to reveal a signature of proteins that are upregulated on cells transformed with KRAS G12V , and driven by MAPK pathway signaling. We next generated a toolkit of recombinant antibodies to seven of these RAS-induced proteins. We found that five of these proteins are broadly distributed on cancer cell lines harboring RAS mutations. In parallel, a cell-surface CRISPRi screen identified integrin and Wnt signaling proteins as critical to RAS-transformed cells. We show that antibodies targeting CDCP1, a protein common to our proteomics and CRISPRi datasets, can be leveraged to deliver cytotoxic and immunotherapeutic payloads to RAS-transformed cancer cells and report for RAS signaling status in vivo. Taken together, this work presents a technological platform for attacking RAS from outside the cell. © 2018, Martinko et al.

  1. Studying Cancer Stem Cell Dynamics on PDMS Surfaces for Microfluidics Device Design

    PubMed Central

    Zhang, Weijia; Choi, Dong Soon; Nguyen, Yen H.; Chang, Jenny; Qin, Lidong

    2013-01-01

    This systematic study clarified a few interfacial aspects of cancer cell phenotypes on polydimethylsiloxane (PDMS) substrates and indicated that the cell phenotypic equilibrium greatly responds to cell-to-surface interactions. We demonstrated that coatings of fibronectin, bovine serum albumin (BSA), or collagen with or without oxygen-plasma treatments of the PDMS surfaces dramatically impacted the phenotypic equilibrium of breast cancer stem cells, while the variations of the PDMS elastic stiffness had much less such effects. Our results showed that the surface coatings of collagen and fibronectin on PDMS maintained breast cancer cell phenotypes to be nearly identical to the cultures on commercial polystyrene Petri dishes. The surface coating of BSA provided a weak cell-substrate adhesion that stimulated the increase in stem-cell-like subpopulation. Our observations may potentially guide surface modification approaches to obtain specific cell phenotypes. PMID:23900274

  2. Dynamics of water droplets detached from porous surfaces of relevance to PEM fuel cells.

    PubMed

    Theodorakakos, A; Ous, T; Gavaises, M; Nouri, J M; Nikolopoulos, N; Yanagihara, H

    2006-08-15

    The detachment of liquid droplets from porous material surfaces used with proton exchange membrane (PEM) fuel cells under the influence of a cross-flowing air is investigated computationally and experimentally. CCD images taken on a purpose-built transparent fuel cell have revealed that the water produced within the PEM is forming droplets on the surface of the gas-diffusion layer. These droplets are swept away if the velocity of the flowing air is above a critical value for a given droplet size. Static and dynamic contact angle measurements for three different carbon gas-diffusion layer materials obtained inside a transparent air-channel test model have been used as input to the numerical model; the latter is based on a Navier-Stokes equations flow solver incorporating the volume of fluid (VOF) two-phase flow methodology. Variable contact angle values around the gas-liquid-solid contact-line as well as their dynamic change during the droplet shape deformation process, have allowed estimation of the adhesion force between the liquid droplet and the solid surface and successful prediction of the separation line at which droplets loose their contact from the solid surface under the influence of the air stream flowing around them. Parametric studies highlight the relevant importance of various factors affecting the detachment of the liquid droplets from the solid surface.

  3. Storage Medium Affects the Surface Porosity of Dental Cements.

    PubMed

    Saghiri, M Ali; Shabani, Asal; Asatourian, Armen; Sheibani, Nader

    2017-08-01

    Calcium silicate-based cements physical properties is influenced by environmental changes. Here, we intended to evaluate the effect of storage medium on surface porosity of root Mineral Trioxide Aggregate (MTA) and Biodentine cement. A total of 40 polyethylene tubes were selected and divided into two groups: Group A (MTA) and Group B (Biodentine). Each group was subdivided into two subgroups (n=10). In subgroups A1 and B1, tubes were transferred to Distilled Water (DW), while samples of subgroup A2 and B2 were transferred to Synthetic Tissue Fluid (STF) as storage medium and samples were stored for three days. All specimens were then placed in a desiccator for 24 hours and then subject to surface porosity evaluation by Scanning Electron Microscopy (SEM) at ×500, ×1000, ×2000 and ×5000 magnifications. The number and the surface porosities were determined by Image J analysis. Data were analyzed by ANOVA at level of significance of p<0.05. The lowest surface porosity was observed in MTA samples stored in STF and the highest was in Biodentine samples stored in DW. Significant differences were noted between groups and subgroups of each group (p< 0.05). MTA samples stored in DW and STF showed significantly lower surface porosities compared to Biodentine samples (p < 0.05). Storage medium can drastically affect the surface porosity of tested calcium silicate-based cements. However, MTA showed lower surface porosity compared to Biodentine cement, which can result in lower microleakage in applied area.

  4. Storage Medium Affects the Surface Porosity of Dental Cements

    PubMed Central

    Shabani, Asal; Asatourian, Armen; Sheibani, Nader

    2017-01-01

    Introduction Calcium silicate-based cements physical properties is influenced by environmental changes. Aim Here, we intended to evaluate the effect of storage medium on surface porosity of root Mineral Trioxide Aggregate (MTA) and Biodentine cement. Materials and Methods A total of 40 polyethylene tubes were selected and divided into two groups: Group A (MTA) and Group B (Biodentine). Each group was subdivided into two subgroups (n=10). In subgroups A1 and B1, tubes were transferred to Distilled Water (DW), while samples of subgroup A2 and B2 were transferred to Synthetic Tissue Fluid (STF) as storage medium and samples were stored for three days. All specimens were then placed in a desiccator for 24 hours and then subject to surface porosity evaluation by Scanning Electron Microscopy (SEM) at ×500, ×1000, ×2000 and ×5000 magnifications. The number and the surface porosities were determined by Image J analysis. Data were analyzed by ANOVA at level of significance of p<0.05. Results The lowest surface porosity was observed in MTA samples stored in STF and the highest was in Biodentine samples stored in DW. Significant differences were noted between groups and subgroups of each group (p< 0.05). MTA samples stored in DW and STF showed significantly lower surface porosities compared to Biodentine samples (p < 0.05). Conclusion Storage medium can drastically affect the surface porosity of tested calcium silicate-based cements. However, MTA showed lower surface porosity compared to Biodentine cement, which can result in lower microleakage in applied area. PMID:28969288

  5. Biofunctionalization of a titanium surface with a nano-sawtooth structure regulates the behavior of rat bone marrow mesenchymal stem cells

    PubMed Central

    Zhang, Wenjie; Li, Zihui; Liu, Yan; Ye, Dongxia; Li, Jinhua; Xu, Lianyi; Wei, Bin; Zhang, Xiuli; Liu, Xuanyong; Jiang, Xinquan

    2012-01-01

    Background: The topography of an implant surface can serve as a powerful signaling cue for attached cells and can enhance the quality of osseointegration. A series of improved implant surfaces functionalized with nanoscale structures have been fabricated using various methods. Methods: In this study, using an H2O2 process, we fabricated two size-controllable sawtooth-like nanostructures with different dimensions on a titanium surface. The effects of the two nano-sawtooth structures on rat bone marrow mesenchymal stem cells (BMMSCs) were evaluated without the addition of osteoinductive chemical factors. Results: These new surface modifications did not adversely affect cell viability, and rat BMMSCs demonstrated a greater increase in proliferation ability on the surfaces of the nano-sawtooth structures than on a control plate. Furthermore, upregulated expression of osteogenic-related genes and proteins indicated that the nano-sawtooth structures promote osteoblastic differentiation of rat BMMSCs. Importantly, the large nano-sawtooth structure resulted in the greatest cell responses, including increased adhesion, proliferation, and differentiation. Conclusion: The enhanced adhesion, proliferation, and osteogenic differentiation abilities of rat BMMSCs on the nano-sawtooth structures suggest the potential to induce improvements in bone-titanium integration in vivo. Our study reveals the key role played by the nano-sawtooth structures on a titanium surface for the fate of rat BMMSCs and provides insights into the study of stem cell-nanostructure relationships and the related design of improved biomedical implant surfaces. PMID:22927760

  6. Waving and skewing: how gravity and the surface of growth media affect root development in Arabidopsis.

    PubMed

    Oliva, Michele; Dunand, Christophe

    2007-01-01

    Arabidopsis seedlings growing on inclined agar surfaces exhibit characteristic root behaviours called 'waving' and 'skewing': the former consists of a series of undulations, whereas the latter is a deviation from the direction of gravity. Even though the precise basis of these growth patterns is not well understood, both gravity and the contact between the medium and the root are considered to be the major players that result in these processes. The influence of these forces on root surface-dependent behaviours can be verified by growing seedlings at different gel pitches: plants growing on vertical plates present roots with slight waving and skewing when compared with seedlings grown on plates held at minor angles of < 90 degrees . However, other factors are thought to modulate root growth on agar; for instance, it has been demonstrated that the presence and concentration of certain compounds in the medium (such as sucrose) and of drugs able to modify the plant cell cytoskeleton also affect skewing and waving. The recent discovery of an active role of ethylene on surface-dependent root behaviour, and the finding of new mutants showing anomalous growth, pave the way for a more detailed description of these phenomena.

  7. Cell-surface prion protein interacts with glycosaminoglycans.

    PubMed Central

    Pan, Tao; Wong, Boon-Seng; Liu, Tong; Li, Ruliang; Petersen, Robert B; Sy, Man-Sun

    2002-01-01

    We used ELISA and flow cytometry to study the binding of prion protein PrP to glycosaminoglycans (GAGs). We found that recombinant human PrP (rPrP) binds GAGs including chondroitin sulphate A, chondroitin sulphate B, hyaluronic acid, and heparin. rPrP binding to GAGs occurs via the N-terminus, a region known to bind divalent cations. Additionally, rPrP binding to GAGs is enhanced in the presence of Cu2+ and Zn2+, but not Ca2+ and Mn2+. rPrP binds heparin strongest, and the binding is inhibited by certain heparin analogues, including heparin disaccharide and sulphate-containing monosaccharides, but not by acetylated heparin. Full-length normal cellular prion protein (PrPC), but not N-terminally truncated PrPC species, from human brain bind GAGs in a similar Cu2+/Zn2+-enhanced fashion. We found that GAGs specifically bind to a synthetic peptide corresponding to amino acid residues 23-35 in the N-terminus of rPrP. We further demonstrated that while both wild-type PrPC and an octapeptide-repeat-deleted mutant PrP produced by transfected cells bound heparin at the cell surface, the PrP N-terminal deletion mutant and non-transfectant control failed to bind heparin. Binding of heparin to wild-type PrPC on the cell surface results in a reduction of the level of cell-surface PrPC. These results provide strong evidence that PrPC is a surface receptor for GAGs. PMID:12186633

  8. Uptake and bio-reactivity of polystyrene nanoparticles is affected by surface modifications, ageing and LPS adsorption: in vitro studies on neural tissue cells

    NASA Astrophysics Data System (ADS)

    Murali, Kumarasamy; Kenesei, Kata; Li, Yang; Demeter, Kornél; Környei, Zsuzsanna; Madarász, Emilia

    2015-02-01

    Because of their capacity of crossing an intact blood-brain barrier and reaching the brain through an injured barrier or via the nasal epithelium, nanoparticles have been considered as vehicles to deliver drugs and as contrast materials for brain imaging. The potential neurotoxicity of nanoparticles, however, is not fully explored. Using particles with a biologically inert polystyrene core material, we investigated the role of the chemical composition of particle surfaces in the in vitro interaction with different neural cell types. PS NPs within a size-range of 45-70 nm influenced the metabolic activity of cells depending on the cell-type, but caused toxicity only at extremely high particle concentrations. Neurons did not internalize particles, while microglial cells ingested a large amount of carboxylated but almost no PEGylated NPs. PEGylation reduced the protein adsorption, toxicity and cellular uptake of NPs. After storage (shelf-life >6 months), the toxicity and cellular uptake of NPs increased. The altered biological activity of ``aged'' NPs was due to particle aggregation and due to the adsorption of bioactive compounds on NP surfaces. Aggregation by increasing the size and sedimentation velocity of NPs results in increased cell-targeted NP doses. The ready endotoxin adsorption which cannot be prevented by PEG coating, can render the particles toxic. The age-dependent changes in otherwise harmless NPs could be the important sources for variability in the effects of NPs, and could explain the contradictory data obtained with ``identical'' NPs.Because of their capacity of crossing an intact blood-brain barrier and reaching the brain through an injured barrier or via the nasal epithelium, nanoparticles have been considered as vehicles to deliver drugs and as contrast materials for brain imaging. The potential neurotoxicity of nanoparticles, however, is not fully explored. Using particles with a biologically inert polystyrene core material, we investigated the

  9. Encapsulant Adhesion to Surface Metallization on Photovoltaic Cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tracy, Jared; Bosco, Nick; Dauskardt, Reinhold

    Delamination of encapsulant materials from PV cell surfaces often appears to originate at regions with metallization. Using a fracture mechanics based metrology, the adhesion of ethylene vinyl acetate (EVA) encapsulant to screen-printed silver metallization was evaluated. At room temperature, the fracture energy Gc [J/m2] of the EVA/silver interface (952 J/m2) was ~70% lower than that of the EVA/antireflective (AR) coating (>2900 J/m2) and ~60% lower than that of the EVA to the surface of cell (2265 J/m2). After only 300 h of damp heat aging, the adhesion energy of the silver interface dropped to and plateaued at ~50-60 J/m2 whilemore » that of the EVA/AR coating and EVA/cell remained mostly unchanged. Elemental surface analysis showed that the EVA separates from the silver in a purely adhesive manner, indicating that bonds at the interface were likely displaced in the presence of humidity and chemical byproducts at elevated temperature, which in part accounts for the propensity of metalized surfaces to delaminate in the field.« less

  10. Cell-surface display of enzymes by the yeast Saccharomyces cerevisiae for synthetic biology.

    PubMed

    Tanaka, Tsutomu; Kondo, Akihiko

    2015-02-01

    In yeast cell-surface displays, functional proteins, such as cellulases, are genetically fused to an anchor protein and expressed on the cell surface. Saccharomyces cerevisiae, which is often utilized as a cell factory for the production of fuels, chemicals, and proteins, is the most commonly used yeast for cell-surface display. To construct yeast cells with a desired function, such as the ability to utilize cellulose as a substrate for bioethanol production, cell-surface display techniques for the efficient expression of enzymes on the cell membrane need to be combined with metabolic engineering approaches for manipulating target pathways within cells. In this Minireview, we summarize the recent progress of biorefinery fields in the development and application of yeast cell-surface displays from a synthetic biology perspective and discuss approaches for further enhancing cell-surface display efficiency. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.

  11. Accelerated cell-surface interlocking on plasma polymer-modified porous ceramics.

    PubMed

    Rebl, Henrike; Finke, Birgit; Schmidt, Jürgen; Mohamad, Heba S; Ihrke, Roland; Helm, Christiane A; Nebe, J Barbara

    2016-12-01

    Excellent osseointegration of permanent implants is crucial for the long lasting success of the implantation. To improve the osseointegrative potential, bio-inert titanium alloy surfaces (Ti6Al4V) are modified by plasma chemical oxidation (PCO®) of the titanium-oxide layer to a non-stoichiometric, amorphous calcium phosphate layer. The native titanium-oxide film measuring only a few nanometers is converted by PCO® to a thick porous calcium phosphate layer of about 10μm. In a second step the PCO surface is combined with a cell adhesive plasma-polymerized allylamine (PPAAm) nano film (5 and 50nm). Independent of the PPAAm coating homogeneity, the human osteoblast-like MG-63 cells show a remarkable increase in cell size and well-developed filopodia. Analyses of the actin cytoskeleton reveal that the cells mold to the pore shape of the PPAAm-covered PCO, thereby establishing a strong attachment to the surface. Interestingly, we could demonstrate that even though our untreated PCO shows excellent hydrophilicity, this alone is not sufficient to facilitate fast cell spreading, but the positive surface charges mediated by PPAAm. This multilayer composite material guarantees enhanced interlocking of the cells with the porous surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Surface properties and graphitization of polyacrylonitrile based fiber electrodes affecting the negative half-cell reaction in vanadium redox flow batteries

    NASA Astrophysics Data System (ADS)

    Langner, J.; Bruns, M.; Dixon, D.; Nefedov, A.; Wöll, Ch.; Scheiba, F.; Ehrenberg, H.; Roth, C.; Melke, J.

    2016-07-01

    Carbon felt electrodes for vanadium redox flow batteries are obtained by the graphitization of polyacrylonitrile based felts at different temperatures. Subsequently, the surface of the felts is modified via thermal oxidation at various temperatures. A single-cell experiment shows that the voltage efficiency is increased by this treatment. Electrode potentials measured with reference electrode setup show that this voltage efficiency increase is caused mainly by a reduction of the overpotential of the negative half-cell reaction. Consequently, this reaction is investigated further by cyclic voltammetry and the electrode activity is correlated with structural and surface chemical properties of the carbon fibers. By Raman, X-ray photoelectron and near edge X-ray absorption fine structure spectroscopy the role of edge sites and oxygen containing functional groups (OCFs) for the electrochemical activity are elucidated. A significant activity increase is observed in correlation with these two characteristics. The amount of OCFs is correlated with structural defects (e.g. edge sites) of the carbon fibers and therefore decreases with an increasing graphitization degree. Thus, for the same thermal oxidation temperature carbon fibers graphitized at a lower temperature show higher activities than those graphitized at a higher temperature.

  13. Establishment of cell surface engineering and its development.

    PubMed

    Ueda, Mitsuyoshi

    2016-07-01

    Cell surface display of proteins/peptides has been established based on mechanisms of localizing proteins to the cell surface. In contrast to conventional intracellular and extracellular (secretion) expression systems, this method, generally called an arming technology, is particularly effective when using yeasts as a host, because the control of protein folding that is often required for the preparation of proteins can be natural. This technology can be employed for basic and applied research purposes. In this review, I describe various strategies for the construction of engineered yeasts and provide an outline of the diverse applications of this technology to industrial processes such as the production of biofuels and chemicals, as well as bioremediation and health-related processes. Furthermore, this technology is suitable for novel protein engineering and directed evolution through high-throughput screening, because proteins/peptides displayed on the cell surface can be directly analyzed using intact cells without concentration and purification. Functional proteins/peptides with improved or novel functions can be created using this beneficial, powerful, and promising technique.

  14. Rho-associated kinase (ROCK) inhibition reverses low cell activity on hydrophobic surfaces.

    PubMed

    Tian, Yu Shun; Kim, Hyun Jung; Kim, Hyun-Man

    2009-08-28

    Hydrophobic polymers do not offer an adequate scaffold surface for cells to attach, migrate, proliferate, and differentiate. Thus, hydrophobic scaffolds for tissue engineering have traditionally been physicochemically modified to enhance cellular activity. However, modifying the surface by chemical or physical treatment requires supplementary engineering procedures. In the present study, regulation of a cell signal transduction pathway reversed the low cellular activity on a hydrophobic surface without surface modification. Inhibition of Rho-associated kinase (ROCK) by Y-27632 markedly enhanced adhesion, migration, and proliferation of osteoblastic cells cultured on a hydrophobic polystyrene surface. ROCK inhibition regulated cell-cycle-related molecules on the hydrophobic surface. This inhibition also decreased expression of the inhibitors of cyclin-dependent kinases such as p21(cip1) and p27(kip1) and increased expression of cyclin A and D. These results indicate that defective cellular activity on the hydrophobic surface can be reversed by the control of a cell signal transduction pathway without physicochemical surface modification.

  15. Targeted identification of metastasis-associated cell-surface sialoglycoproteins in prostate cancer.

    PubMed

    Yang, Lifang; Nyalwidhe, Julius O; Guo, Siqi; Drake, Richard R; Semmes, O John

    2011-06-01

    Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC(4)ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells.

  16. Surface modification of closed plastic bags for adherent cell cultivation

    NASA Astrophysics Data System (ADS)

    Lachmann, K.; Dohse, A.; Thomas, M.; Pohl, S.; Meyring, W.; Dittmar, K. E. J.; Lindenmeier, W.; Klages, C.-P.

    2011-07-01

    In modern medicine human mesenchymal stem cells are becoming increasingly important. However, a successful cultivation of this type of cells is only possible under very specific conditions. Of great importance, for instance, are the absence of contaminants such as foreign microbiological organisms, i.e., sterility, and the chemical functionalization of the ground on which the cells are grown. As cultivation of these cells makes high demands, a new procedure for cell cultivation has been developed in which closed plastic bags are used. For adherent cell growth chemical functional groups have to be introduced on the inner surface of the plastic bag. This can be achieved by a new, atmospheric-pressure plasma-based method presented in this paper. The method which was developed jointly by the Fraunhofer IST and the Helmholtz HZI can be implemented in automated equipment as is also shown in this contribution. Plasma process gases used include helium or helium-based gas mixtures (He + N2 + H2) and vapors of suitable film-forming agents or precursors such as APTMS, DACH, and TMOS in helium. The effect of plasma treatment is investigated by FTIR-ATR spectroscopy as well as surface tension determination based on contact angle measurements and XPS. Plasma treatment in nominally pure helium increases the surface tension of the polymer foil due to the presence of oxygen traces in the gas and oxygen diffusing through the gas-permeable foil, respectively, reacting with surface radical centers formed during contact with the discharge. Primary amino groups are obtained on the inner surface by treatment in mixtures with nitrogen and hydrogen albeit their amount is comparably small due to diffusion of oxygen through the gas-permeable bag, interfering with the plasma-amination process. Surface modifications introducing amino groups on the inner surface turned out to be most efficient in the promotion of cell growth.

  17. Biofilm formation affects surface properties of novel bioactive glass-containing composites.

    PubMed

    Hyun, Hong-Keun; Salehi, Satin; Ferracane, Jack L

    2015-12-01

    This study investigated the effects of bacterial biofilm on the surface properties of novel bioactive glass (BAG)-containing composites of different initial surface roughness. BAG (65 mol% Si; 4% P; 31% Ca) and BAG-F (61% Si; 31% Ca; 4% P; 3% F; 1% B) were synthesized by the sol-gel method and micronized (size ∼0.1-10 μm). Composites with 72wt% total filler load were prepared by replacing 15% of the silanized Sr glass with BAG, BAG-F, or silanized silica. Specimens (n=10/group) were light-cured and divided into 4 subgroups of different surface roughness by wet polishing with 600 and then up to 1200, 2400, or 4000 grit SiC. Surface roughness (SR), gloss, and Knoop microhardness were measured before and after incubating in media with or without a Streptococcus mutans (UA 159) biofilm for 2 weeks. Results were analyzed with ANOVA/Tukey's test (α=0.05). The SR of the BAG-containing composites with the smoothest surfaces (2400/4000 grit) increased in media or bacteria; the SR of the roughest composites (600 grit) decreased. The gloss of the smoothest BAG-containing composites decreased in bacteria and media-only, but more in media-alone. The microhardness of all of the composites decreased with exposure to media or bacteria, with BAG-containing composites affected more than the control. Exposure to bacterial biofilm and its media produced enhanced roughness and reduced gloss and surface microhardness of highly polished dental composites containing a bioactive glass additive, which could affect further biofilm formation, as well as the esthetics, of restorations made from such a material. Copyright © 2015 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  18. Organic Electrochemical Transistors for the Detection of Cell Surface Glycans.

    PubMed

    Chen, Lizhen; Fu, Ying; Wang, Naixiang; Yang, Anneng; Li, Yuanzhe; Wu, Jie; Ju, Huangxian; Yan, Feng

    2018-05-23

    Cell surface glycans play critical roles in diverse biological processes, such as cell-cell communication, immunity, infection, development, and differentiation. Their expressions are closely related to cancer growth and metastasis. This work demonstrates an organic electrochemical transistor (OECT)-based biosensor for the detection of glycan expression on living cancer cells. Herein, mannose on human breast cancer cells (MCF-7) as the target glycan model, poly dimethyl diallyl ammonium chloride-multiwall carbon nanotubes (PDDA-MWCNTs) as the loading interface, concanavalin A (Con A) with active mannose binding sites, aptamer and horseradish peroxidase co-immobilized gold nanoparticles (HRP-aptamer-Au NPs) as specific nanoprobes are used to fabricate the OECT biosensor. In this strategy, PDDA-MWCNT interfaces can enhance the loading of Con A, and the target cells can be captured through Con A via active mannose binding sites. Thus, the expression of cell surface can be reflected by the amount of cells captured on the gate. Specific nanoprobes are introduced to the captured cells to produce an OECT signal because of the reduction of hydrogen peroxide catalyzed by HRP conjugated on Au nanoparticles, while the aptamer on nanoprobes can selectively recognize the MCF-7 cells. It is reasonable that more target cells are captured on the gate electrode, more HRP-nanoprobes are loaded thus a larger signal response. The device shows an obvious response to MCF-7 cells down to 10 cells/μL and can be used to selectively monitor the change of mannose expression on cell surfaces upon a treatment with the N-glycan inhibitor. The OECT-based biosensor is promising for the analysis of glycan expressions on the surfaces of different types of cells.

  19. Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms

    PubMed Central

    van Gestel, Jordi; Weissing, Franz J; Kuipers, Oscar P; Kovács, Ákos T

    2014-01-01

    In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express ‘cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation. PMID:24694715

  20. Density of founder cells affects spatial pattern formation and cooperation in Bacillus subtilis biofilms.

    PubMed

    van Gestel, Jordi; Weissing, Franz J; Kuipers, Oscar P; Kovács, Akos T

    2014-10-01

    In nature, most bacteria live in surface-attached sedentary communities known as biofilms. Biofilms are often studied with respect to bacterial interactions. Many cells inhabiting biofilms are assumed to express 'cooperative traits', like the secretion of extracellular polysaccharides (EPS). These traits can enhance biofilm-related properties, such as stress resilience or colony expansion, while being costly to the cells that express them. In well-mixed populations cooperation is difficult to achieve, because non-cooperative individuals can reap the benefits of cooperation without having to pay the costs. The physical process of biofilm growth can, however, result in the spatial segregation of cooperative from non-cooperative individuals. This segregation can prevent non-cooperative cells from exploiting cooperative neighbors. Here we examine the interaction between spatial pattern formation and cooperation in Bacillus subtilis biofilms. We show, experimentally and by mathematical modeling, that the density of cells at the onset of biofilm growth affects pattern formation during biofilm growth. At low initial cell densities, co-cultured strains strongly segregate in space, whereas spatial segregation does not occur at high initial cell densities. As a consequence, EPS-producing cells have a competitive advantage over non-cooperative mutants when biofilms are initiated at a low density of founder cells, whereas EPS-deficient cells have an advantage at high cell densities. These results underline the importance of spatial pattern formation for competition among bacterial strains and the evolution of microbial cooperation.

  1. The behavior of MC3T3-E1 cells on chitosan/poly-L-lysine composite films: effect of nanotopography, surface chemistry, and wettability.

    PubMed

    Zheng, Zhenhuan; Zhang, Ling; Kong, Lijun; Wang, Aijun; Gong, Yandao; Zhang, Xiufang

    2009-05-01

    In the present work, a series of composite films were produced from chitosan/poly-L-lysine blend solutions. The surface topography, chemistry, and wettability of composite films were characterized by atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle assay, respectively. For all composite films, blending with poly-L-lysine induced changes in surface chemistry and wettability. Interestingly, it was also found that increasing poly-L-lysine weight fraction in blend solutions could result in different nanoscaled surface topographic features, which displayed particle-, granule-, or fiber-dominant morphologies. MC3T3-E1 osteoblast-like cells were cultured on all composite films to evaluate the effects of surface nanotopography, chemistry, and wettability on cell behavior. The observations indicated that MC3T3-E1 cell behavior was affected by surface topography, chemistry, and wettability simultaneously and that cells showed strong responses to surface topography. On fiber-dominant surface, cells fully spread with obvious cytoskeleton organization and exhibited significantly higher level of adhesion and proliferation compared with particle- or granule-dominant surfaces. Furthermore, fiber-dominant surface also induced greater expression of mature osteogenic marker osteocalcin and higher mineralization based on RT-PCR and von Kossa staining. The results suggest that topographic modification of chitosan substratum at the nanoscale may be exploited in regulating cell behavior for its applications in tissue engineering.

  2. The structurally effect of surface coated rhamnogalacturonan I on response of the osteoblast-like cell line SaOS-2.

    PubMed

    Svava, Rikke; Gurzawska, Katarzyna; Yihau, Yu; Haugshøj, Kenneth Brian; Dirscherl, Kai; Levery, Steven B; Jørgensen, Niklas Rye; Gotfredsen, Klaus; Damager, Iben; Ulvskov, Peter; Jørgensen, Bodil

    2014-06-01

    Osseointegration is important when implants are inserted into the bone and can be improved by biochemical surface coating of the implant. In this paper enzymatically modified rhamnogalacturonan I (RG-I) from apple and lupin was used for biochemical coating of aminated surfaces and the importance of the quality of RG-I, the nature of the binding, the fine structure of RG-I, and its effect on SaOS-2 cell line cultured on coated surfaces was investigated. SaOS-2 cells are osteoblast-like cells and a well-established in vitro model of bone-matrix forming osteoblasts. Purification by gel filtration could remove small fragments of galacturonic acid (GalA) and binding studies showed that the purity of the RG-I molecules was important for the quality of the coating. The structure of RG-I and osteoblast-like cells' viability were positively correlated so that high content of 1,4-linked galactose (Gal) and a low content of arabinose in the RG-I molecules favored cell viability. These results indicate that coating of implants with RG-I affect osseointegration positively. Copyright © 2013 Wiley Periodicals, Inc.

  3. Microarray of neuroblastoma cells on the selectively functionalized nanocrystalline diamond thin film surface

    NASA Astrophysics Data System (ADS)

    Park, Young-Sang; Son, Hyeong-Guk; Kim, Dae-Hoon; Oh, Hong-Gi; Lee, Da-Som; Kim, Min-Hye; Lim, Ki-Moo; Song, Kwang-Soup

    2016-01-01

    Nanocrystalline diamond (NCD) film surfaces were modified with fluorine or oxygen by plasma treatment in an O2 or C3F8 gas environment in order to induce wettability. The oxygenated-NCD (O-NCD) film surface was hydrophilic and the fluorinated-NCD (F-NCD) surface was hydrophobic. The efficiency of early cell adhesion, which is dependent on the wettability of the cell culture plate and necessary for the growth and proliferation of cells, was 89.62 ± 3.92% on the O-NCD film and 7.78 ± 0.77% on the F-NCD film surface after 3 h of cell culture. The wettability of the NCD film surface was artificially modified using a metal mask and plasma treatment to fabricate a micro-pattern. Four types of micro-patterns were fabricated (line, circle, mesh, and word) on the NCD film surface. We precisely arrayed the neuroblastoma cells on the micro-patterned NCD film surfaces by controlling the surface wettability and cell seeding density. The neuroblastoma cells adhered and proliferated along the O-NCD film surface.

  4. Cell-surface markers for colon adenoma and adenocarcinoma

    PubMed Central

    Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S.; Wojtkowiak, Jonathan W.; Stark, Valerie E.; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L.

    2016-01-01

    Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC. PMID:26894861

  5. Cell-surface markers for colon adenoma and adenocarcinoma.

    PubMed

    Sewda, Kamini; Coppola, Domenico; Enkemann, Steven; Yue, Binglin; Kim, Jongphil; Lopez, Alexis S; Wojtkowiak, Jonathan W; Stark, Valerie E; Morse, Brian; Shibata, David; Vignesh, Shivakumar; Morse, David L

    2016-04-05

    Early detection of colorectal cancer (CRC) is crucial for effective treatment. Among CRC screening techniques, optical colonoscopy is widely considered the gold standard. However, it is a costly and invasive procedure with a low rate of compliance. Our long-term goal is to develop molecular imaging agents for the non-invasive detection of CRC by molecular imaging-based colonoscopy using CT, MRI or fluorescence. To achieve this, cell surface targets must be identified and validated. Here, we report the discovery of cell-surface markers that distinguish CRC from surrounding tissues that could be used as molecular imaging targets. Profiling of mRNA expression microarray data from patient tissues including adenoma, adenocarcinoma, and normal gastrointestinal tissues was used to identify potential CRC specific cell-surface markers. Of the identified markers, six were selected for further validation (CLDN1, GPR56, GRM8, LY6G6D/F, SLCO1B3 and TLR4). Protein expression was confirmed by immunohistochemistry of patient tissues. Except for SLCO1B3, diffuse and low expression was observed for each marker in normal colon tissues. The three markers with the greatest protein overexpression were CLDN1, LY6G6D/F and TLR4, where at least one of these markers was overexpressed in 97% of the CRC samples. GPR56, LY6G6D/F and SLCO1B3 protein expression was significantly correlated with the proximal tumor location and with expression of mismatch repair genes. Marker expression was further validated in CRC cell lines. Hence, three cell-surface markers were discovered that distinguish CRC from surrounding normal tissues. These markers can be used to develop imaging or therapeutic agents targeted to the luminal surface of CRC.

  6. Cell Surface Changes Associated with Cellular Immune Reactions in Drosophila

    NASA Astrophysics Data System (ADS)

    Nappi, Anthony J.; Silvers, Michael

    1984-09-01

    In Drosophila melanogaster a temperature-induced change in immune competence accompanies cell surface alterations that cause its blood cells to adhere and to encapsulate a parasite. At 29 degrees C the blood cells of the tumorous-lethal (Tuml) mutant show a high degree of immune competence and encapsulate the eggs of the parasitic wasp Leptopilina heterotoma. At 21 degrees C the blood cells are essentially immune incompetent. High percentages of lectin binding cells were found under conditions which potentiated cellular encapsulation responses. Some immune reactive blood cells did not bind lectin. The low percentages of lectin binding cells in susceptible hosts suggest that developing parasites alter the cell surface of the blood cells of immune reactive hosts.

  7. Downregulation of cell surface molecules during noncytopathic infection of T cells with human immunodeficiency virus.

    PubMed Central

    Stevenson, M; Zhang, X H; Volsky, D J

    1987-01-01

    Noncytopathic infection of human T-lymphoid cell line CR-10 with human immunodeficiency virus (HIV) (CEM-N1T isolate) resulted in a gradual loss of cell surface receptors for OKT4/OKT4A (HIV receptor), OKT8, OKT3, and OKT11 but not for OKT9 (transferrin receptor) within 10 days after infection. Surface receptor decline was accompanied by a rapid increase in HIV antigens and mRNA expression. Multireceptor downregulation was also observed in three T-lymphoid cell lines (MT-4, CEM, and HBD-1) cytopathically infected with the HIV/N1T virus and in HUT-78 cells infected with the HIV/SF-2 isolate. HIV-infected and uninfected CR-10 cells contained similar levels of mRNAs coding for T3, T8, T9, T11, HLA-A2, and HLA-B7 proteins. By densitometry, fully infected CR-10 cells showed approximately 75% reduction in T4 and tubulin (beta chain) mRNA levels when compared with uninfected CR-10 cells. No such reduction was detected in HIV-infected MT-4 and HBD-1 cells. A T-cell receptor gene (beta chain) rearrangement study revealed that no distinct CR-10 subpopulation was selected out upon infection with HIV. Our results suggest that the reduction in cell surface receptors observed between 1 and 2 weeks postinfection cannot be directly attributed to similar reductions in mRNA levels coding for these receptor proteins. We conclude that HIV infection induces posttranscriptional downregulation of several T-cell surface receptors. Images PMID:3500327

  8. Cell surface characteristics enable encrustation-free survival of neutrophilic iron-oxidizing bacteria

    NASA Astrophysics Data System (ADS)

    Saini, G.; Chan, C. S.

    2011-12-01

    Microbial growth in mineralizing environments depends on the cells' ability to evade surface precipitation. Cell-mineral interactions may be required for metabolism, but if unmoderated, cells could become encrusted, which would limit diffusion of nutrients and waste across cell walls. A combination of cell surface charge and hydrophobicity could enable the survival of microbes in such environments by inhibiting mineral attachment. To investigate this mechanism, we characterized the surfaces of two neutrophilic iron-oxidizing bacteria (FeOB): Mariprofundus ferrooxydans, a Zetaproteobacterium from Fe(II)-rich submarine hydrothermal vents and a Betaproteobacterium Gallionellales strain R-1, recently isolated from a ferrous groundwater seep. Both bacteria produce iron oxyhydroxides, yet successfully escape surface encrustation while inhabiting milieu where iron minerals are also produced by abiotic processes. SEM-EDX and TEM-EELS analyses of cultured bacteria revealed no iron on the cell surfaces. Zeta potential measurements showed that these bacteria have very small negative surface charge (0 to -4 mV) over a pH range of 4-9, indicating near-neutrally charged surfaces. Water contact angle measurements and thermodynamic calculations demonstrate that both bacteria and abiotically-formed Fe oxhydroxides are hydrophilic. Extended-DLVO calculations showed that hydrophilic repulsion between cells and minerals dominates over electrostatic and Lifshitz-van der Waals interactions. This leads to overall repulsion between microbes and minerals, thus preventing surface encrustation. Low surface charge and hydrophilicity (determined by microbial adhesion to hydrocarbon assay) were common features for both live and azide-inhibited cells, which shows that surface characteristics do not depend on active metabolism. It is remarkable that these two phylogenetically-distant bacteria from different environments employ similar adaptations to prevent surface mineralization. Our results

  9. An unscaled parameter to measure the order of surfaces: a new surface elaboration to increase cells adhesion.

    PubMed

    Bigerelle, M; Anselme, K; Dufresne, E; Hardouin, P; Iost, A

    2002-08-01

    We present a new parameter to quantify the order of a surface. This parameter is scale-independent and can be used to compare the organization of a surface at different scales of range and amplitude. To test the accuracy of this roughness parameter versus a hundred existing ones, we created an original statistical bootstrap method. In order to assess the physical relevance of this new parameter, we elaborated a great number of surfaces with various roughness amplitudes on titanium and titanium-based alloys using different physical processes. Then we studied the influence of the roughness amplitude on in vitro adhesion and proliferation of human osteoblasts. It was then shown that our new parameter best discriminates among the cell adhesion phenomena than others' parameters (Average roughness (Ra em leader )): cells adhere better on isotropic surfaces with a low order, provided this order is quantified on a scale that is more important than that of the cells. Additionally, on these low ordered metallic surfaces, the shape of the cells presents the same morphological aspect as that we can see on the human bone trabeculae. The method used to prepare these isotropic surfaces (electroerosion) could be undoubtedly and easily applied to prepare most biomaterials with complex geometries and to improve bone implant integration. Moreover, the new order parameter we developed may be particularly useful for the fundamental understanding of the mechanism of bone cell installation on a relief and of the formation of bone cell-material interface.

  10. Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

    PubMed Central

    Owen, Caroline A.

    2008-01-01

    A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

  11. Fabrication of cell container arrays with overlaid surface topographies.

    PubMed

    Truckenmüller, Roman; Giselbrecht, Stefan; Escalante-Marun, Maryana; Groenendijk, Max; Papenburg, Bernke; Rivron, Nicolas; Unadkat, Hemant; Saile, Volker; Subramaniam, Vinod; van den Berg, Albert; van Blitterswijk, Clemens; Wessling, Matthias; de Boer, Jan; Stamatialis, Dimitrios

    2012-02-01

    This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.

  12. Triiodothyronine Acutely Stimulates Glucose Transport into L6 Muscle Cells Without Increasing Surface GLUT4, GLUT1, or GLUT3

    PubMed Central

    Teixeira, Silvania Silva; Tamrakar, Akhilesh K.; Goulart-Silva, Francemilson; Serrano-Nascimento, Caroline; Klip, Amira

    2012-01-01

    Background Thyroid hormones (THs) act genomically to stimulate glucose transport by elevating glucose transporter (Slc2a) expression and glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T3) acutely affects glucose transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T3 and insulin action. Methods Differentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T3, Tx plus insulin, and Tx plus insulin and T3. Results Glucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T3 treatment for 30 minutes increased glucose transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the groups studied. The surface GLUT1 content of L6-GLUT1myc cells also remained unaltered after T3 treatment; however, in these cells glucose transport was not stimulated by T3. In wild-type L6 cells, although T3 treatment increased the total amount of GLUT3, it did not change the surface GLUT3 content. Moreover, within 30 minutes, T3 stimulation of glucose uptake was additive to that of insulin in L6-GLUT4myc cells. As expected, insulin elevated surface GLUT4 content and glucose uptake. However, interestingly, surface GLUT4 content remained unchanged or even dropped with T3 plus insulin. Conclusions These data reveal that T3 rapidly increases glucose uptake in L6-GLUT4myc cells, which, at least for 30 minutes, did not depend on an increment in GLUT4 at the cell surface yet potentiates insulin action. We propose that this rapid T3 effect involves activation of GLUT4 transporters at the cell surface, but cannot discount the involvement of an unknown GLUT. PMID:22663547

  13. Clustering of adhesion receptors following exposure of insect blood cells to foreign surfaces.

    PubMed

    Nardi, James B; Zhuang, Shufei; Pilas, Barbara; Bee, Charles Mark; Kanost, Michael R

    2005-05-01

    Cell-mediated immune responses of insects involve interactions of two main classes of blood cells (hemocytes) known as granular cells and plasmatocytes. In response to a foreign surface, these hemocytes suddenly transform from circulating, non-adherent cells to cells that interact and adhere to each other and the foreign surface. This report presents evidence that during this adhesive transformation the extracellular matrix (ECM) proteins lacunin and a ligand for peanut agglutinin (PNA) lectin are released by granular cells and bind to surfaces of both granular cells and plasmatocytes. ECM protein co-localizes on cell surfaces with the adhesive receptors integrin and neuroglian, a member of the immunoglobulin superfamily. The ECM protein(s) secreted by granular cells are hypothesized to interact with adhesion receptors such as neuroglian and integrin by cross linking and clustering them on hemocyte surfaces. This clustering of receptors is known to enhance the adhesiveness (avidity) of interacting mammalian immune cells. The formation of ring-shaped clusters of these adhesion receptors on surfaces of insect immune cells represents an evolutionary antecedent of the mammalian immunological synapse.

  14. Autotransporter-based cell surface display in Gram-negative bacteria.

    PubMed

    Nicolay, Toon; Vanderleyden, Jos; Spaepen, Stijn

    2015-02-01

    Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

  15. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses

    PubMed Central

    Montanuy, Imma; Alejo, Ali; Alcami, Antonio

    2011-01-01

    Eradication of smallpox was accomplished 30 yr ago, but poxviral infections still represent a public health concern due to the potential release of variola virus or the emergence of zoonotic poxviruses, such as monkeypox virus. A critical determinant of poxvirus virulence is the inhibition of interferons (IFNs) by the virus-encoded type I IFN-binding protein (IFNα/βBP). This immunomodulatory protein is secreted and has the unique property of interacting with the cell surface in order to prevent IFN-mediated antiviral responses. However, the mechanism of its attachment to the cell surface remains unknown. Using surface plasmon resonance and cell-binding assays, we report that the IFNα/βBP from vaccinia virus, the smallpox vaccine, interacts with cell surface glycosaminoglycans (GAGs). Analysis of the contribution of different regions of the protein to cell surface binding demonstrated that clusters of basic residues in the first immunoglobulin domain mediate GAG interactions. Furthermore, mutation of the GAG-interaction motifs does not affect its IFN-binding and -blocking capacity. Functional conservation of GAG-binding sites is demonstrated for the IFNα/βBP from variola and monkeypox viruses, extending our understanding of immune modulation by the most virulent human poxviruses. These results are relevant for the design of improved vaccines and intervention strategies.—Montanuy, I., Alejo, A., Alcami, A. Glycosaminoglycans mediate retention of the poxvirus type I interferon binding protein at the cell surface to locally block interferon antiviral responses. PMID:21372110

  16. Targeted Identification of Metastasis-associated Cell-surface Sialoglycoproteins in Prostate Cancer*

    PubMed Central

    Yang, Lifang; Nyalwidhe, Julius O.; Guo, Siqi; Drake, Richard R.; Semmes, O. John

    2011-01-01

    Covalent attachment of carbohydrates to proteins is one of the most common post-translational modifications. At the cell surface, sugar moieties of glycoproteins contribute to molecular recognition events involved in cancer metastasis. We have combined glycan metabolic labeling with mass spectrometry analysis to identify and characterize metastasis-associated cell surface sialoglycoproteins. Our model system used syngeneic prostate cancer cell lines derived from PC3 (N2, nonmetastatic, and ML2, highly metastatic). The metabolic incorporation of AC4ManNAz and subsequent specific labeling of cell surface sialylation was confirmed by flow cytometry and confocal microscopy. Affinity isolation of the modified sialic-acid containing cell surface proteins via click chemistry was followed by SDS-PAGE separation and liquid chromatography-tandem MS analysis. We identified 324 proteins from N2 and 372 proteins of ML2. Using conservative annotation, 64 proteins (26%) from N2 and 72 proteins (29%) from ML2 were classified as extracellular or membrane-associated glycoproteins. A selective enrichment of sialoglycoproteins was confirmed. When compared with global proteomic analysis of the same cells, the proportion of identified glycoprotein and cell-surface proteins were on average threefold higher using the selective capture approach. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the vast majority of glycoproteins overexpressed in the metastatic ML2 subline were involved in cell motility, migration, and invasion. Our approach effectively targeted surface sialoglycoproteins and efficiently identified proteins that underlie the metastatic potential of the ML2 cells. PMID:21447706

  17. Graphite anode surface modification with controlled reduction of specific aryl diazonium salts for improved microbial fuel cells power output.

    PubMed

    Picot, Matthieu; Lapinsonnière, Laure; Rothballer, Michael; Barrière, Frédéric

    2011-10-15

    Graphite electrodes were modified with reduction of aryl diazonium salts and implemented as anodes in microbial fuel cells. First, reduction of 4-aminophenyl diazonium is considered using increased coulombic charge density from 16.5 to 200 mC/cm(2). This procedure introduced aryl amine functionalities at the surface which are neutral at neutral pH. These electrodes were implemented as anodes in "H" type microbial fuel cells inoculated with waste water, acetate as the substrate and using ferricyanide reduction at the cathode and a 1000 Ω external resistance. When the microbial anode had developed, the performances of the microbial fuel cells were measured under acetate saturation conditions and compared with those of control microbial fuel cells having an unmodified graphite anode. We found that the maximum power density of microbial fuel cell first increased as a function of the extent of modification, reaching an optimum after which it decreased for higher degree of surface modification, becoming even less performing than the control microbial fuel cell. Then, the effect of the introduction of charged groups at the surface was investigated at a low degree of surface modification. It was found that negatively charged groups at the surface (carboxylate) decreased microbial fuel cell power output while the introduction of positively charged groups doubled the power output. Scanning electron microscopy revealed that the microbial anode modified with positively charged groups was covered by a dense and homogeneous biofilm. Fluorescence in situ hybridization analyses showed that this biofilm consisted to a large extent of bacteria from the known electroactive Geobacter genus. In summary, the extent of modification of the anode was found to be critical for the microbial fuel cell performance. The nature of the chemical group introduced at the electrode surface was also found to significantly affect the performance of the microbial fuel cells. The method used for

  18. Heterogeneity of chemokine cell-surface receptor expression in triple-negative breast cancer

    PubMed Central

    Norton, Kerri-Ann; Popel, Aleksander S; Pandey, Niranjan B

    2015-01-01

    Introduction: Tumor heterogeneity is a well-established concept in cancer research. In this paper, we examine an additional type of tumor cell heterogeneity - tumor cell-surface receptor heterogeneity. Methods: We use flow cytometry to measure the frequency and numbers of cell-surface receptors on triple negative breast cancer cell lines. Results: We find two distinct populations of human triple-negative breast cancer cells MDA-MB-231 when they are grown in culture, one with low surface levels of various chemokine receptors and a second with much higher levels. The population with high surface levels of these receptors is increased in the more metastatic MDA-MB-231-luc-d3h2ln cell line. Conclusion: We hypothesize that this high cell-surface receptor population is involved in metastasis. We find that the receptor high populations can be modulated by tumor conditioned media and IL6 treatment indicating that the tumor microenvironment is important for the maintenance and sizes of these populations. PMID:26101698

  19. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    PubMed Central

    Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

    2015-01-01

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

  20. Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

    PubMed

    Ziraksaz, Zarrintaj; Nomani, Alireza; Ruponen, Marika; Soleimani, Masoud; Tabbakhian, Majid; Haririan, Ismaeil

    2013-01-23

    Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step

  1. High-Throughput Lectin Microarray-Based Analysis of Live Cell Surface Glycosylation

    PubMed Central

    Li, Yu; Tao, Sheng-ce; Zhu, Heng; Schneck, Jonathan P.

    2011-01-01

    Lectins, plant-derived glycan-binding proteins, have long been used to detect glycans on cell surfaces. However, the techniques used to characterize serum or cells have largely been limited to mass spectrometry, blots, flow cytometry, and immunohistochemistry. While these lectin-based approaches are well established and they can discriminate a limited number of sugar isomers by concurrently using a limited number of lectins, they are not amenable for adaptation to a high-throughput platform. Fortunately, given the commercial availability of lectins with a variety of glycan specificities, lectins can be printed on a glass substrate in a microarray format to profile accessible cell-surface glycans. This method is an inviting alternative for analysis of a broad range of glycans in a high-throughput fashion and has been demonstrated to be a feasible method of identifying binding-accessible cell surface glycosylation on living cells. The current unit presents a lectin-based microarray approach for analyzing cell surface glycosylation in a high-throughput fashion. PMID:21400689

  2. Structure and functions of fungal cell surfaces

    NASA Technical Reports Server (NTRS)

    Nozawa, Y.

    1984-01-01

    A review with 24 references on the biochemistry, molecular structure, and function of cell surfaces of fungi, especially dermatophytes: the chemistry and structure of the cell wall, the effect of polyene antibiotics on the morphology and function of cytoplasmic membranes, and the chemical structure and function of pigments produced by various fungi are discussed.

  3. Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

    PubMed Central

    Deppert, W; Hanke, K; Henning, R

    1980-01-01

    Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

  4. Triclosan and bisphenol a affect decidualization of human endometrial stromal cells.

    PubMed

    Forte, Maurizio; Mita, Luigi; Cobellis, Luigi; Merafina, Verdiana; Specchio, Raffaella; Rossi, Sergio; Mita, Damiano Gustavo; Mosca, Lavinia; Castaldi, Maria Antonietta; De Falco, Maria; Laforgia, Vincenza; Crispi, Stefania

    2016-02-15

    In recent years, impaired fertility and endometrium related diseases are increased. Many evidences suggest that environmental pollution might be considered a risk factor for endometrial physiopathology. Among environmental pollutants, endocrine disrupting chemicals (EDCs) act on endocrine system, causing hormonal imbalance which, in turn, leads to female and male reproductive dysfunctions. In this work, we studied the effects of triclosan (TCL) and bisphenol A (BPA), two widespread EDCs, on human endometrial stromal cells (ESCs), derived from endometrial biopsies from woman not affected by endometriosis. Cell proliferation, cell cycle, migration and decidualization mechanisms were investigated. Treatments have been performed with both the EDCs separately or in presence and in absence of progesterone used as decidualization stimulus. Both TCL and BPA did not affect cell proliferation, but they arrested ESCs at G2/M phase of cell cycle enhancing cell migration. TCL and BPA also increased gene expression and protein levels of some decidualization markers, such as insulin growth factor binding protein 1 (IGFBP1) and prolactin (PRL), amplifying the effect of progesterone alone. All together, our data strongly suggest that TCL and BPA might alter human endometrium physiology so affecting fertility and pregnancy outcome. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Surface roughness and packaging tightness affect calcium lactate crystallization on Cheddar cheese.

    PubMed

    Rajbhandari, P; Kindstedt, P S

    2014-01-01

    Calcium lactate crystals that sometimes form on Cheddar cheese surfaces are a significant expense to manufacturers. Researchers have identified several postmanufacture conditions such as storage temperature and packaging tightness that contribute to crystal formation. Anecdotal reports suggest that physical characteristics at the cheese surface, such as roughness, cracks, and irregularities, may also affect crystallization. The aim of this study was to evaluate the combined effects of surface roughness and packaging tightness on crystal formation in smoked Cheddar cheese. Four 20-mm-thick cross-section slices were cut perpendicular to the long axis of a retail block (~300g) of smoked Cheddar cheese using a wire cutting device. One cut surface of each slice was lightly etched with a cheese grater to create a rough, grooved surface; the opposite cut surface was left undisturbed (smooth). The 4 slices were vacuum packaged at 1, 10, 50, and 90kPa (very tight, moderately tight, loose, very loose, respectively) and stored at 1°C. Digital images were taken at 1, 4, and 8 wk following the first appearance of crystals. The area occupied by crystals and number of discrete crystal regions (DCR) were quantified by image analysis. The experiment was conducted in triplicate. Effects of storage time, packaging tightness, surface roughness, and their interactions were evaluated by repeated-measures ANOVA. Surface roughness, packaging tightness, storage time, and their 2-way interactions significantly affected crystal area and DCR number. Extremely heavy crystallization occurred on both rough and smooth surfaces when slices were packaged loosely or very loosely and on rough surfaces with moderately tight packaging. In contrast, the combination of rough surface plus very tight packaging resulted in dramatic decreases in crystal area and DCR number. The combination of smooth surface plus very tight packaging virtually eliminated crystal formation, presumably by eliminating available

  6. Quantification of surface tension and internal pressure generated by single mitotic cells

    NASA Astrophysics Data System (ADS)

    Fischer-Friedrich, Elisabeth; Hyman, Anthony A.; Jülicher, Frank; Müller, Daniel J.; Helenius, Jonne

    2014-08-01

    During mitosis, adherent cells round up, by increasing the tension of the contractile actomyosin cortex while increasing the internal hydrostatic pressure. In the simple scenario of a liquid cell interior, the surface tension is related to the local curvature and the hydrostatic pressure difference by Laplace's law. However, verification of this scenario for cells requires accurate measurements of cell shape. Here, we use wedged micro-cantilevers to uniaxially confine single cells and determine confinement forces while concurrently determining cell shape using confocal microscopy. We fit experimentally measured confined cell shapes to shapes obeying Laplace's law with uniform surface tension and find quantitative agreement. Geometrical parameters derived from fitting the cell shape, and the measured force were used to calculate hydrostatic pressure excess and surface tension of cells. We find that HeLa cells increase their internal hydrostatic pressure excess and surface tension from ~ 40 Pa and 0.2 mNm-1 during interphase to ~ 400 Pa and 1.6 mNm-1 during metaphase. The method introduced provides a means to determine internal pressure excess and surface tension of rounded cells accurately and with minimal cellular perturbation, and should be applicable to characterize the mechanical properties of various cellular systems.

  7. Nanosecond pulsed electric field induced changes in cell surface charge density.

    PubMed

    Dutta, Diganta; Palmer, Xavier-Lewis; Asmar, Anthony; Stacey, Michael; Qian, Shizhi

    2017-09-01

    This study reports that the surface charge density changes in Jurkat cells with the application of single 60 nanosecond pulse electric fields, using atomic force microscopy. Using an atomic force microscope tip and Jurkat cells on silica in a 0.01M KCl ionic concentration, we were able to measure the interfacial forces, while also predicting surface charge densities of both Jurkat cell and silica surfaces. The most important finding is that the pulsing conditions varyingly reduced the cells' surface charge density. This offers a novel way in which to examine cellular effects of pulsed electric fields that may lead to the identification of unique mechanical responses. Compared to a single low field strength NsPEF (15kV/cm) application, exposure of Jurkat cells to a single high field strength NsPEF (60kV/cm) resulted in a further reduction in charge density and major morphological changes. The structural, physical, and chemical properties of biological cells immensely influence their electrostatic force; we were able to investigate this through the use of atomic force microscopy by measuring the surface forces between the AFM's tip and the Jurkat cells under different pulsing conditions as well as the interfacial forces in ionic concentrations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes

    PubMed Central

    Kanabar, Varsha; Tedaldi, Lauren; Jiang, Jingqian; Nie, Xiaodan; Panina, Irina; Descroix, Karine; Man, Francis; Pitchford, Simon C; Page, Clive P; Wagner, Gerd K

    2016-01-01

    P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell–cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1 also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1, which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development. PMID:27233805

  9. Cell surface engineering with polyelectrolyte multilayer thin films.

    PubMed

    Wilson, John T; Cui, Wanxing; Kozlovskaya, Veronika; Kharlampieva, Eugenia; Pan, Di; Qu, Zheng; Krishnamurthy, Venkata R; Mets, Joseph; Kumar, Vivek; Wen, Jing; Song, Yuhua; Tsukruk, Vladimir V; Chaikof, Elliot L

    2011-05-11

    Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells. © 2011 American Chemical Society

  10. Rapid spectrophotometric method for determining surface free energy of microalgal cells.

    PubMed

    Zhang, Xinru; Jiang, Zeyi; Li, Mengyin; Zhang, Xinxin; Wang, Ge; Chou, Aihui; Chen, Liang; Yan, Hai; Zuo, Yi Y

    2014-09-02

    Microalgae are one of the most promising renewable energy sources with environmental sustainability. The surface free energy of microalgal cells determines their biofouling and bioflocculation behavior and hence plays an important role in microalgae cultivation and harvesting. To date, the surface energetic properties of microalgal cells are still rarely studied. We developed a novel spectrophotometric method for directly determining the surface free energy of microalgal cells. The principles of this method are based on analyzing colloidal stability of microalgae suspensions. We have shown that this method can effectively differentiate the surface free energy of four microalgal strains, i.e., marine Chlorella sp., marine Nannochloris oculata, freshwater autotrophic Chlorella sp., and freshwater heterotrophic Chlorella sp. With advantages of high-throughput and simplicity, this new spectrophotometric method has the potential to evolve into a standard method for measuring the surface free energy of cells and abiotic particles.

  11. Investigating biomolecular recognition at the cell surface using atomic force microscopy.

    PubMed

    Wang, Congzhou; Yadavalli, Vamsi K

    2014-05-01

    Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for understanding diverse biological processes. Force spectroscopy has been a widely used dynamic analytical technique, allowing measurement of such interactions at the molecular and cellular level. The capabilities of working under near physiological environments, combined with excellent force and lateral resolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to measure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cell surfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into how biomolecules on cell surfaces interact with each other and induce relevant biological processes. In this review, we focus on describing the technique of force spectroscopy using the AFM, specifically in the context of probing cell surfaces. We summarize recent progress in understanding the recognition and interactions between macromolecules that may be found at cell surfaces from a force spectroscopy perspective. We further discuss the challenges and future prospects of the application of this versatile technique. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Effects of DDT and Triclosan on Tumor-cell Binding Capacity and Cell-Surface Protein Expression of Human Natural Killer Cells

    PubMed Central

    Hurd-Brown, Tasia; Udoji, Felicia; Martin, Tamara; Whalen, Margaret M.

    2012-01-01

    1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) and triclosan (TCS) are organochlorine (OC) compounds that contaminate the environment, are found in human blood, and have been shown to decrease the tumor-cell killing (lytic) function of human natural killer (NK) cells. NK cells defend against tumor cells and virally infected cells. They bind to these targets, utilizing a variety of cell surface proteins. This study examined concentrations of DDT and TCS that decrease lytic function for alteration of NK binding to tumor targets. Levels of either compound that caused loss of binding function were then examined for effects on expression of cell-surface proteins needed for binding. NK cells exposed to 2.5 μM DDT for 24 h (which caused a greater than 55% loss of lytic function) showed a decrease in NK binding function of about 22%, and a decrease in CD16 cell-surface protein of 20%. NK cells exposed to 5 μM TCS for 24 h showed a decrease in ability to bind tumor cells of 37% and a decrease in expression of CD56 of about 34%. This same treatment caused a decrease in lytic function of greater than 87%. These results indicated that only a portion of the loss of NK lytic function seen with exposures to these compounds could be accounted for by loss of binding function. They also showed that loss of binding function is accompanied by a loss cell-surface proteins important in binding function. PMID:22729613

  13. The influence of surface integrin binding patterns on specific biomaterial-cell interactions

    NASA Astrophysics Data System (ADS)

    Beranek, Maggi Marie

    As the future of biomaterials progresses toward bioactivity, the biomaterial surface must control non-specific protein adsorption and encourage selective protein and cell adsorption. Integrins alphavbeta3, alpha 1beta1, alpha5beta1 and alpha Mbeta2 are expressed on cells involved in endothelialization, inflammation, and intimal hyperplasia. These cellular events play a vital role in biomaterial biocompatibility, especially in the vascular environment. The overall hypothesis of these studies is that biomaterial surfaces exhibit selective integrin binding, which then specifies differential cell binding. To test this hypothesis, four specific aims were developed. The first aim was designed to determine whether metal and polymeric biomaterials exhibit selective integrin binding. The tested materials included 316L stainless steel, nitinol, gold, Elgiloy RTM, poly(D, L-lactide-co-glycolide), polycarbonate urethane and expanded polytetrafluoroethylene. Discrete integrin binding patterns were detected microscopically using integrin specific fluorescent antibodies. Stainless steel exhibited high level integrin alpha1beta 1 and low level integrin alphaMbeta2 binding pattern. This suggests that this metal surface should selectively encourage endothelial cell to inflammatory cell binding. In contrast, gold bound ten times the amount of integrin alphaMbeta2 compared to integrin alpha1beta1, which should encourage inflammatory cell adhesion. The 65/35 poly(D, L-lactide-co-glycolide) was the only polymeric biomaterial tested that had integrin binding levels comparable to metal biomaterials. Based on these observations, a combinational biomaterial with a surface pattern of 65/35 poly(D, L-lactide-co-glycolide) dots on a 316L stainless steel background was created. A pattern of high level integrin alpha1beta1 binding and low level integrin alpha Mbeta2 binding on this combinational surface indicates that this surface should selectively favor endothelial cell binding. In the second

  14. Nanoindentation characterisation of human colorectal cancer cells considering cell geometry, surface roughness and hyperelastic constitutive behaviour

    NASA Astrophysics Data System (ADS)

    Boccaccio, Antonio; Uva, Antonio E.; Papi, Massimiliano; Fiorentino, Michele; De Spirito, Marco; Monno, Giuseppe

    2017-01-01

    Characterisation of the mechanical behaviour of cancer cells is an issue of crucial importance as specific cell mechanical properties have been measured and utilized as possible biomarkers of cancer progression. Atomic force microscopy certainly occupies a prominent place in the field of the mechanical characterisation devices. We developed a hybrid approach to characterise different cell lines (SW620 and SW480) of the human colon carcinoma submitted to nanoindentation measurements. An ad hoc algorithm was written that compares the force-indentation curves experimentally retrieved with those predicted by a finite element model that simulates the nanoindentation process and reproduces the cell geometry and the surface roughness. The algorithm perturbs iteratively the values of the cell mechanical properties implemented in the finite element model until the difference between the experimental and numerical force-indentation curves reaches the minimum value. The occurrence of this indicates that the implemented material properties are very close to the real ones. Different hyperelastic constitutive models, such as Arruda-Boyce, Mooney-Rivlin and Neo-Hookean were utilized to describe the structural behaviour of indented cells. The algorithm was capable of separating, for all the cell lines investigated, the mechanical properties of cell cortex and cytoskeleton. Material properties determined via the algorithm were different with respect to those obtained with the Hertzian contact theory. This demonstrates that factors such as: the cell geometry/anatomy and the hyperelastic constitutive behaviour, which are not contemplated in the Hertz’s theory hypotheses, do affect the nanoindentation measurements. The proposed approach represents a powerful tool that, only on the basis of nanoindentation measurements, is capable of characterising material at the subcellular level.

  15. Ocular surface reconstruction with a tissue-engineered nasal mucosal epithelial cell sheet for the treatment of severe ocular surface diseases.

    PubMed

    Kobayashi, Masakazu; Nakamura, Takahiro; Yasuda, Makoto; Hata, Yuiko; Okura, Shoki; Iwamoto, Miyu; Nagata, Maho; Fullwood, Nigel J; Koizumi, Noriko; Hisa, Yasuo; Kinoshita, Shigeru

    2015-01-01

    Severe ocular surface diseases (OSDs) with severe dry eye can be devastating and are currently some of the most challenging eye disorders to treat. To investigate the feasibility of using an autologous tissue-engineered cultivated nasal mucosal epithelial cell sheet (CNMES) for ocular surface reconstruction, we developed a novel technique for the culture of nasal mucosal epithelial cells expanded ex vivo from biopsy-derived human nasal mucosal tissues. After the protocol, the CNMESs had 4-5 layers of stratified, well-differentiated cells, and we successfully generated cultured epithelial sheets, including numerous goblet cells. Immunohistochemistry confirmed the presence of keratins 3, 4, and 13; mucins 1, 16, and 5AC; cell junction and basement membrane assembly proteins; and stem/progenitor cell marker p75 in the CNMESs. We then transplanted the CNMESs onto the ocular surfaces of rabbits and confirmed the survival of this tissue, including the goblet cells, up to 2 weeks. The present report describes an attempt to overcome the problems of treating severe OSDs with the most severe dry eye by treating them using tissue-engineered CNMESs to supply functional goblet cells and to stabilize and reconstruct the ocular surface. The present study is a first step toward assessing the use of tissue-engineered goblet-cell transplantation of nonocular surface origin for ocular surface reconstruction. ©AlphaMed Press.

  16. Estrogen deficiency heterogeneously affects tissue specific stem cells in mice

    PubMed Central

    Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

    2015-01-01

    Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

  17. Engineering live cell surfaces with functional polymers via cytocompatible controlled radical polymerization

    NASA Astrophysics Data System (ADS)

    Niu, Jia; Lunn, David J.; Pusuluri, Anusha; Yoo, Justin I.; O'Malley, Michelle A.; Mitragotri, Samir; Soh, H. Tom; Hawker, Craig J.

    2017-06-01

    The capability to graft synthetic polymers onto the surfaces of live cells offers the potential to manipulate and control their phenotype and underlying cellular processes. Conventional grafting-to strategies for conjugating preformed polymers to cell surfaces are limited by low polymer grafting efficiency. Here we report an alternative grafting-from strategy for directly engineering the surfaces of live yeast and mammalian cells through cell surface-initiated controlled radical polymerization. By developing cytocompatible PET-RAFT (photoinduced electron transfer-reversible addition-fragmentation chain-transfer polymerization), synthetic polymers with narrow polydispersity (Mw/Mn < 1.3) could be obtained at room temperature in 5 minutes. This polymerization strategy enables chain growth to be initiated directly from chain-transfer agents anchored on the surface of live cells using either covalent attachment or non-covalent insertion, while maintaining high cell viability. Compared with conventional grafting-to approaches, these methods significantly improve the efficiency of grafting polymer chains and enable the active manipulation of cellular phenotypes.

  18. Diffusion of MMPs on the Surface of Collagen Fibrils: The Mobile Cell Surface – Collagen Substratum Interface

    PubMed Central

    Collier, Ivan E.; Legant, Wesley; Marmer, Barry; Lubman, Olga; Saffarian, Saveez; Wakatsuki, Tetsuro; Elson, Elliot; Goldberg, Gregory I.

    2011-01-01

    Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)2/TIMP-2/MMP-2 represents a Mobile Cell Surface – Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions. PMID:21912660

  19. Tuning cell adhesion on polymeric and nanocomposite surfaces: Role of topography versus superhydrophobicity.

    PubMed

    Zangi, Sepideh; Hejazi, Iman; Seyfi, Javad; Hejazi, Ehsan; Khonakdar, Hossein Ali; Davachi, Seyed Mohammad

    2016-06-01

    Development of surface modification procedures which allow tuning the cell adhesion on the surface of biomaterials and devices is of great importance. In this study, the effects of different topographies and wettabilities on cell adhesion behavior of polymeric surfaces are investigated. To this end, an improved phase separation method was proposed to impart various wettabilities (hydrophobic and superhydrophobic) on polypropylene surfaces. Surface morphologies and compositions were characterized by scanning electron microscopy and X-ray photoelectron spectroscopy, respectively. Cell culture was conducted to evaluate the adhesion of 4T1 mouse mammary tumor cells. It was found that processing conditions such as drying temperature is highly influential in cell adhesion behavior due to the formation of an utterly different surface topography. It was concluded that surface topography plays a more significant role in cell adhesion behavior rather than superhydrophobicity since the nano-scale topography highly inhibited the cell adhesion as compared to the micro-scale topography. Such cell repellent behavior could be very useful in many biomedical devices such as those in drug delivery and blood contacting applications as well as biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Development of exosome surface display technology in living human cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated themore » successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.« less

  1. Inducement of a spontaneously wrinkled polydimethylsiloxane surface and its potential as a cell culture substrate.

    PubMed

    Kim, Da Som; Lee, Ho Won; Lee, Jong Hyun; Kwon, Hyuck Gi; Lee, Sang Wook; Han, Seung Jin; Jeong, Ok Chan

    2018-06-18

    Spontaneous wrinkling of a polydimethylsiloxane (PDMS) surface was induced by repeated thermal shrinkage of liquid PDMS coated onto a cured PDMS layer. We investigated and evaluated the potential of the resulting surface as a cell culture substrate by monitoring the viability, spreading area, and proliferation rate of MG-63 cells cultured on native, wrinkled, and poly-L-lysine (PLL)-coated PDMS surfaces. Cells seeded on the wrinkled and PLL-coated PDMS surfaces spread and adhered better than those on native surfaces. The numbers of attached cells growing on wrinkled and PLL-coated PDMS surfaces were higher than those of cells on a native PDMS surface. The spreading area of cells on the wrinkled surface was similar to that of cells on the PLL-coated surface, and was much larger than that on native PDMS. The proliferation rate of cells on the wrinkled surface was more than double that of cells on native PDMS. Reverse-transcription polymerase chain reaction (RT-PCR) analysis of integrin mRNA expression showed that cells on the wrinkled surface were more tightly attached due to higher expression of the protein than exhibited in cells on native PDMS. Thus, the novel findings of this study are that the induction of a wrinkled PDMS surface through a simple curing process produces a suitable cell culture substrate without need of surface modification, and that its effectiveness is comparable to that of a PLL-coated PDMS surface. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Bioinspired Pollen-Like Hierarchical Surface for Efficient Recognition of Target Cancer Cells.

    PubMed

    Wang, Wenshuo; Yang, Gao; Cui, Haijun; Meng, Jingxin; Wang, Shutao; Jiang, Lei

    2017-08-01

    The efficient recognition and isolation of rare cancer cells holds great promise for cancer diagnosis and prognosis. In nature, pollens exploit spiky structures to realize recognition and adhesion to stigma. Herein, a bioinspired pollen-like hierarchical surface is developed by replicating the assembly of pollen grains, and efficient and specific recognition to target cancer cells is achieved. The pollen-like surface is fabricated by combining filtering-assisted assembly and soft lithography-based replication of pollen grains of wild chrysanthemum. After modification with a capture agent specific to cancer cells, the pollen-like surface enables the capture of target cancer cells with high efficiency and specificity. In addition, the pollen-like surface not only assures high viability of captured cells but also performs well in cell mixture system and at low cell density. This study represents a good example of constructing cell recognition biointerfaces inspired by pollen-stigma adhesion. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Shape-memory surfaces for cell mechanobiology

    PubMed Central

    Ebara, Mitsuhiro

    2015-01-01

    Shape-memory polymers (SMPs) are a new class of smart materials, which have the capability to change from a temporary shape ‘A’ to a memorized permanent shape ‘B’ upon application of an external stimulus. In recent years, SMPs have attracted much attention from basic and fundamental research to industrial and practical applications due to the cheap and efficient alternative to well-known metallic shape-memory alloys. Since the shape-memory effect in SMPs is not related to a specific material property of single polymers, the control of nanoarchitecture of polymer networks is particularly important for the smart functions of SMPs. Such nanoarchitectonic approaches have enabled us to further create shape-memory surfaces (SMSs) with tunable surface topography at nano scale. The present review aims to bring together the exciting design of SMSs and the ever-expanding range of their uses as tools to control cell functions. The goal for these endeavors is to mimic the surrounding mechanical cues of extracellular environments which have been considered as critical parameters in cell fate determination. The untapped potential of SMSs makes them one of the most exciting interfaces of materials science and cell mechanobiology. PMID:27877747

  4. Vector vortex beam generation with dolphin-shaped cell meta-surface.

    PubMed

    Yang, Zhuo; Kuang, Deng-Feng; Cheng, Fang

    2017-09-18

    We present a dolphin-shaped cell meta-surface, which is a combination of dolphin-shaped metallic cells and dielectric substrate, for vector vortex beam generation with the illumination of linearly polarized light. Surface plasmon polaritons are excited at the boundary of the metallic cells, then guided by the metallic structures, and finally squeezed to the tips to form highly localized strong electromagnetic fields, which generate the intensity of vector vortex beams at z component. Synchronously, the abrupt phase change produced by the meta-surface is utilized to explain the vortex phase generated by elements. The new kind of structure can be utilized for communication, bioscience, and materiality.

  5. An efficient delivery of DAMPs on the cell surface by the unconventional secretion pathway

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan

    2011-01-21

    Research highlights: {yields} Hsp60 transported to cell surface through the classical secretory pathway was modified with N-glycosylation. {yields} HSAPB-N18 could efficiently deliver Hsp60 to the cell surface via the unconventional secretory pathway. {yields} Cell surface Hsp60 delivered by HASPB-N18 has a proper conformation. {yields} HASPB-N18 is an efficient delivery signal for other DAMP molecules such as Hsp70 and HMGB1. -- Abstract: Damage-associated molecular patterns (DAMPs) are signals released from dying cells evoking the immune system response in several inflammatory disorders. In normal situations, many of DAMPs are nuclear or cytosolic proteins with defined intracellular function, but they could be foundmore » on the cell surface following tissue injury. The biological function of the translocated DAMPs is still not well known and an efficient delivery of these molecules on the cell surface is required to clarify their biological effects. In this study, we demonstrated that an unclassical secretory signal peptide, N-terminal 18 amino acids of HASPB (HASPB-N18), could efficiently deliver Hsp60, Hsp70, and HMGB1 on the cell surface. Furthermore, the delivery of these molecules on the cell surface by HASPB-N18 is not limited to a special cell line because several cell lines could use this delivery signal to deliver these molecules on the cell surface. Moreover, we demonstrated that Hsp60 on the cell surface delivered by HASPB-N18 could be recognized by a soluble form of LOX-1, which implies that DAMPs on the cell surface delivered by HASPB-N18 have a proper conformation during transport. Therefore, delivery of DAMPs by HASPB-N18 is a reliable model to further understand the biological significance of DAMPs on the cell surface.« less

  6. NOVEL POLYPHENOLS THAT INHIBIT COLON CANCER CELL GROWTH AFFECTING CANCER CELL METABOLISM.

    PubMed

    Gomez de Cedron, Marta; Vargas, Teodoro; Madrona, Andres; Jimenez, Aranza; Perez Perez, Maria Jesus; Quintela, Jose Carlos; Reglero, Guillermo; San-Felix, Ana Rosa; Ramirez de Molina, Ana

    2018-06-05

    New series of polyphenols with a hydrophilic galloyl based "head" and a hydrophobic N-acyl "tail", linked through a serinol moiety, have been synthesized and tested against colon cancer cell growth. Our structure activity relationship studies revealed that galloyl moieties are essential for growth inhibition. Moreover, the length of the N-acyl chain is crucial for the activity. Introduction of a (Z) double bond in the acyl chain increased the anti-cancer properties. Our findings demonstrate that 16, the most potent compound within this series, has inhibitory effects on colon cancer cell growth and metabolism (glycolysis and mitochondrial respiration) at the same time that activates AMPK and induces apoptotic cell death. Based on these results we propose that 16 might reprogram colon cancer cell metabolism through AMPK activation. This might lead to alterations on cancer cell bioenergy compromising cancer cell viability. Importantly, these anti-proliferative and pro-apoptotic effects are selective for cancer cells. Accordingly, these results indicate that 16, with an unsaturated C18 chain, might be a useful prototype for the development of novel colon cancer cell growth inhibitors affecting cell metabolism. The American Society for Pharmacology and Experimental Therapeutics.

  7. Robotic Patterning a Superhydrophobic Surface for Collective Cell Migration Screening.

    PubMed

    Pang, Yonggang; Yang, Jing; Hui, Zhixin; Grottkau, Brian E

    2018-04-01

    Collective cell migration, in which cells migrate as a group, is fundamental in many biological and pathological processes. There is increasing interest in studying the collective cell migration in high throughput. Cell scratching, insertion blocker, and gel-dissolving techniques are some methodologies used previously. However, these methods have the drawbacks of cell damage, substrate surface alteration, limitation in medium exchange, and solvent interference. The superhydrophobic surface, on which the water contact angle is greater than 150 degrees, has been recently utilized to generate patterned arrays. Independent cell culture areas can be generated on a substrate that functions the same as a conventional multiple well plate. However, so far there has been no report on superhydrophobic patterning for the study of cell migration. In this study, we report on the successful development of a robotically patterned superhydrophobic array for studying collective cell migration in high throughput. The array was developed on a rectangular single-well cell culture plate consisting of hydrophilic flat microwells separated by the superhydrophobic surface. The manufacturing process is robotic and includes patterning discrete protective masks to the substrate using 3D printing, robotic spray coating of silica nanoparticles, robotic mask removal, robotic mini silicone blocker patterning, automatic cell seeding, and liquid handling. Compared with a standard 96-well plate, our system increases the throughput by 2.25-fold and generates a cell-free area in each well non-destructively. Our system also demonstrates higher efficiency than conventional way of liquid handling using microwell plates, and shorter processing time than manual operating in migration assays. The superhydrophobic surface had no negative impact on cell viability. Using our system, we studied the collective migration of human umbilical vein endothelial cells and cancer cells using assays of endpoint

  8. How internal drainage affects evaporation dynamics from soil surfaces ?

    NASA Astrophysics Data System (ADS)

    Or, D.; Lehmann, P.; Sommer, M.

    2017-12-01

    Following rainfall, infiltrated water may be redistributed internally to larger depths or lost to the atmosphere by evaporation (and by plant uptake from depths at longer time scales). A large fraction of evaporative losses from terrestrial surfaces occurs during stage1 evaporation during which phase change occurs at the wet surface supplied by capillary flow from the soil. Recent studies have shown existence of a soil-dependent characteristic length below which capillary continuity is disrupted and a drastic shift to slower stage 2 evaporation ensues. Internal drainage hastens this transition and affect evaporative losses. To predict the transition to stage 2 and associated evaporative losses, we developed an analytical solution for evaporation dynamics with concurrent internal drainage. Expectedly, evaporative losses are suppressed when drainage is considered to different degrees depending on soil type and wetness. We observe that high initial water content supports rapid drainage and thus promotes the sheltering of soil water below the evaporation depth. The solution and laboratory experiments confirm nonlinear relationship between initial water content and total evaporative losses. The concept contributes to establishing bounds on regional surface evaporation considering rainfall characteristics and soil types.

  9. Polymer surface functionalities that control human embryoid body cell adhesion revealed by high throughput surface characterization of combinatorial material microarrays

    PubMed Central

    Yang, Jing; Mei, Ying; Hook, Andrew L.; Taylor, Michael; Urquhart, Andrew J.; Bogatyrev, Said R.; Langer, Robert; Anderson, Daniel G.; Davies, Martyn C.; Alexander, Morgan R.

    2010-01-01

    High throughput materials discovery using combinatorial polymer microarrays to screen for new biomaterials with new and improved function is established as a powerful strategy. Here we combine this screening approach with high throughput surface characterisation (HT-SC) to identify surface structure-function relationships. We explore how this combination can help to identify surface chemical moieties that control protein adsorption and subsequent cellular response. The adhesion of human embryoid body (hEB) cells to a large number (496) of different acrylate polymers synthesized in a microarray format is screened using a high throughput procedure. To determine the role of the polymer surface properties on hEB cell adhesion, detailed HT-SC of these acrylate polymers is carried out using time of flight secondary ion mass spectrometry (ToF SIMS), x-ray photoelectron spectroscopy (XPS), pico litre drop sessile water contact angle (WCA) measurement and atomic force microscopy (AFM). A structure-function relationship is identified between the ToF SIMS analysis of the surface chemistry after a fibronectin (Fn) pre-conditioning step and the cell adhesion to each spot using the multivariate analysis technique partial least squares (PLS) regression. Secondary ions indicative of the adsorbed Fn correlate with increased cell adhesion whereas glycol and other functionalities from the polymers are identified that reduce cell adhesion. Furthermore, a strong relationship between the ToF SIMS spectra of bare polymers and the cell adhesion to each spot is identified using PLS regression. This identifies a role for both the surface chemistry of the bare polymer and the pre-adsorbed Fn, as-represented in the ToF SIMS spectra, in controlling cellular adhesion. In contrast, no relationship is found between cell adhesion and wettability, surface roughness, elemental or functional surface composition. The correlation between ToF SIMS data of the surfaces and the cell adhesion demonstrates

  10. Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces.

    PubMed

    Rideout, D C; Lambert, M; Kendall, D A; Moe, G R; Osterman, D G; Tao, H P; Weinstein, I B; Kaiser, E T

    1985-09-01

    Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.

  11. Altered Stem Cell Receptor Activity in the Ovarian Surface Epithelium by Exogenous Zinc and/or Progesterone.

    PubMed

    Oktem, G; Sahin, C; Dilsiz, O Y; Demiray, S B; Goker, E N T; Tavmergen, E

    2015-05-01

    Ovarian surface epithelium (OSE) has the characteristics of a stem cell and the potential for differentiation. Previous studies on this subject have succeeded in deriving oocytes from OSE stem cells, leading to the belief that OSE could be used for infertility treatment. Each rat (n = 10) was subjected to zinc and/or progesterone injection for 5 days after conception. After a 6-day implantation period, ovarian tissues were removed and comprehensive immunohistochemical analysis of stem cell markers was conducted: Sox2, Klf4, Oct3/4, c-Myc, CD117, CD90, SSEA-1 and Notch pathway analysis; Notch1, Jagged1, and Delta1 in the OSE and ovarian stromal cells were evaluated after treatment with zinc, progesterone, or both. Progesterone moderately affected Sox2 expression (p < 0.001), while zinc application strongly affected Klf4 and Oct3/4 and immunoreactivity (p < 0.001). CD90 immunoreactivity was decreased in the OSE and stroma of the progesterone group (p = 0.006) compared with the zinc (p = 0.244) and zinc/progesterone groups (p = 0.910). On the other hand, SSEA-1 showed moderate staining in the OSE and weak staining in stromal cells in animals treated with zinc (p = 0.727), progesterone (p = 0.626), and zinc/progesterone (p = 0.371), with no differences compared with control. Zinc application affected Notch pathway immunoreactivity, with a significant increase in Notch1 (p = 0.0015) and Jagged1 (p < 0.001). The expression of putative stem cell markers in the OSE was verified and stem cell receptor activity was raised in the OSE and ovarian stromal cells by zinc and progesterone. Thus, this increased expression allows the therapeutic use of zinc and progesterone in ovary-related infertility and brings a different perspective to reproductive medicine. © Georg Thieme Verlag KG Stuttgart · New York.

  12. Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hang, Howard C.; Bertozzi, Carolyn R.

    2000-08-22

    Novel chemical reactivity can be engendered on cell surfaces by the metabolic incorporation of unnatural sugars into cell surface glycoconjuagtes. 2-N-Acetamido sugars such as GalNAc and GlcNAc are abundant components of cell surface glycoconjugates, and hence attractive targets for metabolic cell surface engineering. Here we report (1) the synthesis of isosteric analogs bearing a ketone group in place of the N-acetamido group, and (2) evaluation of their metabolic incorporation into mammalian cell surface glycans. A ketone isostere of GalNAc was metabolized by CHO cells through the salvage pathway and delivered to O-linked glycoproteins on the cell surface. Its residence atmore » the core position of O-linked glycans is suggested by studies with a-benzyl GalNAc, an inhibitor of O-linked oligosaccharide extension. A mutant CHO cell line lacking endogenous UDP-GalNAc demonstrated enhanced metabolism of the GalNAc analog, suggesting that competition with native intermediates might limits enzymatic transformation in mammalian cells. A ketone isostere of GlcNAc could not be detected on CHO or human cell surfaces after incubation. Thus, the enzymes in the GlcNAc salvage pathway might be less permissive of unnatural substrates than those comprising the GalNAc salvage pathway. Alternatively, high levels of endogenous GlcNAc derivatives might compete with the ketone isostere and prevent its incorporation into oligosaccharides.« less

  13. Effect of surface characteristics on retention and removal of Escherichia coli O157:H7 on surfaces of spinach

    USDA-ARS?s Scientific Manuscript database

    The topography and the spatial heterogeneity of produce surfaces may impact the attachment of microbial cells onto produce surfaces and affect disinfection efficacy. In this study, the effects of produce surface characteristics on the removal of bacteria were studied. Fresh spinach leaves were sp...

  14. Microbial cell surface characteristics: Elucidating attachment/detachment using hydrophobicity and electrokinetic measurements

    EPA Science Inventory

    The surface properties of microorganisms play an important role in their behavior within the environment. Electrophoretic mobility and cell surface hydrophobicity of bacterial cells influence their initial interaction with surfaces and mediate their stability within an aqueous su...

  15. γδ T cells affect IL-4 production and B-cell tolerance

    PubMed Central

    Huang, Yafei; Heiser, Ryan A.; Detanico, Thiago O.; Getahun, Andrew; Kirchenbaum, Greg A.; Casper, Tamara L.; Aydintug, M. Kemal; Carding, Simon R.; Ikuta, Koichi; Huang, Hua; Cambier, John C.; Wysocki, Lawrence J.; O’Brien, Rebecca L.; Born, Willi K.

    2015-01-01

    γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4–producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4–regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4–inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance. PMID:25535377

  16. γδ T cells affect IL-4 production and B-cell tolerance.

    PubMed

    Huang, Yafei; Heiser, Ryan A; Detanico, Thiago O; Getahun, Andrew; Kirchenbaum, Greg A; Casper, Tamara L; Aydintug, M Kemal; Carding, Simon R; Ikuta, Koichi; Huang, Hua; Cambier, John C; Wysocki, Lawrence J; O'Brien, Rebecca L; Born, Willi K

    2015-01-06

    γδ T cells can influence specific antibody responses. Here, we report that mice deficient in individual γδ T-cell subsets have altered levels of serum antibodies, including all major subclasses, sometimes regardless of the presence of αβ T cells. One strain with a partial γδ deficiency that increases IgE antibodies also displayed increases in IL-4-producing T cells (both residual γδ T cells and αβ T cells) and in systemic IL-4 levels. Its B cells expressed IL-4-regulated inhibitory receptors (CD5, CD22, and CD32) at diminished levels, whereas IL-4-inducible IL-4 receptor α and MHCII were increased. They also showed signs of activation and spontaneously formed germinal centers. These mice displayed IgE-dependent features found in hyper-IgE syndrome and developed antichromatin, antinuclear, and anticytoplasmic autoantibodies. In contrast, mice deficient in all γδ T cells had nearly unchanged Ig levels and did not develop autoantibodies. Removing IL-4 abrogated the increases in IgE, antichromatin antibodies, and autoantibodies in the partially γδ-deficient mice. Our data suggest that γδ T cells, controlled by their own cross-talk, affect IL-4 production, B-cell activation, and B-cell tolerance.

  17. Human NK Cell Subset Functions Are Differentially Affected by Adipokines

    PubMed Central

    Huebner, Lena; Engeli, Stefan; Wrann, Christiane D.; Goudeva, Lilia; Laue, Tobias; Kielstein, Heike

    2013-01-01

    Background Obesity is a risk factor for various types of infectious diseases and cancer. The increase in adipose tissue causes alterations in both adipogenesis and the production of adipocyte-secreted proteins (adipokines). Since natural killer (NK) cells are the host’s primary defense against virus-infected and tumor cells, we investigated how adipocyte-conditioned medium (ACM) affects functions of two distinct human NK cell subsets. Methods Isolated human peripheral blood mononuclear cells (PBMCs) were cultured with various concentrations of human and murine ACM harvested on two different days during adipogenesis and analyzed by fluorescent-activated cell sorting (FACS). Results FACS analyses showed that the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granzyme A (GzmA) and interferon (IFN)-γ in NK cells was regulated in a subset-specific manner. ACM treatment altered IFN-γ expression in CD56dim NK cells. The production of GzmA in CD56bright NK cells was differentially affected by the distinct adipokine compositions harvested at different states of adipogenesis. Comparison of the treatment with either human or murine ACM revealed that adipokine-induced effects on NK cell expression of the leptin receptor (Ob-R), TRAIL and IFN-γ were species-specific. Conclusion Considering the growing prevalence of obesity and the various disorders related to it, the present study provides further insights into the roles human NK cell subsets play in the obesity-associated state of chronic low-grade inflammation. PMID:24098717

  18. Increasing binding density of yeast cells by control of surface charge with allylamine grafting to ion modified polymer surfaces.

    PubMed

    Tran, Clara T H; Kondyurin, Alexey; Chrzanowski, Wojciech; Bilek, Marcela M M; McKenzie, David R

    2014-10-01

    Plasma immersion ion implantation (PIII) treatment of polymers creates a biointerface capable of direct covalent immobilization of biomolecules. The immobilization of protein molecules is achieved by covalent bonds formed between embedded radicals on the treated surface and amino acid side chains and cells can be immobilized through cell-wall proteins. The attachment density of negatively charged entities on a PIII treated surface is inhibited by its negative surface charge at neutral pH. To reduce the negative charge of PIII treated surfaces in phosphate buffer (pH 7.4, 11mM), we develop an effective approach of grafting allylamine monomers onto the treated surface. The results reveal reactions between allylamine and radicals on the PIII treated surface. One of these triggers polymerization, increasing the number of amine groups grafted. As a consequence, the PIII treated polystyrene surface after allylamine exposure becomes more hydrophobic and less negatively charged in phosphate buffer. Using yeast cells as an example, we have shown a significant improvement (6-15 times) of cell density immobilized on the PIII treated surface after exposure to allylamine. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. Flagellin based biomimetic coatings: From cell-repellent surfaces to highly adhesive coatings.

    PubMed

    Kovacs, Boglarka; Patko, Daniel; Szekacs, Inna; Orgovan, Norbert; Kurunczi, Sandor; Sulyok, Attila; Khanh, Nguyen Quoc; Toth, Balazs; Vonderviszt, Ferenc; Horvath, Robert

    2016-09-15

    Biomimetic coatings with cell-adhesion-regulating functionalities are intensively researched today. For example, cell-based biosensing for drug development, biomedical implants, and tissue engineering require that the surface adhesion of living cells is well controlled. Recently, we have shown that the bacterial flagellar protein, flagellin, adsorbs through its terminal segments to hydrophobic surfaces, forming an oriented monolayer and exposing its variable D3 domain to the solution. Here, we hypothesized that this nanostructured layer is highly cell-repellent since it mimics the surface of the flagellar filaments. Moreover, we proposed flagellin as a carrier molecule to display the cell-adhesive RGD (Arg-Gly-Asp) peptide sequence and induce cell adhesion on the coated surface. The D3 domain of flagellin was replaced with one or more RGD motifs linked by various oligopeptides modulating flexibility and accessibility of the inserted segment. The obtained flagellin variants were applied to create surface coatings inducing cell adhesion and spreading to different levels, while wild-type flagellin was shown to form a surface layer with strong anti-adhesive properties. As reference surfaces synthetic polymers were applied which have anti-adhesive (PLL-g-PEG poly(l-lysine)-graft-poly(ethylene glycol)) or adhesion inducing properties (RGD-functionalized PLL-g-PEG). Quantitative adhesion data was obtained by employing optical biochips and microscopy. Cell-adhesion-regulating coatings can be simply formed on hydrophobic surfaces by using the developed flagellin-based constructs. The developed novel RGD-displaying flagellin variants can be easily obtained by bacterial production and can serve as alternatives to create cell-adhesion-regulating biomimetic coatings. In the present work, we show for the first time that. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  20. Curcumin affects cell survival and cell volume regulation in human renal and intestinal cells

    PubMed Central

    Kössler, Sonja; Nofziger, Charity; Jakab, Martin; Dossena, Silvia; Paulmichl, Markus

    2012-01-01

    Curcumin (1,7-bis(4-hydroxy-3-methoxyphenyl)-1E,6E-heptadiene-3,5-dione or diferuloyl methane) is a polyphenol derived from the Curcuma longa plant, commonly known as turmeric. This substance has been used extensively in Ayurvedic medicine for centuries for its anti-oxidant, analgesic, anti-inflammatory and antiseptic activity. More recently curcumin has been found to possess anti-cancer properties linked to its pro-apoptotic and anti-proliferative actions. The underlying mechanisms of these diverse effects are complex, not fully elucidated and subject of intense scientific debate. Despite increasing evidence indicating that different cation channels can be a molecular target for curcumin, very little is known about the effect of curcumin on chloride channels. Since, (i) the molecular structure of curcumin indicates that the substance could potentially interact with chloride channels, (ii) chloride channels play a role during the apoptotic process and regulation of the cell volume, and (iii) apoptosis is a well known effect of curcumin, we set out to investigate whether or not curcumin could (i) exert a modulatory effect (direct or indirect) on the swelling activated chloride current IClswell in a human cell system, therefore (ii) affect cell volume regulation and (iii) ultimately modulate cell survival. The IClswell channels, which are essential for regulating the cell volume after swelling, are also known to be activated under isotonic conditions as an early event in the apoptotic process. Here we show that long-term exposure of a human kidney cell line to extracellular 0.1–10 μM curcumin modulates IClswell in a dose-dependent manner (0.1 μM curcumin is ineffective, 0.5–5.0 μM curcumin increase, while 10 μM curcumin decrease the current), and short-term exposure to micromolar concentrations of curcumin does not affect IClswell neither if applied from the extracellular nor from the intracellular side – therefore, a direct effect of curcumin on

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Xiaojun; Department of Biotechnology, Nanchang University, Nanchang, Jiangxi 330031; Chen, Yuan

    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)more » 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.« less

  2. The effect of MTAD, an endodontic irrigant, on fibroblast attachment to periodontally affected root surfaces: A SEM analysis.

    PubMed

    Ghandi, Mostafa; Houshmand, Behzad; Nekoofar, Mohammad H; Tabor, Rachel K; Yadeghari, Zahra; Dummer, Paul M H

    2013-03-01

    Root surface debridement (RSD) is necessary to create an environment suitable for reattachment of the periodontium. Root surface conditioning may aid the formation of a biocompatible surface suitable for cell reattachment. BioPure™ MTAD (mixture of Doxycycline, citric acid and a detergent) is an endodontic irrigant with antibacterial properties and the ability to remove smear layer. It was hypothesized that MTAD may be useful for root surface conditioning. The efficacy of MTAD as a conditioner was measured by examining fibroblast attachment to root surfaces. Thirty-two specimens of human teeth with advanced periodontal disease were used. The surfaces were root planed until smooth. Half of the specimens were treated with 0.9% saline and the other samples with Biopure MTAD. As a negative control group, five further samples were left unscaled with surface calculus. Human gingival fibroblast cells HGF1-PI1 were cultured and poured over the tooth specimens and incubated. After fixation, the samples were sputter-coated with gold and examined with a SEM. The morphology and number of attached, fixed viable cells were examined. The data was analysed using the Mann-Whitney-U statistical test. There was no significant difference between the numbers of attached cells in the experimental group treated with MTAD and the control group treated with saline. Little or no attached cells were seen in the negative control group. RSD created an environment suitable for cell growth and attachment in a laboratory setting. The use of MTAD did not promote the attachment and growth of cells on the surface of human roots following RSD.

  3. Processes Affecting the Annual Surface Energy Budget at High-Latitude Terrestrial Sites

    NASA Astrophysics Data System (ADS)

    Persson, P. O. G.; Stone, R. S.; Grachev, A.; Matrosova, L.

    2012-04-01

    Instrumentation at four Study of Environmental Arctic Change (SEARCH) sites (Barrow, Eureka, Alert, and Tiksi) have been enhanced in the past 6 years, including during the 2007-2008 IPY. Data from these sites are used to investigate the annual cycle of the surface energy budget (SEB), its coupling to atmospheric processes, and for Alert, its interannual variability. The comprehensive data sets are useful for showing interactions between the atmosphere, surface, and soil at high temporal resolution throughout the annual cycle. Processes that govern the SEB variability at each site are identified, and their impacts on the SEB are quantified. For example, mesoscale modulation of the SEB caused by forcing from the local terrain (downslope wind events) and coastlines (sea and land breezes) are significant at Alert and Eureka, with these processes affecting both radiative, turbulent, and ground heat flux terms in the SEB. Sub-seasonal and interannual variations in atmospheric processes and SEB impact soil thermal structures, such as the depth and timing of the summer active layer. These analyses provide an improved understanding of the processes producing changes in surface and soil temperature, linking them through the SEB as affected by atmospheric processes.

  4. Pregnancy persistently affects memory T cell populations.

    PubMed

    Kieffer, Tom E C; Faas, Marijke M; Scherjon, Sicco A; Prins, Jelmer R

    2017-02-01

    Pregnancy is an immune challenge to the maternal immune system. The effects of pregnancy on maternal immunity and particularly on memory T cells during and after pregnancy are not fully known. This observational study aims to show the short term and the long term effects of pregnancy on the constitution, size and activation status of peripheral human memory T-lymphocyte populations. Effector memory (EM) and central memory (CM) T-lymphocytes were analyzed using flow cytometry of peripheral blood from 14 nulligravid, 12 primigravid and 15 parous women that were on average 18 months postpartum. The short term effects were shown by the significantly higher CD4+ EM cell and activated CD4+ memory cell proportions in primigravid women compared to nulligravid women. The persistent effects found in this study were the significantly higher proportions of CD4+ EM, CD4+ CM and activated memory T cells in parous women compared to nulligravid women. In contrast to CD4+ cells, activation status of CD8+ memory cells did not differ between the groups. This study shows that pregnancy persistently affects the pre-pregnancy CD4+ memory cell pool in human peripheral blood. During pregnancy, CD4+ T-lymphocytes might differentiate into EM cells followed by persistent higher proportions of CD4+ CM and EM cells postpartum. The persistent effects of pregnancy on memory T cells found in this study support the hypothesis that memory T cells are generated during pregnancy and that these cells could be involved in the lower complication risks in multiparous pregnancies in humans. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  5. Particles induced surface nanoroughness of titanium surface and its influence on adhesion of osteoblast-like MG-63 cells

    NASA Astrophysics Data System (ADS)

    Solař, P.; Kylián, O.; Marek, A.; Vandrovcová, M.; Bačáková, L.; Hanuš, J.; Vyskočil, J.; Slavínská, D.; Biederman, H.

    2015-01-01

    Titanium is one of the most common materials employed for production of implants, which is due to its good biocompatibility. However, the colonization of titanium surface by osteoblast cells may be influenced by its roughness and therefore precise control of roughness of titanium surface as well as identification of its optimal value for growth of cells is of high importance. In this study the nanorough titanium surfaces were prepared on polished disks of TiAlV by two step method of deposition. In the first step TiAlV were coated by nanoparticles generated by gas aggregation sources. Such prepared films of nanoparticles were subsequently covered with a titanium overlayer. Different values of surface roughness in the range 1-100 nm were achieved by variation of the size and number of the nanoparticles. Such prepared surfaces were subsequently used for investigation of influence of roughness of titanium surfaces on the adhesion of human osteoblast-like MG-63 cells. It was found out that 7 days after seeding the highest number of adhering cells was observed for samples with root-mean-square roughness of 30 nm.

  6. Engineered Aptamers to Probe Molecular Interactions on the Cell Surface

    PubMed Central

    Batool, Sana; Bhandari, Sanam; George, Shanell; Okeoma, Precious; Van, Nabeela; Zümrüt, Hazan E.; Mallikaratchy, Prabodhika

    2017-01-01

    Significant progress has been made in understanding the nature of molecular interactions on the cell membrane. To decipher such interactions, molecular scaffolds can be engineered as a tool to modulate these events as they occur on the cell membrane. To guarantee reliability, scaffolds that function as modulators of cell membrane events must be coupled to a targeting moiety with superior chemical versatility. In this regard, nucleic acid aptamers are a suitable class of targeting moieties. Aptamers are inherently chemical in nature, allowing extensive site-specific chemical modification to engineer sensing molecules. Aptamers can be easily selected using a simple laboratory-based in vitro evolution method enabling the design and development of aptamer-based functional molecular scaffolds against wide range of cell surface molecules. This article reviews the application of aptamers as monitors and modulators of molecular interactions on the mammalian cell surface with the aim of increasing our understanding of cell-surface receptor response to external stimuli. The information gained from these types of studies could eventually prove useful in engineering improved medical diagnostics and therapeutics. PMID:28850067

  7. Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells.

    PubMed

    Albertsmeyer, Ann-Christin; Kakkassery, Vinodh; Spurr-Michaud, Sandra; Beeks, Olivia; Gipson, Ilene K

    2010-03-01

    Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-alpha (TNF-alpha), and Interferon-gamma (IFN-gamma), or a combination of TNF-alpha and IFN-gamma, or IFN-gamma and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-alpha and IFN-gamma each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-gamma, affected all three parameters-gene expression, cellular protein, and ectodomain release-for MUC16. Combined treatment with TNF-alpha and IFN-gamma showed effects similar to IFN-gamma alone, except that ectodomain release followed that of TNF-alpha, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

  8. Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

    PubMed Central

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

  9. Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

    PubMed

    Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

    2011-01-01

    Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

  10. Engineering micropatterned surfaces to modulate the function of vascular stem cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Li, Jennifer; Wu, Michelle; Chu, Julia

    2014-02-21

    Highlights: • We examine vascular stem cell function on microgrooved and micropost patterned polymer substrates. • 10 μm microgrooved surfaces significantly lower VSC proliferation but do not modulate calcified matrix deposition. • Micropost surfaces significantly lower VSC proliferation and decrease calcified matrix deposition. - Abstract: Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymermore » surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10 μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces.« less

  11. Metabolite Depletion Affects Flux Profiling of Cell Lines.

    PubMed

    Nilsson, A; Haanstra, J R; Teusink, B; Nielsen, J

    2018-06-01

    Quantifying the rate of consumption and release of metabolites (i.e., flux profiling) has become integral to the study of cancer. The fluxes as well as the growth of the cells may be affected by metabolite depletion during cultivation. Copyright © 2018 Elsevier Ltd. All rights reserved.

  12. Surface chemistry of gold nanorods: origin of cell membrane damage and cytotoxicity

    NASA Astrophysics Data System (ADS)

    Wang, Liming; Jiang, Xiumei; Ji, Yinglu; Bai, Ru; Zhao, Yuliang; Wu, Xiaochun; Chen, Chunying

    2013-08-01

    We investigated how surface chemistry influences the interaction between gold nanorods (AuNRs) and cell membranes and the subsequent cytotoxicity arising from them in a serum-free cell culture system. Our results showed that the AuNRs coated with cetyl trimethylammonium bromide (CTAB) molecules can generate defects in the cell membrane and induce cell death, mainly due to the unique bilayer structure of CTAB molecules on the surface of the rods rather than their charge. Compared to CTAB-capped nanorods, positively charged polyelectrolyte-coated, i.e. poly(diallyldimethyl ammonium chloride) (PDDAC), AuNRs show improved biocompatibility towards cells. Thus, the present results indicate that the nature of surface molecules, especially their packing structures on the surface of AuNRs rather than surface charge, play a more crucial role in determining cytotoxicity. These findings about interfacial interactions could also explain the effects of internalized AuNRs on the structures or functions of organelles. This study will help understanding of the toxic nature of AuNRs and guide rational design of the surface chemistry of AuNRs for good biocompatibility in pharmaceutical therapy.

  13. Cell surface physiology and outer cell envelope impermeability for hydrophobic substances in Burkholderia multivorans.

    PubMed

    Ruskoski, Sallie A; Champlin, Franklin R

    2017-07-01

    The purpose of the present study was to obtain a better understanding of the relationship between cell surface physiology and outer cellular envelope permeability for hydrophobic substances in mucoid and non-mucoid B. multivorans strains, as well as in two capsule-deficient derivatives of a mucoid parental strain. Cell surface hydrophobicity properties were determined using the hydrocarbon adherence method, while outer cell envelope accessibility and permeability for non-polar compounds were measured using hydrophobic antimicrobial agent susceptibility and fluorescent probe assays. Extracellular polysaccharide (EPS) production was assessed by cultivating strains of disparate origin on yeast extract agar (YEA) containing different sugars, while the resultant colonial and cellular morphological parameters were assessed macro- and microscopically, respectively.Results/Key findings. The cell surfaces of all the strains were hydrophilic, impermeable to mechanistically disparate hydrophobic antibacterial agents and inaccessible to the hydrophobic probe N-phenyl-1-napthylamine, regardless of EPS phenotype. Supplementation of basal YEA with eight different sugars enhanced macroscopic EPS expression for all but one non-mucoid strain, with mannose potentiating the greatest effect. Despite acquisition of the mucoid phenotype, non-mucoid strains remained non-capsulated and capsulation of a hyper-mucoid strain and its two non-mucoid derivative strains was unaffected, as judged by microscopic observation. These data support the conclusion that EPS expression and the consistent mucoid phenotype are not necessarily associated with the ability of the outer cell surface to associate with non-polar substances or cellular capsulation.

  14. Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles.

    PubMed

    Asati, Atul; Santra, Santimukul; Kaittanis, Charalambos; Perez, J Manuel

    2010-09-28

    Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticle's surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles' cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles' toxicity profile.

  15. Short-chain ubiquitination is associated with the degradation rate of a cell-surface-resident bile salt export pump (BSEP/ABCB11).

    PubMed

    Hayashi, Hisamitsu; Sugiyama, Yuichi

    2009-01-01

    The reduced expression of the bile salt export pump (BSEP/ABCB11) at the canalicular membrane is associated with cholestasis-induced hepatotoxicity due to the accumulation of bile acids in hepatocytes. We demonstrated previously that 4-phenylbutyrate (4PBA) treatment, a U.S. Food and Drug Administration-approved drug for the treatment of urea cycle disorders, induces the cell-surface expression of BSEP by prolonging the degradation rate of cell-surface-resident BSEP. On the other hand, BSEP mutations, E297G and D482G, found in progressive familial intrahepatic cholestasis type 2 (PFIC2), reduced it by shortening the degradation rate of cell-surface-resident BSEP. Therefore, to help the development of the medical treatment of cholestasis, we investigated the underlying mechanism by which 4PBA and PFIC2-type mutations affect the BSEP degradation from cell surface, focusing on short-chain ubiquitination. In Madin-Darby canine kidney II (MDCK II) cells expressing BSEP and rat canalicular membrane vesicles, the molecular mass of the mature form of BSEP/Bsep shifted from 170 to 190 kDa after ubiquitin modification (molecular mass, 8 kDa). Ubiquitination susceptibility of BSEP/Bsep was reduced in vitro and in vivo by 4PBA treatment and, conversely, was enhanced by BSEP mutations E297G and D482G. Moreover, biotin-labeling studies using MDCK II cells demonstrated that the degradation of cell-surface-resident chimeric protein fusing ubiquitin to BSEP was faster than that of BSEP itself. In conclusion, BSEP/Bsep is modified with two to three ubiquitins, and its ubiquitination is modulated by 4PBA treatment and PFIC2-type mutations. Modulation of short-chain ubiquitination can regulate the change in the degradation rate of cell-surface-resident BSEP by 4PBA treatment and PFIC2-type mutations.

  16. Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin.

    PubMed

    Bastounis, Effie E; Yeh, Yi-Ting; Theriot, Julie A

    2018-05-02

    Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been previously studied, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens was hitherto unexplored. To address this open question, we manufactured hydrogels of varying physiologically-relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm), and found that adhesion of Lm onto host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.

  17. Dexamethasone upregulates ANP C-receptor protein in human mesangial cells without affecting mRNA.

    PubMed

    Ardaillou, N; Blaise, V; Placier, S; Amestoy, F; Ardaillou, R

    1996-03-01

    The objective of this study was to examine the role of dexamethasone on the expression of natriuretic peptide B-type and C-type receptors (ANPR-B and ANPR-C) in cultured human mesangial cells, which only possess these two subtypes. Dexamethasone caused concentration- and time-dependent increases in 125I-labeled ANP binding, which were prevented by glucocorticoid receptor inhibition with RU-38486. A lag time of 24 h and a concentration of dexamethasone of at least 1 nmol/l were necessary for this effect to occur. Dexamethasone-induced upregulation of 125I-ANP binding resulted from increased receptor density. No change in dissociation constant (Kd) was observed. Only ANPR-C were affected by dexamethasone. Indeed, dexamethasone did not modify C-type natriuretic peptide (i.e., CNP)-dependent cGMP production by mesangial cells. Moreover, dexamethasone upregulated ANPR-C protein expression as shown by Western blot analysis and by an increase in ANPR-C immunoreactivity at the cell surface. In contrast, dexamethasone did not modify ANPR-C mRNA expression. In conclusion, glucocorticoids increase ANPR-C density on mesangial cells through a mechanism implying, successively, interaction with the glucocorticoid receptor and increase of ANPR-C protein synthesis at a posttranscriptional stage. Thus dexamethasone may influence availability of natriuretic peptides at their glomerular target sites.

  18. Influence of carbon source on cell surface topology of Thermomonospora curvata.

    PubMed Central

    Hostalka, F; Moultrie, A; Stutzenberger, F

    1992-01-01

    The appearance of cell surface protuberances in Thermomonospora curvata correlated with cell-bound exoenzymes which could be removed by brief sonication. Mycelia grown on cellulose or xylan had numerous protuberances and retained 20 to 25% of endoglucanase and endoxylanase at cell surfaces, while those grown on pectin or starch had few protuberances and negligible bound pectinase or amylase. Images PMID:1400256

  19. The effect of MTAD, an endodontic irrigant, on fibroblast attachment to periodontally affected root surfaces: A SEM analysis

    PubMed Central

    Ghandi, Mostafa; Houshmand, Behzad; Nekoofar, Mohammad H.; Tabor, Rachel K.; Yadeghari, Zahra; Dummer, Paul M. H.

    2013-01-01

    Background: Root surface debridement (RSD) is necessary to create an environment suitable for reattachment of the periodontium. Root surface conditioning may aid the formation of a biocompatible surface suitable for cell reattachment. BioPure™ MTAD (mixture of Doxycycline, citric acid and a detergent) is an endodontic irrigant with antibacterial properties and the ability to remove smear layer. It was hypothesized that MTAD may be useful for root surface conditioning. The efficacy of MTAD as a conditioner was measured by examining fibroblast attachment to root surfaces. Materials and Methods: Thirty-two specimens of human teeth with advanced periodontal disease were used. The surfaces were root planed until smooth. Half of the specimens were treated with 0.9% saline and the other samples with Biopure MTAD. As a negative control group, five further samples were left unscaled with surface calculus. Human gingival fibroblast cells HGF1-PI1 were cultured and poured over the tooth specimens and incubated. After fixation, the samples were sputter-coated with gold and examined with a SEM. The morphology and number of attached, fixed viable cells were examined. The data was analysed using the Mann-Whitney-U statistical test. Results: There was no significant difference between the numbers of attached cells in the experimental group treated with MTAD and the control group treated with saline. Little or no attached cells were seen in the negative control group. Conclusion: RSD created an environment suitable for cell growth and attachment in a laboratory setting. The use of MTAD did not promote the attachment and growth of cells on the surface of human roots following RSD. PMID:23869124

  20. Mitigated reactive oxygen species generation leads to an improvement of cell proliferation on poly[glycidyl methacrylate-co-poly(ethylene glycol) methacrylate] functionalized polydimethylsiloxane surfaces.

    PubMed

    Yu, Ling; Shi, ZhuanZhuan; Gao, LiXia; Li, ChangMing

    2015-09-01

    In vitro cell-based analysis is strongly affected by material's surface chemical properties. The cell spreading, migration, and proliferation on a substrate surface are initiated and controlled by successful adhesion, particularly for anchor-dependent cells. Unfortunately, polydimethylsiloxane (PDMS), one of the most used polymeric materials for construction of microfluidic and miniaturized biomedical analytic devices, is not a cell-friendly surface because of its inherent hydrophobic property. Herein, a poly[glycidyl methacrylate-co-poly(ethylene glycol) methacrylate] (poly(GMA-co-pEGMA)) polymer brush was synthesized on a PDMS surface through a surface-initiated atom-transfer radical polymerization method. Contact angle and Fourier transform infrared characterization show that the poly (GMA-co-pEGMA) polymer brush functionalization can increase wettability of PDMS and introduce epoxy, hydroxyl, and ether groups into PDMS surface. In vitro cell growth assay demonstrates that cell adhesion and proliferation on poly(GMA-co-pEGMA) polymer brush-functionalized PDMS (poly(GMA-co-pEGMA)@PDMS) are better than on pristine PDMS. Additionally, immobilization of collagen type I (CI) and fibronectin (FN) on poly(GMA-co-pEGMA)@PDMS is better than direct coating of CI and FN on pristine PDMS to promote cell adhesion. Furthermore, increased intracellular reactive oxygen species and cell mitochondrial membrane depolarization, two indicators of cell oxidative stress, are observed from cells growing on pristine PDMS, but not from those on poly(GMA-co-pEGMA)@PDMS. Collectively, we demonstrate that poly(GMA-co-pEGMA) functionalization can enhance cell adhesion and proliferation on PDMS, and thus can be potentially used for microfluidic cell assay devices for cellular physiology study or drug screening. © 2015 Wiley Periodicals, Inc.

  1. Presence of closely spaced protein thiols on the surface of mammalian cells.

    PubMed Central

    Donoghue, N.; Yam, P. T.; Jiang, X. M.; Hogg, P. J.

    2000-01-01

    It has been proposed that certain cell-surface proteins undergo redox reactions, that is, transfer of hydrogens and electrons between closely spaced cysteine thiols that can lead to reduction, formation, or interchange of disulfide bonds. This concept was tested using a membrane-impermeable trivalent arsenical to identify closely spaced thiols in cell-surface proteins. We attached the trivalent arsenical, phenylarsenoxide, to the thiol of reduced glutathione to produce 4-(N-(S-glutathionylacetyl)amino)phenylarsenoxide (GSAO). GSAO bound tightly to synthetic, peptide, and protein dithiols like thioredoxin, but not to monothiols. To identify cell-surface proteins that contain closely spaced thiols, we attached a biotin moiety through a spacer arm to the primary amino group of the gamma-glutamyl residue of GSAO (GSAO-B). Incorporation of GSAO-B into proteins was assessed by measuring the biotin using streptavidin-peroxidase. Up to 12 distinct proteins were labeled with GSAO-B on the surface of endothelial and fibrosarcoma cells. The pattern of labeled proteins differed between the different cell types. Protein disulfide isomerase was one of the proteins on the endothelial and fibrosarcoma cell surface that incorporated GSAO-B. These findings demonstrate that the cell-surface environment can support the existence of closely spaced protein thiols and suggest that at least some of these thiols are redox active. PMID:11206065

  2. HOS cell adhesion on Ti6Al4V surfaces texturized by laser engraving

    NASA Astrophysics Data System (ADS)

    Sandoval Amador, A.; Carreño Garcia, H.; Escobar Rivero, P.; Peña Ballesteros, D. Y.; Estupiñán Duran, H. A.

    2016-02-01

    The cell adhesion of the implant is determinate by the chemical composition, topography, wettability, surface energy and biocompatibility of the biomaterial. In this work the interaction between human osteosarcoma HOS cells and textured Ti6Al4V surfaces were evaluated. Ti6Al4V surfaces were textured using a CO2 laser in order to obtain circular spots on the surfaces. Test surfaces were uncoated (C1) used as a control surface, and surfaces with points obtained by laser engraving, with 1mm spacing (C2) and 0.5mm (C3). The HOS cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% antibiotics. No cells toxicity after one month incubation time occurred. The increased cell adhesion and cell spreading was observed after 1, 3 and 5 days without significant differences between the sample surfaces (C2 and C3) and control (uncoated) at the end of the experiment.

  3. Nanoscale analysis of caspofungin-induced cell surface remodelling in Candida albicans

    NASA Astrophysics Data System (ADS)

    El-Kirat-Chatel, Sofiane; Beaussart, Audrey; Alsteens, David; Jackson, Desmond N.; Lipke, Peter N.; Dufrêne, Yves F.

    2013-01-01

    The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the cells, which correlate with a decrease of the cell wall mechanical strength. Moreover, we find that the drug induces the massive exposure of the cell adhesion protein Als1 on the cell surface and leads to increased cell surface hydrophobicity, two features that trigger cell aggregation. This behaviour is not observed in yeast species lacking Als1, demonstrating the key role that the protein plays in determining the aggregation phenotype of C. albicans. The results show that AFM opens up new avenues for understanding the molecular bases of microbe-drug interactions and for developing new therapeutic agents.The advent of fungal pathogens that are resistant to the classic repertoire of antifungal drugs has increased the need for new therapeutic agents. A prominent example of such a novel compound is caspofungin, known to alter cell wall biogenesis by inhibiting β-1,3-d-glucan synthesis. Although much progress has been made in understanding the mechanism of action of caspofungin, little is known about its influence on the biophysical properties of the fungal cells. Here, we use atomic force microscopy (AFM) to demonstrate that caspofungin induces major remodelling of the cell surface properties of Candida albicans. Caspofungin causes major morphological and structural alterations of the

  4. Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

    PubMed

    Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

    2013-08-06

    Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

  5. 3D patterned substrates for bioartificial blood vessels - The effect of hydrogels on aligned cells on a biomaterial surface.

    PubMed

    Zhao, Xinxin; Irvine, Scott Alexander; Agrawal, Animesh; Cao, Ye; Lim, Pei Qi; Tan, Si Ying; Venkatraman, Subbu S

    2015-10-01

    The optimal bio-artificial blood vessel construct is one that has a compliant tubular core with circumferentially aligned smooth muscle cells (SMCs). Obtaining this well-aligned pattern of SMCs on a scaffold is highly beneficial as this cellular orientation preserves the SMC contractile phenotype. We used 3D patterning to create channels on a polycaprolactone (PCL) scaffold; SMCs were then found to be aligned within the microchannels. To preserve this alignment, and to provide a protective coating that could further incorporate cells, we evaluated the use of two hydrogels, one based on poly(ethylene glycol) diacrylate (PEGDA) and the other based on gelatin. Hydrogels were either physically coated on the PCL surfaces or covalently linked via suitable surface modification of PCL. For covalent immobilization of PEGDA hydrogel, alkene groups were introduced on PCL, while for gelatin covalent linkage, serum proteins were introduced. It is, however, crucial that the hydrogel coating does not disrupt the cellular patterning and distribution. We show in this work that both the process of coating as well as the nature of the coating are critical to preservation of the aligned SMCs. The covalent coating methods involving the crosslinking of hydrogels with the surface of PCL films promoted hydrogel retention time on the film as compared with physical deposition. Furthermore, subsequent hydrogel degradation is affected by the components of the cell culture medium, hinting at a possible route to in vivo biodegradation. Surface features control cellular orientation and subsequently influence their functionality, a useful effect for cellularized biomedical devices. Such devices also can benefit from protective and cell friendly hydrogel coatings. However, literature is lacking on the fate of cells that have endured hydrogel coating whilst orientated on a biomaterial surface. In particular, elucidation of the cells ability to remain adherent and orientated post hydrogel addition

  6. A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation*

    PubMed Central

    Graessel, Anke; Hauck, Stefanie M.; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B.; Suttner, Kathrin

    2015-01-01

    Naive CD4+ T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4+ T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4+ T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4+ T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4+ T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. PMID:25991687

  7. Base-modified UDP-sugars reduce cell surface levels of P-selectin glycoprotein 1 (PSGL-1) on IL-1β-stimulated human monocytes.

    PubMed

    Kanabar, Varsha; Tedaldi, Lauren; Jiang, Jingqian; Nie, Xiaodan; Panina, Irina; Descroix, Karine; Man, Francis; Pitchford, Simon C; Page, Clive P; Wagner, Gerd K

    2016-10-01

    P-selectin glycoprotein ligand-1 (PSGL-1, CD162) is a cell-surface glycoprotein that is expressed, either constitutively or inducibly, on all myeloid and lymphoid cell lineages. PSGL-1 is implicated in cell-cell interactions between platelets, leukocytes and endothelial cells, and a key mediator of inflammatory cell recruitment and transmigration into tissues. Here, we have investigated the effects of the β-1,4-galactosyltransferase inhibitor 5-(5-formylthien-2-yl) UDP-Gal (5-FT UDP-Gal, compound 1: ) and two close derivatives on the cell surface levels of PSGL-1 on human peripheral blood mononuclear cells (hPBMCs). PSGL-1 levels were studied both under basal conditions, and upon stimulation of hPBMCs with interleukin-1β (IL-1β). Between 1 and 24 hours after IL-1β stimulation, we observed initial PSGL-1 shedding, followed by an increase in PSGL-1 levels on the cell surface, with a maximal window between IL-1β-induced and basal levels after 72 h. All three inhibitors reduce PSGL-1 levels on IL-1β-stimulated cells in a concentration-dependent manner, but show no such effect in resting cells. Compound 1: also affects the cell surface levels of adhesion molecule CD11b in IL-1β-stimulated hPBMCs, but not of glycoproteins CD14 and CCR2. This activity profile may be linked to the inhibition of global Sialyl Lewis presentation on hPBMCs by compound 1: , which we have also observed. Although this mechanistic explanation remains hypothetical at present, our results show, for the first time, that small molecules can discriminate between IL-1β-induced and basal levels of cell surface PSGL-1. These findings open new avenues for intervention with PSGL-1 presentation on the cell surface of primed hPBMCs and may have implications for anti-inflammatory drug development. © The Author 2016. Published by Oxford University Press.

  8. Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.

    PubMed

    Dorrell, Craig; Abraham, Stephanie L; Lanxon-Cookson, Kelsea M; Canaday, Pamela S; Streeter, Philip R; Grompe, Markus

    2008-09-01

    We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.

  9. Biophysical model of bacterial cell interactions with nanopatterned cicada wing surfaces.

    PubMed

    Pogodin, Sergey; Hasan, Jafar; Baulin, Vladimir A; Webb, Hayden K; Truong, Vi Khanh; Phong Nguyen, The Hong; Boshkovikj, Veselin; Fluke, Christopher J; Watson, Gregory S; Watson, Jolanta A; Crawford, Russell J; Ivanova, Elena P

    2013-02-19

    The nanopattern on the surface of Clanger cicada (Psaltoda claripennis) wings represents the first example of a new class of biomaterials that can kill bacteria on contact based solely on their physical surface structure. The wings provide a model for the development of novel functional surfaces that possess an increased resistance to bacterial contamination and infection. We propose a biophysical model of the interactions between bacterial cells and cicada wing surface structures, and show that mechanical properties, in particular cell rigidity, are key factors in determining bacterial resistance/sensitivity to the bactericidal nature of the wing surface. We confirmed this experimentally by decreasing the rigidity of surface-resistant strains through microwave irradiation of the cells, which renders them susceptible to the wing effects. Our findings demonstrate the potential benefits of incorporating cicada wing nanopatterns into the design of antibacterial nanomaterials. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Perron, Amelie; Sharif, Nadder; Gendron, Louis

    2006-05-12

    In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores,more » as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.« less

  11. ELECTRON MICROSCOPE STUDY OF SURFACE IMMUNOGLOBULIN-BEARING HUMAN TONSIL CELLS

    PubMed Central

    Zucker-Franklin, Dorothea; Berney, Steven

    1972-01-01

    Surface immunoglobulin-bearing cells were selected from suspensions of human tonsil cells by the reverse immune cytoadherence technique. The method employed a hybrid antibody directed against Ig on lymphoid cells and against ferritin bound to sheep red blood cells (SRBC). Only 6% of the cells formed rosettes. When subjected to electron microscopy they were shown to consist of a morphologically heterogeneous population of cells. However, most cells in the center of rosettes showed ribosome-associated endoplasmic reticulum (RER) and polyribosomes. Usually these organelles were located in close proximity to membrane sites where a 400–600 A bridge was resolved between the lymphocyte and the ferritin particle on the SRBC. The bridge is postulated to consist at least in part of Ig. Only 50% of the plasma cells formed rosettes and bridges could not be resolved. The surface of the plasma cells within rosettes differed from that of plasma cells which had not reacted with ferritin-coated sheep erythrocytes. The incidence of plasma cells and γ-globulin-bearing lymphoid cells was corroborated with the help of fluorescent antibody techniques. PMID:5061976

  12. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance.

    PubMed

    Chen, Xianzhong

    2017-03-04

    The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed.

  13. Yeast cell surface display: An efficient strategy for improvement of bioethanol fermentation performance

    PubMed Central

    Chen, Xianzhong

    2017-01-01

    ABSTRACT The cell surface serves as a functional interface between the inside and the outside of the cell. Within the past 20 y the ability of yeast (Saccharomyces cerevisiae) to display heterologous proteins on the cell surface has been demonstrated. Furthermore, S. cerevisiae has been both developed and applied in expression of various proteins on the cell surface. Using this novel and useful strategy, proteins and peptides of various kinds can be displayed on the yeast cell surface by fusing the protein of interest with the glycosylphosphatidylinositol (GPI)-anchoring system. Consolidated bioprocessing (CBP) using S. cerevisiae represents a promising technology for bioethanol production. However, further work is needed to improve the fermentation performance. There is some excellent previous research regarding construction of yeast biocatalyst using the surface display system to decrease cost, increase efficiency of ethanol production and directly utilize starch or biomass for fuel production. In this commentary, we reviewed the yeast surface display system and highlighted recent work. Additionally, the strategy for decrease of phytate phosphate content in dried distillers grains with solubles (DDGS) by display of phytase on the yeast cell surface is discussed. PMID:27459271

  14. Silane-modified surfaces in specific antibody-mediated cell recognition.

    PubMed

    Sterzynska, Karolina; Budna, Joanna; Frydrych-Tomczak, Emilia; Hreczycho, Grzegorz; Malinska, Agnieszka; Maciejewski, Hieronim; Zabel, Maciej

    2014-01-01

    The immobilization of antibodies on various surfaces has been the subject of advanced research in various immunoassay-based diagnostic devices. The physical and chemical stabilities of the immobilized antibodies on a solid surface still cause many problems because upon immobilizing antibody molecules, the antigen-binding ability usually decreases. The silanization of surfaces with organosilanes carrying chemically active groups such as (3-aminopropyl) triethoxysilane (APTES) can accommodate these antigen-binding molecules in an appropriate orientation so that their functionality and binding activity are essentially retained. In this study, n-butyltrimethoxysilane (BMS) and 3-(octafluoropentyloxy)-propyltriethoxysilane (OFPOS) were used as "blocking silanes". The aims of this study were to compare the effectiveness of specific antibody binding of APTES, APTES + BMS and APTES + OFPOS and to characterize the modified surfaces by contact angle measurements and immunofluorescence measurements prior to and after immobilizing proteins. Additionally, we have evaluated the functionality of the immobilized antibodies by their abilities to bind EpCAM-positive human colon adenocarcinoma cell line (LoVo) and EpCAM-negative mouse embryonic fibroblast cell line (3T3). Cell enumeration was conducted on the basis of DAPI-positive signals and recorded using a confocal laser scanning biological microscope. The results of our study showed that the immobilization capability and reactivity of APTES, APTES + BMS and APTES + OFPOS differ. The modification of APTES with unreactive silanes (BMS,OFPOS) is recommended to improve the antibody binding efficiency. However, using OFPOS resulted in more effective antibody and cell binding, and it appears to be the most useful compound in specific antibody-mediated cell recognition.

  15. Determining surface areas of marine alga cells by acid-base titration method.

    PubMed

    Wang, X; Ma, Y; Su, Y

    1997-09-01

    A new method for determining the surface area of living marine alga cells was described. The method uses acid-base titration to measure the surface acid/base amount on the surface of alga cells and uses the BET (Brunauer, Emmett, and Teller) equation to estimate the maximum surface acid/base amount, assuming that hydrous cell walls have carbohydrates or other structural compounds which can behave like surface Brönsted acid-base sites due to coordination of environmental H2O molecules. The method was applied to 18 diverse alga species (including 7 diatoms, 2 flagellates, 8 green algae and 1 red alga) maintained in seawater cultures. For the species examined, the surface areas of individual cells ranged from 2.8 x 10(-8) m2 for Nannochloropsis oculata to 690 x 10(-8) m2 for Dunaliella viridis, specific surface areas from 1,030 m2.g-1 for Dunaliella salina to 28,900 m2.g-1 for Pyramidomonas sp. Measurement accuracy was 15.2%. Preliminary studies show that the method may be more promising and accurate than light/electron microscopic measurements for coarse estimation of the surface area of living algae.

  16. Analysis of Solar Cell Efficiency for Venus Atmosphere and Surface Missions

    NASA Technical Reports Server (NTRS)

    Landis, Geoffrey A.; Haag, Emily

    2013-01-01

    A simplified model of solar power in the Venus environment is developed, in which the solar intensity, solar spectrum, and temperature as a function of altitude is applied to a model of photovoltaic performance, incorporating the temperature and intensity dependence of the open-circuit voltage and the temperature dependence of the bandgap and spectral response of the cell. We use this model to estimate the performance of solar cells for both the surface of Venus and for atmospheric probes at altitudes from the surface up to 60 km. The model shows that photovoltaic cells will produce power even at the surface of Venus.

  17. Electrochemical Responsive Superhydrophilic Surfaces of Polythiophene Derivatives towards Cell Capture and Release.

    PubMed

    Hao, Yuwei; Li, Yingying; Zhang, Feilong; Cui, Haijun; Hu, Jinsong; Meng, Jingxin; Wang, Shutao

    2018-03-23

    Highly efficient cell capture and release with low background are urgently required for early diagnosis of diseases such as cancer. Herein, we report an electrochemical responsive superhydrophilic surface exhibiting specific cell capture and release with high yields and extremely low nonspecific adhesion. Through electrochemical deposition, 3-substituted thiophene derivatives are deposited onto indium tin oxide (ITO) nanowire arrays with 4-n-nonylbenzeneboronic acid (BA) as dopant, fabricating the electrochemical responsive superhydrophilic surfaces. The molecular recognition between sialic acids over-expressed on the cell membrane and doped BAs endows the electrochemical responsive surfaces with the ability to capture and release targeted cancer cells. By adjusting the substituent group of thiophene derivatives, the surface wettability can be readily regulated and further utilized for reducing nonspecific cell adhesion. Significantly, the released cells still maintain a high proliferation ability, which indicates that the applied potential does not significantly harm the cells. Therefore, these results may provide a new strategy to achieve advanced functions of biomedical materials, such as low nonspecific adhesion. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering.

    PubMed

    O'Brien, Paul J; Elahipanah, Sina; Rogozhnikov, Dmitry; Yousaf, Muhammad N

    2017-05-24

    The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types

  19. Analyses of cell surface molecules on hepatic stem/progenitor cells in mouse fetal liver.

    PubMed

    Kakinuma, Sei; Ohta, Haruhiko; Kamiya, Akihide; Yamazaki, Yuji; Oikawa, Tsunekazu; Okada, Ken; Nakauchi, Hiromitsu

    2009-07-01

    Hepatic stem/progenitor cells possess active proliferative ability and the capacity for differentiation into hepatic and cholangiocytic lineages. Our group and others have shown that a prospectively defined population in mid-gestational fetal liver contains hepatic stem/progenitor cells. However, the phenotypes of such cells are incompletely elucidated. We analyzed the profile of cell-surface molecules on primary hepatic stem/progenitor cells. Expression of cell surface molecules on primary hepatic stem/progenitor cells in mouse mid-gestational fetal liver was analyzed using flow cytometric multicolor analyses and colony-formation assays. The potential of the cells for liver repopulation was examined by transplantation assay. We found that CD13 (aminopeptidase N) was detected on the cells of the previously reported (Dlk/Pref-1(+)) hepatic stem/progenitor fraction. Colony-formation assays revealed that the CD13(+) fraction, compared with the Dlk(+) fraction, of non-hematopoietic cells in fetal liver was enriched in hepatic stem/progenitor cells. Transplantation assay showed the former fraction exhibited repopulating potential in regenerating liver. Moreover, flow cytometric analysis for over 90 antigens demonstrated enrichment of hepatic stem/progenitor cells using several positive selection markers, including (hitherto unknown) CD13, CD73, CD106, and CD133. Our data indicated that CD13 is a positive selection marker for hepatic stem/progenitor cells in mid-gestational fetal liver.

  20. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions.

    PubMed

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A; Brown, Elizabeth E; Sanderson, Ralph D

    2016-01-22

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Fibronectin on the Surface of Myeloma Cell-derived Exosomes Mediates Exosome-Cell Interactions*

    PubMed Central

    Purushothaman, Anurag; Bandari, Shyam Kumar; Liu, Jian; Mobley, James A.; Brown, Elizabeth E.; Sanderson, Ralph D.

    2016-01-01

    Exosomes regulate cell behavior by binding to and delivering their cargo to target cells; however, the mechanisms mediating exosome-cell interactions are poorly understood. Heparan sulfates on target cell surfaces can act as receptors for exosome uptake, but the ligand for heparan sulfate on exosomes has not been identified. Using exosomes isolated from myeloma cell lines and from myeloma patients, we identify exosomal fibronectin as a key heparan sulfate-binding ligand and mediator of exosome-cell interactions. We discovered that heparan sulfate plays a dual role in exosome-cell interaction; heparan sulfate on exosomes captures fibronectin, and on target cells it acts as a receptor for fibronectin. Removal of heparan sulfate from the exosome surface releases fibronectin and dramatically inhibits exosome-target cell interaction. Antibody specific for the Hep-II heparin-binding domain of fibronectin blocks exosome interaction with tumor cells or with marrow stromal cells. Regarding exosome function, fibronectin-mediated binding of exosomes to myeloma cells activated p38 and pERK signaling and expression of downstream target genes DKK1 and MMP-9, two molecules that promote myeloma progression. Antibody against fibronectin inhibited the ability of myeloma-derived exosomes to stimulate endothelial cell invasion. Heparin or heparin mimetics including Roneparstat, a modified heparin in phase I trials in myeloma patients, significantly inhibited exosome-cell interactions. These studies provide the first evidence that fibronectin binding to heparan sulfate mediates exosome-cell interactions, revealing a fundamental mechanism important for exosome-mediated cross-talk within tumor microenvironments. Moreover, these results imply that therapeutic disruption of fibronectin-heparan sulfate interactions will negatively impact myeloma tumor growth and progression. PMID:26601950

  2. Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

    PubMed

    Vabbilisetty, Pratima; Boron, Mallorie; Nie, Huan; Ozhegov, Evgeny; Sun, Xue-Long

    2018-02-28

    Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell's functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine-poly(ethylene glycol)-dibenzocyclooctyne (DSPE-PEG 2000 -DBCO) and cholesterol-PEG-dibenzocyclooctyne (CHOL-PEG 2000 -DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

  3. Elastic modulus affects the growth and differentiation of neural stem cells

    PubMed Central

    Jiang, Xian-feng; Yang, Kai; Yang, Xiao-qing; Liu, Ying-fu; Cheng, Yuan-chi; Chen, Xu-yi; Tu, Yue

    2015-01-01

    It remains poorly understood if carrier hardness, elastic modulus, and contact area affect neural stem cell growth and differentiation. Tensile tests show that the elastic moduli of Tiansu and SMI silicone membranes are lower than that of an ordinary dish, while the elastic modulus of SMI silicone membrane is lower than that of Tiansu silicone membrane. Neural stem cells from the cerebral cortex of embryonic day 16 Sprague-Dawley rats were seeded onto ordinary dishes as well as Tiansu silicone membrane and SMI silicone membrane. Light microscopy showed that neural stem cells on all three carriers show improved adherence. After 7 days of differentiation, neuron specific enolase, glial fibrillary acidic protein, and myelin basic protein expression was detected by immunofluorescence. Moreover, flow cytometry revealed a higher rate of neural stem cell differentiation into astrocytes on Tiansu and SMI silicone membranes than on the ordinary dish, which was also higher on the SMI than the Tiansu silicone membrane. These findings confirm that all three cell carrier types have good biocompatibility, while SMI and Tiansu silicone membranes exhibit good mechanical homogenization. Thus, elastic modulus affects neural stem cell differentiation into various nerve cells. Within a certain range, a smaller elastic modulus results in a more obvious trend of cell differentiation into astrocytes. PMID:26604916

  4. Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application

    PubMed Central

    2018-01-01

    Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell’s functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine–poly(ethylene glycol)–dibenzocyclooctyne (DSPE–PEG2000–DBCO) and cholesterol–PEG–dibenzocyclooctyne (CHOL–PEG2000–DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids. PMID:29503972

  5. Surface-engineered substrates for improved human pluripotent stem cell culture under fully defined conditions.

    PubMed

    Saha, Krishanu; Mei, Ying; Reisterer, Colin M; Pyzocha, Neena Kenton; Yang, Jing; Muffat, Julien; Davies, Martyn C; Alexander, Morgan R; Langer, Robert; Anderson, Daniel G; Jaenisch, Rudolf

    2011-11-15

    The current gold standard for the culture of human pluripotent stem cells requires the use of a feeder layer of cells. Here, we develop a spatially defined culture system based on UV/ozone radiation modification of typical cell culture plastics to define a favorable surface environment for human pluripotent stem cell culture. Chemical and geometrical optimization of the surfaces enables control of early cell aggregation from fully dissociated cells, as predicted from a numerical model of cell migration, and results in significant increases in cell growth of undifferentiated cells. These chemically defined xeno-free substrates generate more than three times the number of cells than feeder-containing substrates per surface area. Further, reprogramming and typical gene-targeting protocols can be readily performed on these engineered surfaces. These substrates provide an attractive cell culture platform for the production of clinically relevant factor-free reprogrammed cells from patient tissue samples and facilitate the definition of standardized scale-up friendly methods for disease modeling and cell therapeutic applications.

  6. Psoriatic T cells reduce epidermal turnover time and affect cell proliferation contributed from differential gene expression.

    PubMed

    Li, Junqin; Li, Xinhua; Hou, Ruixia; Liu, Ruifeng; Zhao, Xincheng; Dong, Feng; Wang, Chunfang; Yin, Guohua; Zhang, Kaiming

    2015-09-01

    Psoriasis is mediated primarily by T cells, which reduce epidermal turnover time and affect keratinocyte proliferation. We aimed to identify differentially expressed genes (DEG) in T cells from normal, five pairs of monozygotic twins concordant or discordant for psoriasis, to determine whether these DEG may account for the influence to epidermal turnover time and keratinocyte proliferation. The impact of T cells on keratinocyte proliferation and epidermal turnover time were investigated separately by immunohistochemistry and cultured with (3) H-TdR. mRNA expression patterns were investigated by RNA sequencing and verified by real-time reverse transcription polymerase chain reaction. After co-culture with psoriatic T cells, the expression of Ki-67, c-Myc and p53 increased, while expression of Bcl-2 and epidermal turnover time decreased. There were 14 DEG which were found to participate in the regulation of cell proliferation or differentiation. Psoriatic T cells exhibited the ability to decrease epidermal turnover time and affect keratinocyte proliferation because of the differential expression of PPIL1, HSPH1, SENP3, NUP54, FABP5, PLEKHG3, SLC9A9 and CHCHD4. © 2015 Japanese Dermatological Association.

  7. Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

    PubMed Central

    Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

    2015-01-01

    In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

  8. Label-free detection of surface markers on stem cells by oblique-incidence reflectivity difference microscopy

    PubMed Central

    Lo, Kai-Yin; Sun, Yung-Shin; Landry, James P.; Zhu, Xiangdong; Deng, Wenbin

    2012-01-01

    Conventional fluorescent microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in ways often uncharacterized. The binding between antibody and antigen might not be in the native situation after such conjugation. Here, we present an oblique-incidence reflectivity difference (OI-RD) microscope as an effective method for label-free, real-time detection of cell surface markers and apply such a technique to analysis of Stage-Specific Embryonic Antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies only bind to the surface of the stem cells while not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. The results concluded that OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells. The information could be another indicator to determine the cell stages. PMID:21781038

  9. Altering textural properties of fermented milk by using surface-engineered Lactococcus lactis.

    PubMed

    Tarazanova, Mariya; Huppertz, Thom; Kok, Jan; Bachmann, Herwig

    2018-05-09

    Lactic acid bacteria are widely used for the fermentation of dairy products. While bacterial acidification rates, proteolytic activity and the production of exopolysaccharides are known to influence textural properties of fermented milk products, little is known about the role of the microbial surface on microbe-matrix interactions in dairy products. To investigate how alterations of the bacterial cell surface affect fermented milk properties, 25 isogenic Lactococcus lactis strains that differed with respect to surface charge, hydrophobicity, cell chaining, cell-clumping, attachment to milk proteins, pili expression and EPS production were used to produce fermented milk. We show that overexpression of pili increases surface hydrophobicity of various strains from 3-19% to 94-99%. A profound effect of different cell surface properties was an altered spatial distribution of the cells in the fermented product. Aggregated cells tightly fill the cavities of the protein matrix, while chaining cells seem to be localized randomly. A positive correlation was found between pili overexpression and viscosity and gel hardness of fermented milk. Gel hardness also positively correlated with clumping of cells in the fermented milk. Viscosity of fermented milk was also higher when it was produced with cells with a chaining phenotype or with cells that overexpress exopolysaccharides. Our results show that alteration of cell surface morphology affects textural parameters of fermented milk and cell localization in the product. This is indicative of a cell surface-dependent potential of bacterial cells as structure elements in fermented foods. © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  10. Cell Cycle-Dependent Phosphorylation of Theileria annulata Schizont Surface Proteins

    PubMed Central

    von Schubert, Conrad; Wastling, Jonathan M.; Heussler, Volker T.; Woods, Kerry L.

    2014-01-01

    The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state. PMID:25077614

  11. Antibiotic Algae by Chemical Surface Engineering.

    PubMed

    Kerschgens, Isabel P; Gademann, Karl

    2018-03-02

    Chemical cell-surface engineering is a tool for modifying and altering cellular functions. Herein, we report the introduction of an antibiotic phenotype to the green alga Chlamydomonas reinhardtii by chemically modifying its cell surface. Flow cytometry and confocal microscopy studies demonstrated that a hybrid of the antibiotic vancomycin and a 4-hydroxyproline oligomer binds reversibly to the cell wall without affecting the viability or motility of the cells. The modified cells were used to inhibit bacterial growth of Gram-positive Bacillus subtilis cultures. Delivery of the antibiotic from the microalgae to the bacterial cells was verified by microscopy. Our studies provide compelling evidence that 1) chemical surface engineering constitutes a useful tool for the introduction of new, previously unknown functionality, and 2) living microalgae can serve as new platforms for drug delivery. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Cell-to-Cell Heterogeneity in Cortical Tension Specifies Curvature of Contact Surfaces in Caenorhabditis elegans Embryos

    PubMed Central

    Fujita, Masashi; Onami, Shuichi

    2012-01-01

    In the two-cell stage embryos of Caenorhabditis elegans, the contact surface of the two blastomeres forms a curve that bulges from the AB blastomere to the P1 blastomere. This curve is a consequence of the high intracellular hydrostatic pressure of AB compared with that of P1. However, the higher pressure in AB is intriguing because AB has a larger volume than P1. In soap bubbles, which are a widely used model of cell shape, a larger bubble has lower pressure than a smaller bubble. Here, we reveal that the higher pressure in AB is mediated by its higher cortical tension. The cell fusion experiments confirmed that the curvature of the contact surface is related to the pressure difference between the cells. Chemical and genetic interferences showed that the pressure difference is mediated by actomyosin. Fluorescence imaging indicated that non-muscle myosin is enriched in the AB cortex. The cell killing experiments provided evidence that AB but not P1 is responsible for the pressure difference. Computer simulation clarified that the cell-to-cell heterogeneity of cortical tensions is indispensable for explaining the pressure difference. This study demonstrates that heterogeneity in surface tension results in significant deviations of cell behavior compared to simple soap bubble models, and thus must be taken into consideration in understanding cell shape within embryos. PMID:22253922

  13. Microarrays for the evaluation of cell-biomaterial surface interactions

    NASA Astrophysics Data System (ADS)

    Thissen, H.; Johnson, G.; McFarland, G.; Verbiest, B. C. H.; Gengenbach, T.; Voelcker, N. H.

    2007-01-01

    The evaluation of cell-material surface interactions is important for the design of novel biomaterials which are used in a variety of biomedical applications. While traditional in vitro test methods have routinely used samples of relatively large size, microarrays representing different biomaterials offer many advantages, including high throughput and reduced sample handling. Here, we describe the simultaneous cell-based testing of matrices of polymeric biomaterials, arrayed on glass slides with a low cell-attachment background coating. Arrays were constructed using a microarray robot at 6 fold redundancy with solid pins having a diameter of 375 μm. Printed solutions contained at least one monomer, an initiator and a bifunctional crosslinker. After subsequent UV polymerisation, the arrays were washed and characterised by X-ray photoelectron spectroscopy. Cell culture experiments were carried out over 24 hours using HeLa cells. After labelling with CellTracker ® Green for the final hour of incubation and subsequent fixation, the arrays were scanned. In addition, individual spots were also viewed by fluorescence microscopy. The evaluation of cell-surface interactions in high-throughput assays as demonstrated here is a key enabling technology for the effective development of future biomaterials.

  14. Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae.

    PubMed

    Matsuoka, Hiroyuki; Hashimoto, Kazuya; Saijo, Aki; Takada, Yuki; Kondo, Akihiko; Ueda, Mitsuyoshi; Ooshima, Hiroshi; Tachibana, Taro; Azuma, Masayuki

    2014-02-01

    A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild-type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The relative value (mnn2 deletion mutant/wild-type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.

  15. Back surface studies of Cu(In,Ga)Se2 thin film solar cells

    NASA Astrophysics Data System (ADS)

    Simchi, Hamed

    Cu(In,Ga)Se2 thin film solar cells have attracted a lot of interest because they have shown the highest achieved efficiency (21%) among thin film photovoltaic materials, long-term stability, and straightforward optical bandgap engineering by changing relative amounts of present elements in the alloy. Still, there are several opportunities to further improve the performance of the Cu(In,Ga)Se2 devices. The interfaces between layers significantly affect the device performance, and knowledge of their chemical and electronic structures is essential in identifying performance limiting factors. The main goal of this research is to understand the characteristics of the Cu(In,Ga)Se2-back contact interface in order to design ohmic back contacts for Cu(In,Ga)Se2-based solar cells with a range of band gaps and device configurations. The focus is on developing either an opaque or transparent ohmic back contact via surface modification or introduction of buffer layers in the back surface. In this project, candidate back contact materials have been identified based on modeling of band alignments and surface chemical properties of the absorber layer and back contact. For the first time, MoO3 and WO 3 transparent back contacts were successfully developed for Cu(In,Ga)Se 2 solar cells. The structural, optical, and surface properties of MoO 3 and WO3 were optimized by controlling the oxygen partial pressure during reactive sputtering and post-deposition annealing. Valence band edge energies were also obtained by analysis of the XPS spectra and used to characterize the interface band offsets. As a result, it became possible to illuminate of the device from the back, resulting in a recently developed "backwall superstrate" device structure that outperforms conventional substrate Cu(In,Ga)Se2 devices in the absorber thickness range 0.1-0.5 microm. Further enhancements were achieved by introducing moderate amounts of Ag into the Cu(In,Ga)Se2 lattice during the co-evaporation method

  16. Highly multiplexed and quantitative cell-surface protein profiling using genetically barcoded antibodies.

    PubMed

    Pollock, Samuel B; Hu, Amy; Mou, Yun; Martinko, Alexander J; Julien, Olivier; Hornsby, Michael; Ploder, Lynda; Adams, Jarrett J; Geng, Huimin; Müschen, Markus; Sidhu, Sachdev S; Moffat, Jason; Wells, James A

    2018-03-13

    Human cells express thousands of different surface proteins that can be used for cell classification, or to distinguish healthy and disease conditions. A method capable of profiling a substantial fraction of the surface proteome simultaneously and inexpensively would enable more accurate and complete classification of cell states. We present a highly multiplexed and quantitative surface proteomic method using genetically barcoded antibodies called phage-antibody next-generation sequencing (PhaNGS). Using 144 preselected antibodies displayed on filamentous phage (Fab-phage) against 44 receptor targets, we assess changes in B cell surface proteins after the development of drug resistance in a patient with acute lymphoblastic leukemia (ALL) and in adaptation to oncogene expression in a Myc-inducible Burkitt lymphoma model. We further show PhaNGS can be applied at the single-cell level. Our results reveal that a common set of proteins including FLT3, NCR3LG1, and ROR1 dominate the response to similar oncogenic perturbations in B cells. Linking high-affinity, selective, genetically encoded binders to NGS enables direct and highly multiplexed protein detection, comparable to RNA-sequencing for mRNA. PhaNGS has the potential to profile a substantial fraction of the surface proteome simultaneously and inexpensively to enable more accurate and complete classification of cell states. Copyright © 2018 the Author(s). Published by PNAS.

  17. Nanoarchitectured electrochemical cytosensors for selective detection of leukemia cells and quantitative evaluation of death receptor expression on cell surfaces.

    PubMed

    Zheng, Tingting; Fu, Jia-Ju; Hu, Lihui; Qiu, Fan; Hu, Minjin; Zhu, Jun-Jie; Hua, Zi-Chun; Wang, Hui

    2013-06-04

    The variable susceptibility to the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) treatment observed in various types of leukemia cells is related to the difference in the expression levels of death receptors, DR4 and DR5, on the cell surfaces. Quantifying the DR4/DR5 expression status on leukemia cell surfaces is of vital importance to the development of diagnostic tools to guide death receptor-based leukemia treatment. Taking the full advantages of novel nanobiotechnology, we have developed a robust electrochemical cytosensing approach toward ultrasensitive detection of leukemia cells with detection limit as low as ~40 cells and quantitative evaluation of DR4/DR5 expression on leukemia cell surfaces. The optimization of electron transfer and cell capture processes at specifically tailored nanobiointerfaces and the incorporation of multiple functions into rationally designed nanoprobes provide unique opportunities of integrating high specificity and signal amplification on one electrochemical cytosensor. The high sensitivity and selectivity of this electrochemical cytosensing approach also allows us to evaluate the dynamic alteration of DR4/DR5 expression on the surfaces of living cells in response to drug treatments. Using the TRAIL-resistant HL-60 cells and TRAIL-sensitive Jurkat cells as model cells, we have further verified that the TRAIL susceptibility of various types of leukemia cells is directly correlated to the surface expression levels of DR4/DR5. This versatile electrochemical cytosensing platform is believed to be of great clinical value for the early diagnosis of human leukemia and the evaluation of therapeutic effects on leukemia patients after radiation therapy or drug treatment.

  18. CD24 cell surface expression in Mvt1 mammary cancer cells serves as a biomarker for sensitivity to anti-IGF1R therapy.

    PubMed

    Rostoker, Ran; Ben-Shmuel, Sarit; Rashed, Rola; Shen Orr, Zila; LeRoith, Derek

    2016-05-14

    The pro-tumorigenic effects of the insulin-like growth factor receptor (IGF1R) are well described. IGF1R promotes cancer cell survival and proliferation and prevents apoptosis, and, additionally it was shown that IGF1R levels are significantly elevated in most common human malignancies including breast cancer. However, results from phase 3 clinical trials in unselected patients demonstrated lack of efficacy for anti-IGF1R therapy. These findings suggest that predictive biomarkers are greatly warranted in order to identify patients that will benefit from anti-IGF1R therapeutic strategies. Using the delivery of shRNA vectors into the Mvt1 cell line, we tested the role of the IGF1R in the development of mammary tumors. Based on CD24 cell surface expression, control and IGF1R-knockdown (IGF1R-KD) cells were FACS sorted into CD24(-) and CD24(+) subsets and further characterized in vitro. The tumorigenic capacity of each was determined following orthotopic inoculation into the mammary fat pad of female mice. Tumor cells were FACS characterized upon sacrifice to determine IGF1R effect on the plasticity of this cell's phenotype. Metastatic capacity of the cells was assessed using the tail vein assay. In this study we demonstrate that downregulation of the IGF1R specifically in cancer cells expressing CD24 on the cell surface membrane affect both their morphology (from mesenchymal-like into epithelial-like morphology) and phenotype in vitro. Moreover, we demonstrate that IGF1R-KD abolished both CD24(+) cells capacity to form mammary tumors and lung metastatic lesions. We found in both cells and tumors a marked upregulation in CTFG and a significant reduction of SLP1 expression in the CD24(+)/IGF1R-KD; tumor-suppressor and tumor-promoting genes respectively. Moreover, we demonstrate here that the IGF1R is essential for the maintenance of stem/progenitor-like cancer cells and we further demonstrate that IGF1R-KD induces in vivo differentiation of the CD24(+) cells toward the

  19. Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

    PubMed Central

    Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

    2010-01-01

    Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

  20. Relationship between Surface Modifications of Nanoparticle and Invasion into Suspension Cells

    NASA Astrophysics Data System (ADS)

    Matsui, Y.; Sakai, N.; Tsuda, A.; Yoneda, M.

    2011-07-01

    Nanomaterials have a variety of properties for each material. There is little information available on which kinds of material properties have effects on toxicity and kinetics. This paper presents that a relationship between material properties and hazard data by undertaking a bibliographical survey at first. With respect to cytotoxicity, it probably depends mainly on the particle volume dose and to a certain degree on particle solubility. It can be concluded from these results that there is a relationship between material properties and hazard data. Many activities involving nano risk are occurring all over the world. Secondly, we assayed actually for cellular uptake of three kinds of Quantum dots (15 nm, 5.5×1012 particles/ml) to demonstrate our result of bibliographical survey. Three different surface modification quantum dots (non-modification, -COOH, -NH3) were mixed with floating Jurkat cells in each. After thirty minute, we washed these cells three times and detected fluorescence by flow cytometer. Almost all the carboxylate particles invaded a cell, about 60% aminated them also invaded and few non-modification particles were taken up. Nanomaterials are often very broadly categorized and named based upon their basic material composition or product shape. Our results confirm that we have to examine which physical-chemical properties affect some adverse effects for each nanomaterial.

  1. Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions▿ †

    PubMed Central

    Sherer, Nathan M.; Jin, Jing; Mothes, Walther

    2010-01-01

    The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions. PMID:20089647

  2. Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

    PubMed

    Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

    2013-07-02

    Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

  3. Biocompatible, smooth, plasma-treated nickel-titanium surface--an adequate platform for cell growth.

    PubMed

    Chrzanowski, W; Szade, J; Hart, A D; Knowles, J C; Dalby, M J

    2012-02-01

    High nickel content is believed to reduce the number of biomedical applications of nickel-titanium alloy due to the reported toxicity of nickel. The reduction in nickel release and minimized exposure of the cell to nickel can optimize the biocompatibility of the alloy and increase its use in the application where its shape memory effects and pseudoelasticity are particularly useful, e.g., spinal implants. Many treatments have been tried to improve the biocompatibility of Ni-Ti, and results suggest that a native, smooth surface could provide sufficient tolerance, biologically. We hypothesized that the native surface of nickel-titanium supports cell differentiation and insures good biocompatibility. Three types of surface modifications were investigated: thermal oxidation, alkali treatment, and plasma sputtering, and compared with smooth, ground surface. Thermal oxidation caused a drop in surface nickel content, while negligible chemistry changes were observed for plasma-modified samples when compared with control ground samples. In contrast, alkali treatment caused significant increase in surface nickel concentration and accelerated nickel release. Nickel release was also accelerated in thermally oxidized samples at 600 °C, while in other samples it remained at low level. Both thermal oxidation and alkali treatment increased the roughness of the surface, but mean roughness R(a) was significantly greater for the alkali-treated ones. Ground and plasma-modified samples had 'smooth' surfaces with R(a)=4 nm. Deformability tests showed that the adhesion of the surface layers on samples oxidized at 600 °C and alkali treatment samples was not sufficient; the layer delaminated upon deformation. It was observed that the cell cytoskeletons on the samples with a high nickel content or release were less developed, suggesting some negative effects of nickel on cell growth. These effects were observed primarily during initial cell contact with the surface. The most favorable

  4. Pseudomonas aeruginosa attachment on QCM-D sensors: the role of cell and surface hydrophobicities.

    PubMed

    Marcus, Ian M; Herzberg, Moshe; Walker, Sharon L; Freger, Viatcheslav

    2012-04-17

    While biofilms are ubiquitous in nature, the mechanism by which they form is still poorly understood. This study investigated the process by which bacteria deposit and, shortly after, attach irreversibly to surfaces by reorienting to create a stronger interaction, which leads to biofilm formation. A model for attachment of Pseudomonas aeruginosa was developed using a quartz crystal microbalance with dissipation monitoring (QCM-D) technology, along with a fluorescent microscope and camera to monitor kinetics of adherence of the cells over time. In this model, the interaction differs depending on the force that dominates between the viscous, inertial, and elastic loads. P. aeruginosa, grown to the midexponential growth phase (hydrophilic) and stationary phase (hydrophobic) and two different surfaces, silica (SiO(2)) and polyvinylidene fluoride (PVDF), which are hydrophilic and hydrophobic, respectively, were used to test the model. The bacteria deposited on both of the sensor surfaces, though on the silica surface the cells reached a steady state where there was no net increase in deposition of bacteria, while the quantity of cells depositing on the PVDF surface continued to increase until the end of the experiments. The change in frequency and dissipation per cell were both positive for each overtone (n), except when the cells and surface are both hydrophilic. In the model three factors, specifically, viscous, inertial, and elastic loads, contribute to the change in frequency and dissipation at each overtone when a cell deposits on a sensor. On the basis of the model, hydrophobic cells were shown to form an elastic connection to either surface, with an increase of elasticity at higher overtones. At lower overtones, hydrophilic cells depositing on the hydrophobic surface were shown to also be elastic, but as the overtone increases the connection between the cells and sensor becomes more viscoelastic. In the case of hydrophilic cells interacting with the hydrophilic

  5. Targeted cell adhesion on selectively micropatterned polymer arrays on a poly(dimethylsiloxane) surface.

    PubMed

    Tang, Linzhi; Min, Junhong; Lee, Eun-Cheol; Kim, Jong Sung; Lee, Nae Yoon

    2010-02-01

    Herein, we introduce the fabrication of polymer micropattern arrays on a chemically inert poly(dimethylsiloxane) (PDMS) surface and employ them for the selective adhesion of cells. To fabricate the micropattern arrays, a mercapto-ester-based photocurable adhesive was coated onto a mercaptosilane-coated PDMS surface and photopolymerized using a photomask to obtain patterned arrays at the microscale level. Robust polymer patterns, 380 microm in diameter, were successfully fabricated onto a PDMS surface, and cells were selectively targeted toward the patterned regions. Next, the performance of the cell adhesion was observed by anchoring cell adhesive linker, an RGD oligopeptide, on the surface of the mercapto-ester-based adhesive-cured layer. The successful anchoring of the RGD linker was confirmed through various surface characterizations such as water contact angle measurement, XPS analysis, FT-IR analysis, and AFM measurement. The micropatterning of a photocurable adhesive onto a PDMS surface can provide high structural rigidity, a highly-adhesive surface, and a physical pathway for selective cell adhesion, while the incorporated polymer micropattern arrays inside a PDMS microfluidic device can serve as a microfluidic platform for disease diagnoses and high-throughput drug screening.

  6. High efficiency silicon nanowire/organic hybrid solar cells with two-step surface treatment.

    PubMed

    Wang, Jianxiong; Wang, Hao; Prakoso, Ari Bimo; Togonal, Alienor Svietlana; Hong, Lei; Jiang, Changyun; Rusli

    2015-03-14

    A simple two-step surface treatment process is proposed to boost the efficiency of silicon nanowire/PEDOT:PSS hybrid solar cells. The Si nanowires (SiNWs) are first subjected to a low temperature ozone treatment to form a surface sacrificial oxide, followed by a HF etching process to partially remove the oxide. TEM investigation demonstrates that a clean SiNW surface is achieved after the treatment, in contrast to untreated SiNWs that have Ag nanoparticles left on the surface from the metal-catalyzed etching process that is used to form the SiNWs. The cleaner SiNW surface achieved and the thin layer of residual SiO2 on the SiNWs have been found to improve the performance of the hybrid solar cells. Overall, the surface recombination of the hybrid SiNW solar cells is greatly suppressed, resulting in a remarkably improved open circuit voltage of 0.58 V. The power conversion efficiency has also increased from about 10% to 12.4%. The two-step surface treatment method is promising in enhancing the photovoltaic performance of the hybrid silicon solar cells, and can also be applied to other silicon nanostructure based solar cells.

  7. Novel Anti-CRR9/CLPTM1L Antibodies with Antitumorigenic Activity Inhibit Cell Surface Accumulation, PI3K Interaction, and Survival Signaling

    PubMed Central

    Puskás, László G.; Mán, Imola; Szebeni, Gabor; Tiszlavicz, László; Tsai, Susan; James, Michael A.

    2016-01-01

    We and others have recently shown Cisplatin Resistance-Related Protein 9 (CRR9)/Cleft Lip and Palate Transmembrane 1-Like (CLPTM1L) to affect survival and proliferation in lung and pancreatic tumor cells. Our research has indicated that CLPTM1L affects multiple survival signaling pathways in tumor cells under oncogenic, genotoxic, and microenvironmental stress. We have confirmed the association of CLPTM1L with pancreatic cancer by demonstrating overexpression of CLPTM1L in pancreatic tumors and poor survival in patients with high tumor expression of CLPTM1L. Predicting a transmembrane structure, we determined that CLPTM1L could be targeted at the plasma membrane. Herein, we describe the development of monoclonal antibodies targeting CLPTM1L. Lead antibodies inhibited surface accumulation of CLPTM1L, Akt phosphorylation, anchorage-independent growth, and chemotherapeutic resistance in lung and pancreatic tumor cells. Gemcitabine promoted a physical interaction between CLPTM1L and p110α in pancreatic tumor cells, which was inhibited by anti-CLPTM1L. In-vivo treatment with anti-CLPTM1L robustly inhibited the growth of both lung and pancreatic adenocarcinoma xenografts. The efficacy of anti-CLPTM1L correlated with specific epitopes representing important targets in human cancers, particularly those driven by KRas, for which effective targeted therapies have been elusive. This study is the first to report cell-surface exposure of the tumor survival protein CLPTM1L and inhibition of the function of surface CLPTM1L with novel, systematically developed inhibitory monoclonal antibodies establishing proof of concept of clinically practical agents inhibiting this compelling new tumor survival target in cancer. PMID:26939707

  8. Nutrient-deprived cancer cells preferentially use sialic acid to maintain cell surface glycosylation.

    PubMed

    Badr, Haitham A; AlSadek, Dina M M; Mathew, Mohit P; Li, Chen-Zhong; Djansugurova, Leyla B; Yarema, Kevin J; Ahmed, Hafiz

    2015-11-01

    Cancer is characterized by abnormal energy metabolism shaped by nutrient deprivation that malignant cells experience during various stages of tumor development. This study investigated the response of nutrient-deprived cancer cells and their non-malignant counterparts to sialic acid supplementation and found that cells utilize negligible amounts of this sugar for energy. Instead cells use sialic acid to maintain cell surface glycosylation through complementary mechanisms. First, levels of key metabolites (e.g., UDP-GlcNAc and CMP-Neu5Ac) required for glycan biosynthesis are maintained or enhanced upon Neu5Ac supplementation. In concert, sialyltransferase expression increased at both the mRNA and protein levels, which facilitated increased sialylation in biochemical assays that measure sialyltransferase activity as well as at the whole cell level. In the course of these experiments, several important differences emerged that differentiated the cancer cells from their normal counterparts including resistant to sialic acid-mediated energy depletion, consistently more robust sialic acid-mediated glycan display, and distinctive cell surface vs. internal vesicle display of newly-produced sialoglycans. Finally, the impact of sialic acid supplementation on specific markers implicated in cancer progression was demonstrated by measuring levels of expression and sialylation of EGFR1 and MUC1 as well as the corresponding function of sialic acid-supplemented cells in migration assays. These findings both provide fundamental insight into the biological basis of sialic acid supplementation of nutrient-deprived cancer cells and open the door to the development of diagnostic and prognostic tools. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Relationship between Cell Surface Carbohydrates and Intrastrain Variation on Opsonophagocytosis of Streptococcus pneumoniae

    PubMed Central

    Kim, Jean O.; Romero-Steiner, Sandra; Sørensen, Uffe B. Skov; Blom, Jens; Carvalho, M.; Barnard, S.; Carlone, George; Weiser, Jeffrey N.

    1999-01-01

    Streptococcus pneumoniae undergoes spontaneous phase variation between a transparent and an opaque colony phenotype, the latter being more virulent in a murine model of sepsis. Opaque pneumococci have previously been shown to express lower amounts of C polysaccharide (cell wall teichoic acid) and in this study were shown to have a higher content of capsular polysaccharide by immunoelectron microscopy. This report then examined the relationship between expression of these two cell surface carbohydrate structures and their relative contribution to the increased virulence of opaque variants. Comparison of genetically related strains showed that the differential content of capsular polysaccharide did not affect the amount of teichoic acid as measured by a capture enzyme-linked immunosorbent assay (ELISA). In contrast, when the teichoic acid structure was altered by replacing choline in the growth medium with structural analogs, the quantity of capsular polysaccharide as measured by a capture ELISA was decreased, demonstrating a linkage in the expression of the two surface carbohydrate structures. A standardized assay was used to assess the relative contribution of cell surface carbohydrates to opsonophagocytosis. The opaque variants required 1.2- to 30-fold more immune human serum to achieve 50% opsonophagocytic killing than did related transparent variants (types 6B and 9V). The opsonophagocytic titer was proportional to the quantity of capsular polysaccharide rather than teichoic acid. The major factor in binding of the opsonin, C-reactive protein (CRP), was also the amount of capsular polysaccharide rather than the teichoic acid ligand. Only for the transparent variant (type 6B), which bound more CRP, was there enhanced opsonophagocytic killing in the presence of this serum protein. Increased expression of capsular polysaccharide, therefore, appeared to be the major factor in the decreased opsonophagocytic killing of opaque pneumococci. PMID:10225891

  10. Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

    NASA Astrophysics Data System (ADS)

    Christenson, Wayne B.

    Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering

  11. TOF-SIMS investigation of metallic material surface after culturing cells

    NASA Astrophysics Data System (ADS)

    Aoyagi, Satoka; Hiromoto, Sachiko; Hanawa, Takao; Kudo, Masahiro

    2004-06-01

    Biomolecules such as extracellular matrix and adhesive proteins generated by adhered cells on metallic specimens were characterized by means of time-of-flight secondary ion mass spectrometry (TOF-SIMS) in order to clarify the interaction between cells and metal surfaces. Since composition and structure of the extracellular matrix depends on conditions of cells, characterization of the interaction between cells and metallic specimens is important in order to evaluate the biocompatibility and the degradation behavior of metallic biomaterials and artificial organs. Moreover, the obtained data can contribute to the development of new metallic biomaterials. TOF-SIMS spectra were analyzed by means of mutual information described by information theory and principal components analysis (PCA). The results show that cells have great influence on adsorption of biomolecules on metallic materials because they change surface conditions of the materials. Thus TOF-SIMS is a useful technique to investigate the interaction between metallic biomaterials and cells.

  12. Internal processes affecting surfaces of low-density satellites - Ganymede and Callisto

    NASA Technical Reports Server (NTRS)

    Parmentier, E. M.; Head, J. W.

    1979-01-01

    Possible significant physical processes on low-density (icy) satellites, particularly Ganymede and Callisto, are outlined, and the relations of these interior processes to the formation and evolution of satellite surfaces are discussed. A variety of mechanisms is shown to lead to interior melting in early satellite history and a configuration characterized by a predominantly water ice lithosphere overlying a mantle containing liquid water. Physical processes capable of affecting the lithosphere of an ice-silicate body and thus creating observable surface features are assessed, including tectonic stresses from tidal deformation and volume changes, gravitational effects on density differences and water volcanism. The residence time of surface features on icy bodies produced by the outlined processes and by impact cratering is considered, and a tentative outline of the geologic history of Ganymede and Callisto is presented. Observations from Voyager and Galileo are expected to provide evidence on the evolution and geologic history of low-density satellites.

  13. CARbodies: Human Antibodies Against Cell Surface Tumor Antigens Selected From Repertoires Displayed on T Cell Chimeric Antigen Receptors

    PubMed Central

    Alonso-Camino, Vanesa; Sánchez-Martín, David; Compte, Marta; Nuñez-Prado, Natalia; Diaz, Rosa M; Vile, Richard; Alvarez-Vallina, Luis

    2013-01-01

    A human single-chain variable fragment (scFv) antibody library was expressed on the surface of human T cells after transduction with lentiviral vectors (LVs). The repertoire was fused to a first-generation T cell receptor ζ (TCRζ)-based chimeric antigen receptor (CAR). We used this library to isolate antibodies termed CARbodies that recognize antigens expressed on the tumor cell surface in a proof-of-principle system. After three rounds of activation-selection there was a clear repertoire restriction, with the emergence dominant clones. The CARbodies were purified from bacterial cultures as soluble and active proteins. Furthermore, to validate its potential application for adoptive cell therapy, human T cells were transduced with a LV encoding a second-generation costimulatory CAR (CARv2) bearing the selected CARbodies. Transduced human primary T cells expressed significant levels of the CARbodies-based CARv2 fusion protein on the cell surface, and importantly could be specifically activated, after stimulation with tumor cells. This approach is a promising tool for the generation of antibodies fully adapted to the display format (CAR) and the selection context (cell synapse), which could extend the scope of current adoptive cell therapy strategies with CAR-redirected T cells. PMID:23695536

  14. Performance of epitaxial back surface field cells

    NASA Technical Reports Server (NTRS)

    Brandhorst, H. W., Jr.; Baraona, C. R.; Swartz, C. K.

    1973-01-01

    Epitaxial back surface field structures were formed by depositing a 10 micron thick 10 Omega-cm epitaxial silicon layer onto substrates with resistivities of 0.01, 0.1, 1.0 and 10 Omega-cm. A correlation between cell open-circuit voltage and substrate resistivity was observed and was compared to theory. The cells were also irradiated with 1 MeV electrons to a fluence of 5 X 10 to the 15th power e/cm2. The decrease of cell open-circuit voltage was in excellent agreement with theoretical predictions and the measured short circuit currents were within 2% of the prediction. Calculations are presented of optimum cell performance as functions of epitaxial layer thickness, radiation fluence and substrate diffusion length.

  15. Cell surface engineering of microorganisms towards adsorption of heavy metals.

    PubMed

    Li, Peng-Song; Tao, Hu-Chun

    2015-06-01

    Heavy metal contamination has become a worldwide environmental concern due to its toxicity, non-degradability and food-chain bioaccumulation. Conventional physical and chemical treatment methods for heavy metal removal have disadvantages such as cost-intensiveness, incomplete removal, secondary pollution and the lack of metal specificity. Microbial biomass-based biosorption is one of the approaches gaining increasing attention because it is effective, cheap, and environmental friendly and can work well at low concentrations. To enhance the adsorption properties of microbial cells to heavy metal ions, the cell surface display of various metal-binding proteins/peptides have been performed using a cell surface engineering approach. The surface engineering of Gram-negative bacteria, Gram-positive bacteria and yeast towards the adsorption of heavy metals are reviewed in this article. The problems and future perspectives of this technology are discussed.

  16. Display of adenoregulin with a novel Pichia pastoris cell surface display system.

    PubMed

    Ren, Ren; Jiang, Zhengbing; Liu, Meiyun; Tao, Xinyi; Ma, Yushu; Wei, Dongzhi

    2007-02-01

    Two Pichia pastoris cell surface display vectors were constructed. The vectors consisted of the flocculation functional domain of Flo1p with its own secretion signal sequence or the alpha-factor secretion signal sequence, a polyhistidine (6xHis) tag for detection, an enterokinase recognition site, and the insertion sites for target proteins. Adenoregulin (ADR) is a 33-amino-acid antimicrobial peptide isolated from Phyllomedusa bicolor skin. The ADR was expressed and displayed on the Pichia pastoris KM71 cell surface with the system reported. The displayed recombinant ADR fusion protein was detected by fluorescence microscopy and confocal laser scanning microscopy (CLSM). The antimicrobial activity of the recombinant adenoregulin was detected after proteolytic cleavage of the fusion protein on cell surface. The validity of the Pichia pastoris cell surface display vectors was proved by the displayed ADR.

  17. Formulation Changes Affect Material Properties and Cell Behavior in HA-Based Hydrogels.

    PubMed

    Lawyer, Thomas; McIntosh, Kristen; Clavijo, Cristian; Potekhina, Lydia; Mann, Brenda K

    2012-01-01

    To develop and optimize new scaffold materials for tissue engineering applications, it is important to understand how changes to the scaffold affect the cells that will interact with that scaffold. In this study, we used a hyaluronic acid- (HA-) based hydrogel as a synthetic extracellular matrix, containing modified HA (CMHA-S), modified gelatin (Gtn-S), and a crosslinker (PEGda). By varying the concentrations of these components, we were able to change the gelation time, enzymatic degradation, and compressive modulus of the hydrogel. These changes also affected fibroblast spreading within the hydrogels and differentially affected the proliferation and metabolic activity of fibroblasts and mesenchymal stem cells (MSCs). In particular, PEGda concentration had the greatest influence on gelation time, compressive modulus, and cell spreading. MSCs appeared to require a longer period of adjustment to the new microenvironment of the hydrogels than fibroblasts. Fibroblasts were able to proliferate in all formulations over the course of two weeks, but MSCs did not. Metabolic activity changed for each cell type during the two weeks depending on the formulation. These results highlight the importance of determining the effect of matrix composition changes on a particular cell type of interest in order to optimize the formulation for a given application.

  18. Engineering micropatterned surfaces to modulate the function of vascular stem cells.

    PubMed

    Li, Jennifer; Wu, Michelle; Chu, Julia; Sochol, Ryan; Patel, Shyam

    2014-02-21

    Multipotent vascular stem cells have been implicated in vascular disease and in tissue remodeling post therapeutic intervention. Hyper-proliferation and calcified extracellular matrix deposition of VSC cause blood vessel narrowing and plaque hardening thereby increasing the risk of myocardial infarct. In this study, to optimize the surface design of vascular implants, we determined whether micropatterned polymer surfaces can modulate VSC differentiation and calcified matrix deposition. Undifferentiated rat VSC were cultured on microgrooved surfaces of varied groove widths, and on micropost surfaces. 10μm microgrooved surfaces elongated VSC and decreased cell proliferation. However, microgrooved surfaces did not attenuate calcified extracellular matrix deposition by VSC cultured in osteogenic media conditions. In contrast, VSC cultured on micropost surfaces assumed a dendritic morphology, were significantly less proliferative, and deposited minimal calcified extracellular matrix. These results have significant implications for optimizing the design of cardiovascular implant surfaces. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Corynebacterium diphtheriae invasion-associated protein (DIP1281) is involved in cell surface organization, adhesion and internalization in epithelial cells

    PubMed Central

    2010-01-01

    Background Corynebacterium diphtheriae, the causative agent of diphtheria, is well-investigated in respect to toxin production, while little is known about C. diphtheriae factors crucial for colonization of the host. In this study, we investigated the function of surface-associated protein DIP1281, previously annotated as hypothetical invasion-associated protein. Results Microscopic inspection of DIP1281 mutant strains revealed an increased size of the single cells in combination with an altered less club-like shape and formation of chains of cells rather than the typical V-like division forms or palisades of growing C. diphtheriae cells. Cell viability was not impaired. Immuno-fluorescence microscopy, SDS-PAGE and 2-D PAGE of surface proteins revealed clear differences of wild-type and mutant protein patterns, which were verified by atomic force microscopy. DIP1281 mutant cells were not only altered in shape and surface structure but completely lack the ability to adhere to host cells and consequently invade these. Conclusions Our data indicate that DIP1281 is predominantly involved in the organization of the outer surface protein layer rather than in the separation of the peptidoglycan cell wall of dividing bacteria. The adhesion- and invasion-negative phenotype of corresponding mutant strains is an effect of rearrangements of the outer surface. PMID:20051108

  20. Bacterial Cell Surface Adsorption of Rare Earth Elements

    NASA Astrophysics Data System (ADS)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  1. Seismic signatures of carbonate caves affected by near-surface absorptions

    NASA Astrophysics Data System (ADS)

    Rao, Ying; Wang, Yanghua

    2015-12-01

    The near-surface absorption within a low-velocity zone generally has an exponential attenuation effect on seismic waves. But how does this absorption affect seismic signatures of karstic caves in deep carbonate reservoirs? Seismic simulation and analysis reveals that, although this near-surface absorption attenuates the wave energy of a continuous reflection, it does not alter the basic kinematic shape of bead-string reflections, a special seismic characteristic associated with carbonate caves in the Tarim Basin, China. Therefore, the bead-strings in seismic profiles can be utilized, with a great certainty, for interpreting the existence of caves within the deep carbonate reservoirs and for evaluating their pore spaces. Nevertheless, the difference between the central frequency and the peak frequency is increased along with the increment in the absorption. While the wave energy of bead-string reflections remains strong, due to the interference of seismic multiples generated by big impedance contrast between the infill materials of a cave and the surrounding carbonate rocks, the central frequency is shifted linearly with respect to the near-surface absorption. These two features can be exploited simultaneously, for a stable attenuation analysis of field seismic data.

  2. Factors affecting the establishment of direct-seeded pine on surface-mine spoils

    Treesearch

    William T. Plass

    1974-01-01

    In a greenhouse study the emergence, survival, and growth of seven species of pine were related to chemical and textural characteristics of 12 Kentucky spoils. The results identify three factors that may affect the establishment of direct-seeded pine on surface-mine spoils. First, fine-textured spoil material may restrict seedling emergence. Coarse-textured sandstones...

  3. Preferential invasion of mitotic cells by Salmonella reveals that cell surface cholesterol is maximal during metaphase.

    PubMed

    Santos, António J M; Meinecke, Michael; Fessler, Michael B; Holden, David W; Boucrot, Emmanuel

    2013-07-15

    Cell surface-exposed cholesterol is crucial for cell attachment and invasion of many viruses and bacteria, including the bacterium Salmonella, which causes typhoid fever and gastroenteritis. Using flow cytometry and 3D confocal fluorescence microscopy, we found that mitotic cells, although representing only 1-4% of an exponentially growing population, were much more efficiently targeted for invasion by Salmonella. This targeting was not dependent on the spherical shape of mitotic cells, but was instead SipB and cholesterol dependent. Thus, we measured the levels of plasma membrane and cell surface cholesterol throughout the cell cycle using, respectively, brief staining with filipin and a fluorescent ester of polyethylene glycol-cholesterol that cannot flip through the plasma membrane, and found that both were maximal during mitosis. This increase was due not only to the rise in global cell cholesterol levels along the cell cycle but also to a transient loss in cholesterol asymmetry at the plasma membrane during mitosis. We measured that cholesterol, but not phosphatidylserine, changed from a ∼2080 outerinner leaflet repartition during interphase to ∼5050 during metaphase, suggesting this was specific to cholesterol and not due to a broad change of lipid asymmetry during metaphase. This explains the increase in outer surface levels that make dividing cells more susceptible to Salmonella invasion and perhaps to other viruses and bacteria entering cells in a cholesterol-dependent manner. The change in cholesterol partitioning also favoured the recruitment of activated ERM (Ezrin, Radixin, Moesin) proteins at the plasma membrane and thus supported mitotic cell rounding.

  4. MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

    PubMed Central

    Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

    2012-01-01

    The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

  5. Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance.

    PubMed

    Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

    2017-06-01

    Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method.

  6. Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

    PubMed Central

    Shanehbandi, Dariush; Majidi, Jafar; Kazemi, Tohid; Baradaran, Behzad; Aghebati-Maleki, Leili; Fathi, Farzaneh; Ezzati Nazhad Dolatabadi, Jafar

    2017-01-01

    Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique. Methods: Two distinct strategies were used for immobilization of the anti-CD20 MAb onto gold (Au) chips. MUA (11-mercaptoundecanoic acid) and Staphylococcus aureus protein A (SpA) were the two systems used for this purpose. A suspension of CD20-positive Raji cells was injected in the analyte phase and the resulting interactions were analyzed and compared to those of MOLT-4 cell line as CD20-negative control. Results: Efficient binding of anti-CD20 MAb to the surface antigens of Raji cell line was confirmed by both immobilizing methods, whereas this MAb had not a noticeable affinity to the MOLT-4 cells. Conclusion: According to the outcomes, the investigated MAb had acceptable affinity and specificity to the target antigens on the cell surface and could be utilized for immuno-detection of CD20-positive intact cells by SPR method. PMID:28761820

  7. Laser-modified titanium surfaces enhance the osteogenic differentiation of human mesenchymal stem cells.

    PubMed

    Bressel, Tatiana A B; de Queiroz, Jana Dara Freires; Gomes Moreira, Susana Margarida; da Fonseca, Jéssyca T; Filho, Edson A; Guastaldi, Antônio Carlos; Batistuzzo de Medeiros, Silvia Regina

    2017-11-28

    Titanium surfaces have been modified by various approaches with the aim of improving the stimulation of osseointegration. Laser beam (Yb-YAG) treatment is a controllable and flexible approach to modifying surfaces. It creates a complex surface topography with micro and nano-scaled patterns, and an oxide layer that can improve the osseointegration of implants, increasing their usefulness as bone implant materials. Laser beam irradiation at various fluences (132, 210, or 235 J/cm 2 ) was used to treat commercially pure titanium discs to create complex surface topographies. The titanium discs were investigated by scanning electron microscopy, X-ray diffraction, and measurement of contact angles. The surface generated at a fluence of 235 J/cm 2 was used in the biological assays. The behavior of mesenchymal stem cells from an umbilical cord vein was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, a mineralization assay, and an alkaline phosphatase activity assay and by carrying out a quantitative real-time polymerase chain reaction for osteogenic markers. CHO-k1 cells were also exposed to titanium discs in the MTT assay. The best titanium surface was that produced by laser beam irradiation at 235 J/cm 2 fluence. Cell proliferation analysis revealed that the CHO-k1 and mesenchymal stem cells behaved differently. The laser-processed titanium surface increased the proliferation of CHO-k1 cells, reduced the proliferation of mesenchymal stem cells, upregulated the expression of the osteogenic markers, and enhanced alkaline phosphatase activity. The laser-treated titanium surface modulated cellular behavior depending on the cell type, and stimulated osteogenic differentiation. This evidence supports the potential use of laser-processed titanium surfaces as bone implant materials, and their use in regenerative medicine could promote better outcomes.

  8. Deletion of Adseverin in Osteoclasts Affects Cell Structure But Not Bone Metabolism.

    PubMed

    Cao, Yixuan; Wang, Yongqiang; Sprangers, Sara; Picavet, Daisy I; Glogauer, Michael; McCulloch, Christopher A; Everts, Vincent

    2017-08-01

    Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP + osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm 2 ; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.

  9. Variation of solar cell sensitivity and solar radiation on tilted surfaces

    NASA Technical Reports Server (NTRS)

    Klucher, T. M.

    1978-01-01

    The validity is studied that one of various insolation models used to compute solar radiation incident on tilted surfaces from global data measured on horizontal surfaces. The variation of solar cell sensitivity to solar radiation is determined over a wide range of atmospheric condition. A new model was formulated that reduced the deviations between measured and predicted insolation to less than 3 percent. Evaluation of solar cell sensitivity data indicates small change (2-3 percent) in sensitivity from winter to summer for tilted cells. The feasibility of using such global data as a means for calibrating terrestrial solar cells is discussed.

  10. Mechanisms of surface runoff genesis on a subsurface drained soil affected by surface crusting: A field investigation

    NASA Astrophysics Data System (ADS)

    Augeard, Bénédicte; Kao, Cyril; Chaumont, Cédric; Vauclin, Michel

    Artificial drainage has been subject to widespread criticism because of its impact on water quality and because there is suspicion that it may have detrimental effects on flood genesis. The present work aims at a better understanding of the mechanisms controlling infiltration and surface runoff genesis, particularly in soils with artificial drainage and affected by surface crusting. A field experiment was conducted during one drainage season (November 2003-March 2004) in the Brie region (80 km east of Paris, France) on a subsurface drained silty soil. Water table elevation and surface runoff were monitored above the drain and at midpoint between drains. Soil water pressure head was measured at various depths and locations between the midpoint and the drain. Soil surface characteristics (microtopography and degree of structural and sedimentary crust development) were recorded regularly on the experimental site and on other plots of various drainage intensities. The results show that the first surface runoff events were induced by high water table. However, runoff was higher at midpoint between the drains because water table reached the soil surface at that point, thus considerably reducing infiltration capacity compared to that above the drain. Comparing different plots, the area with older drainage installation (1948) yielded the most surface runoff. Wider drain spacing, smaller drain depth and possible plugging may have led to a greater area of saturated soil between drains. During the winter period, the impact of raindrops induced the formation of a structural crust on the soil surface. Furthermore, the development of the sedimentary crust, which was favored by water actually flowing on the soil surface during the high water table periods could be correlated with surface runoff volume. The formation of this crust had a significant impact on runoff occurrence at the end of the winter. Therefore, poorly drained fields presented more favorable conditions for both

  11. Nanopatterned polystyrene-b-poly(acrylic acid) surfaces to modulate cell-material interaction.

    PubMed

    Lizundia, Erlantz; Sáenz-Pérez, Míriam; Patrocinio, David; Aurrekoetxea, Iskander; dM Vivanco, Maria; Vilas, José Luis

    2017-06-01

    In this work we explore the effect of surface nanoarchitecture of polystyrene (PS) and polystyrene-b-poly(acrylic acid) (PS-b-PAA) diblock copolymer films on cell viability. PS and PS-b-PAA have been nanopatterned at temperatures of 110, 120 and 140°C using nanoporous aluminium oxide membranes (AAO) as a template. Surface architecture strongly depends on the infiltration temperature and the nature of the infiltrated polymer. High patterning temperatures yield hollow fibre shape architecture at the nanoscale level, which substantially modifies the surface hydrophobicity of the resulting materials. Up to date very scarce reports could be found in the literature dealing with the interaction of microstructured/nanostructured polymeric surfaces with cancer cells. Therefore, MCF-7 breast cancer cells have been selected as a model to conduct cell viability assays. The findings reveal that the fine-tuning of the surface nanoarchitecture contributes to the modification of its biocompatibility. Overall, this study highlights the potential of AAO membranes to obtain well-defined tailored morphologies at nanoscale level and its importance to develop novel soft functional surfaces to be used in the biomedical field. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Increased efficiency with surface texturing in ITO/InP solar cells

    NASA Technical Reports Server (NTRS)

    Jenkins, Phillip; Landis, Geoffrey A.; Fatemi, Navid; Li, Xiaonan; Scheiman, David; Bailey, Sheila

    1992-01-01

    Optimization of an InP solar cell with a V-grooved surface is discussed. Total internal reflection in the coverglass reduces surface reflection and can recover light reflected from the front metallization. Results from the first ITO/InP solar cells on low-angle V-grooved substrates are presented, showing a 5.8 percent increase in current.

  13. Adjustment of surface chemical and physical properties with functionalized polymers to control cell adhesion

    NASA Astrophysics Data System (ADS)

    Zhou, Zhaoli

    Cell-surface interaction is crucial in many cellular functions such as movement, growth, differentiation, proliferation and survival. In the present work, we have developed several strategies to design and prepare synthetic polymeric materials with selected cues to control cell attachment. To promote neuronal cell adhesion on the surfaces, biocompatible, non-adhesive PEG-based materials were modified with neurotransmitter acetylcholine functionalities to produce hydrogels with a range of porous structures, swollen states, and mechanical strengths. Mice hippocampal cells cultured on the hydrogels showed differences in number, length of processes and exhibited different survival rates, thereby highlighting the importance of chemical composition and structure in biomaterials. Similar strategies were used to prepare polymer brushes to assess how topographical cues influence neuronal cell behaviors. The brushes were prepared using the "grown from" method through surface-initiated atom transfer radical polymerization (SI-ATRP) reactions and further patterned via UV photolithography. Protein absorption tests and hippocampal neuronal cell culture of the brush patterns showed that both protein and neuronal cells can adhere to the patterns and therefore can be guided by the patterns at certain length scales. We also prepared functional polymers to discourage attachment of undesirable cells on the surfaces. For example, we synthesized PEG-perfluorinated alkyl amphiphilic surfactants to modify polystyrene-block-poly(ethylene-ran-butylene)- block-polyisoprene (SEBI or K3) triblock copolymers for marine antifouling/fouling release surface coatings. Initial results showed that the polymer coated surfaces can facilitate removal of Ulva sporelings on the surfaces. In addition, we prepared both bioactive and dual functional biopassive/bioactive antimicrobial coatings based on SEBI polymers. Incubating the polymer coated surfaces with gram-positive bacteria (S. aureus), gram

  14. Tetraploid cells from cytokinesis failure induce aneuploidy and spontaneous transformation of mouse ovarian surface epithelial cells.

    PubMed

    Lv, Lei; Zhang, Tianwei; Yi, Qiyi; Huang, Yun; Wang, Zheng; Hou, Heli; Zhang, Huan; Zheng, Wei; Hao, Qiaomei; Guo, Zongyou; Cooke, Howard J; Shi, Qinghua

    2012-08-01

    Most ovarian cancers originate from the ovarian surface epithelium and are characterized by aneuploid karyotypes. Aneuploidy, a consequence of chromosome instability, is an early event during the development of ovarian cancers. However, how aneuploid cells are evolved from normal diploid cells in ovarian cancers remains unknown. In the present study, cytogenetic analyses of a mouse syngeneic ovarian cancer model revealed that diploid mouse ovarian surface epithelial cells (MOSECs) experienced an intermediate tetraploid cell stage, before evolving to aneuploid (mainly near-tetraploid) cells. Using long-term live-cell imaging followed by fluorescence in situ hybridization (FISH), we demonstrated that tetraploid cells originally arose from cytokinesis failure of bipolar mitosis in diploid cells, and gave rise to aneuploid cells through chromosome mis-segregation during both bipolar and multipolar mitoses. Injection of the late passage aneuploid MOSECs resulted in tumor formation in C57BL/6 mice. Therefore, we reveal a pathway for the evolution of diploid to aneuploid MOSECs and elucidate a mechanism for the development of near-tetraploid ovarian cancer cells.

  15. How chimeric antigen receptor design affects adoptive T cell therapy

    PubMed Central

    Gacerez, Albert T.; Arellano, Benjamine; Sentman, Charles L.

    2016-01-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR’s function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. PMID:27163336

  16. Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

    PubMed Central

    Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

    2005-01-01

    Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

  17. Co-precipitation of DEAE-dextran coated SPIONs: how synthesis conditions affect particle properties, stem cell labelling and MR contrast.

    PubMed

    Barrow, Michael; Taylor, Arthur; García Carrión, Jaime; Mandal, Pranab; Park, B Kevin; Poptani, Harish; Murray, Patricia; Rosseinsky, Matthew J; Adams, Dave J

    2016-09-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used as contrast agents for stem cell tracking using magnetic resonance imaging (MRI). The total mass of iron oxide that can be internalised into cells without altering their viability or phenotype is an important criterion for the generation of contrast, with SPIONs designed for efficient labelling of stem cells allowing for an increased sensitivity of detection. Although changes in the ratio of polymer and iron salts in co-precipitation reactions are known to affect the physicochemical properties of SPIONs, particularly core size, the effects of these synthesis conditions on stem cell labelling and magnetic resonance (MR) contrast have not been established. Here, we synthesised a series of cationic SPIONs with very similar hydrodynamic diameters and surface charges, but different polymer content. We have investigated how the amount of polymer in the co-precipitation reaction affects core size and modulates not only the magnetic properties of the SPIONs but also their uptake into stem cells. SPIONs with the largest core size and lowest polymer content presented the highest magnetisation and relaxivity. These particles also had the greatest uptake efficiency without any deleterious effect on either the viability or function of the stem cells. However, for all particles internalised in cells, the T 2 and T 2 * relaxivity was independent of the SPION's core size. Our results indicate that the relative mass of iron taken up by cells is the major determinant of MR contrast generation and suggest that the extent of SPION uptake can be regulated by the amount of polymer used in co-precipitation reactions. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

    PubMed

    Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

    2010-07-26

    Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

  19. Pooled Protein Immunization for Identification of Cell Surface Antigens in Streptococcus sanguinis

    PubMed Central

    Ge, Xiuchun; Kitten, Todd; Munro, Cindy L.; Conrad, Daniel H.; Xu, Ping

    2010-01-01

    Background Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. Methods and Findings We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. Conclusions The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases. PMID:20668678

  20. Effect of silica nanoparticles with variable size and surface functionalization on human endothelial cell viability and angiogenic activity

    NASA Astrophysics Data System (ADS)

    Guarnieri, Daniela; Malvindi, Maria Ada; Belli, Valentina; Pompa, Pier Paolo; Netti, Paolo

    2014-02-01

    Silica nanoparticles could be promising delivery vehicles for drug targeting or gene therapy. However, few studies have been undertaken to determine the biological behavior effects of silica nanoparticles on primary endothelial cells. Here we investigated uptake, cytotoxicity and angiogenic properties of silica nanoparticle with positive and negative surface charge and sizes ranging from 25 to 115 nm in primary human umbilical vein endothelial cells. Dynamic light scattering measurements and nanoparticle tracking analysis were used to estimate the dispersion status of nanoparticles in cell culture media, which was a key aspect to understand the results of the in vitro cellular uptake experiments. Nanoparticles were taken up by primary endothelial cells in a size-dependent manner according to their degree of agglomeration occurring after transfer in cell culture media. Functionalization of the particle surface with positively charged groups enhanced the in vitro cellular uptake, compared to negatively charged nanoparticles. However, this effect was contrasted by the tendency of particles to form agglomerates, leading to lower internalization efficiency. Silica nanoparticle uptake did not affect cell viability and cell membrane integrity. More interestingly, positively and negatively charged 25 nm nanoparticles did not influence capillary-like tube formation and angiogenic sprouting, compared to controls. Considering the increasing interest in nanomaterials for several biomedical applications, a careful study of nanoparticle-endothelial cells interactions is of high relevance to assess possible risks associated to silica nanoparticle exposure and their possible applications in nanomedicine as safe and effective nanocarriers for vascular transport of therapeutic agents.

  1. Endosomal acidification by Na+/H+ exchanger NHE5 regulates TrkA cell-surface targeting and NGF-induced PI3K signaling

    PubMed Central

    Diering, Graham H.; Numata, Yuka; Fan, Steven; Church, John; Numata, Masayuki

    2013-01-01

    To facilitate polarized vesicular trafficking and signal transduction, neuronal endosomes have evolved sophisticated mechanisms for pH homeostasis. NHE5 is a member of the Na+/H+ exchanger family and is abundantly expressed in neurons and associates with recycling endosomes. Here we show that NHE5 potently acidifies recycling endosomes in PC12 cells. NHE5 depletion by plasmid-based short hairpin RNA significantly reduces cell surface abundance of TrkA, an effect similar to that observed after treatment with the V-ATPase inhibitor bafilomycin. A series of cell-surface biotinylation experiments suggests that anterograde trafficking of TrkA from recycling endosomes to plasma membrane is the likeliest target affected by NHE5 depletion. NHE5 knockdown reduces phosphorylation of Akt and Erk1/2 and impairs neurite outgrowth in response to nerve growth factor (NGF) treatment. Of interest, although both phosphoinositide 3-kinase–Akt and Erk signaling are activated by NGF-TrkA, NGF-induced Akt-phosphorylation appears to be more sensitively affected by perturbed endosomal pH. Furthermore, NHE5 depletion in rat cortical neurons in primary culture also inhibits neurite formation. These results collectively suggest that endosomal pH modulates trafficking of Trk-family receptor tyrosine kinases, neurotrophin signaling, and possibly neuronal differentiation. PMID:24006492

  2. [Cell surface peroxidase--generator of superoxide anion in wheat root cells under wound stress].

    PubMed

    Chasov, A V; Gordon, L Kh; Kolesnikov, O P; Minibaeva, F V

    2002-01-01

    Development of wound stress in excised wheat roots is known to be accompanied with an increase in reactive oxygen species (ROS) production, fall of membrane potential, release of K+ from cells, alkalization of extracellular solution, changes in respiration and metabolism of structural lipids. Dynamics of superoxide release correlates with changes in other physiological parameters, indicating the cross-reaction of these processes. Activity of peroxidase in extracellular solution after a 1 h incubation and removal of roots was shown to be stimulated by the range of organic acids, detergents, metals, and to be inhibited by cyanide. Superoxide production was sensitive to the addition of Mn2+ and H2O2. Increase in superoxide production correlates with the enhancement of peroxidase activity at the application of organic acids and detergents. The results obtained indicate that cell surface peroxidase is one of the main generators of superoxide in wounded wheat root cells. Different ways of stimulation of the ROS producing activity in root cells is supposed. By controlling superoxide and hydrogen peroxide formation, the cell surface peroxidase can control the adaptation processes in stressed plant cells.

  3. How Escherichia coli lands and forms cell clusters on a surface: a new role of surface topography

    PubMed Central

    Gu, Huan; Chen, Aaron; Song, Xinran; Brasch, Megan E.; Henderson, James H.; Ren, Dacheng

    2016-01-01

    Bacterial response to surface topography during biofilm formation was studied using 5 μm tall line patterns of poly (dimethylsiloxane) (PDMS). Escherichia coli cells attached on top of protruding line patterns were found to align more perpendicularly to the orientation of line patterns when the pattern narrowed. Consistently, cell cluster formation per unit area on 5 μm wide line patterns was reduced by 14-fold compared to flat PDMS. Contrasting the reduced colony formation, cells attached on narrow patterns were longer and had higher transcriptional activities, suggesting that such unfavorable topography may present a stress to attached cells. Results of mutant studies indicate that flagellar motility is involved in the observed preference in cell orientation on narrow patterns, which was corroborated by the changes in cell rotation pattern before settling on different surface topographies. These findings led to a set of new design principles for creating antifouling topographies, which was validated using 10 μm tall hexagonal patterns. PMID:27412365

  4. The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

    PubMed

    Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

    2015-01-01

    Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Advances in the theory and application of BSF cells. [Back Surface Field solar cells

    NASA Technical Reports Server (NTRS)

    Mandelkorn, J.; Lamneck, J. H.

    1975-01-01

    A study to determine the influence of fabrication processes and bulk material properties on the behavior of back surface field (BSF) cells is reported. It is concluded that a photovoltage is generated at the p(+), p back junction of the cell. The concept of majority carrier collection is proposed as a possible mechanism for this generation. Advantages accruing to the advent of BSF cells are outlined.

  6. Cellular membrane enrichment of self-assembling D-peptides for cell surface engineering.

    PubMed

    Wang, Huaimin; Wang, Youzhi; Han, Aitian; Cai, Yanbin; Xiao, Nannan; Wang, Ling; Ding, Dan; Yang, Zhimou

    2014-06-25

    We occasionally found that several self-assembling peptides containing D-amino acids would be preferentially enriched in cellular membranes at self-assembled stages while distributed evenly in the cytoplasma of cells at unassembled stages. Self-assembling peptides containing only Lamino acids distributed evenly in cytoplasma of cells at both self-assembled and unassembled stages. The self-assembling peptides containing D-amino acids could therefore be applied for engineering cell surface with peptides. More importantly, by integrating a protein binding peptide (a PDZ domain binding hexapeptide of WRESAI) with the self-assembling peptide containing D-amino acids, protein could also be introduced to the cell surface. This study not only provided a novel approach to engineer cell surface, but also highlighted the unusual properties and potential applications of self-assembling peptides containing D-amino acids in regenerative medicine, drug delivery, and tissue engineering.

  7. Osteoconductive phosphoserine-modified poly(ε-lysine) dendrons: synthesis, titanium oxide surface functionalization and response of osteoblast-like cell lines

    PubMed Central

    Meikle, S. T.; Bianchi, G.; Olivier, G.; Santin, M.

    2013-01-01

    The lack of direct bonding between the surface of an implant and the mineralized bony tissue is among the main causes of aseptic loosening in titanium-based implants. Surface etching and ceramic coatings have led to improved osteointegration, but their clinical performance is still limited either by partial bonding or by coating delamination. In this work, a solid-phase synthesis method has been optimized to produce poly(ε-lysine) dendrons, the outermost branching generation of which is functionalized by phosphoserine (PS), a known catalyst of the biomineralization process. The dendrons were deposited onto etched titanium oxide surfaces as a near-to-monolayer film able to induce the formation of a homogeneous calcium phosphate phase in a simulated body fluid over 3 days. The dendron films also stimulated MG63 and SAOS-2 osteoblast-like cells to proliferate at a rate significantly higher than etched titanium, with SAOS-2 also showing a higher degree of differentiation over 14 days. PS-tethered dendron films were not affected by various sterilization methods and UV treatment appeared to improve the cell substrate potential of these films, thus suggesting their potential as a surface functionalization method for bone implants. PMID:23193106

  8. Natural bactericidal surfaces: mechanical rupture of Pseudomonas aeruginosa cells by cicada wings.

    PubMed

    Ivanova, Elena P; Hasan, Jafar; Webb, Hayden K; Truong, Vi Khanh; Watson, Gregory S; Watson, Jolanta A; Baulin, Vladimir A; Pogodin, Sergey; Wang, James Y; Tobin, Mark J; Löbbe, Christian; Crawford, Russell J

    2012-08-20

    Natural superhydrophobic surfaces are often thought to have antibiofouling potential due to their self-cleaning properties. However, when incubated on cicada wings, Pseudomonas aeruginosa cells are not repelled; instead they are penetrated by the nanopillar arrays present on the wing surface, resulting in bacterial cell death. Cicada wings are effective antibacterial, as opposed to antibiofouling, surfaces. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Coupling Binding to Catalysis: Using Yeast Cell Surface Display to Select Enzymatic Activities.

    PubMed

    Zhang, Keya; Bhuripanyo, Karan; Wang, Yiyang; Yin, Jun

    2015-01-01

    We find yeast cell surface display can be used to engineer enzymes by selecting the enzyme library for high affinity binding to reaction intermediates. Here we cover key steps of enzyme engineering on the yeast cell surface including library design, construction, and selection based on magnetic and fluorescence-activated cell sorting.

  10. Zwitterion-Coated Iron Oxide Nanoparticles: Surface Chemistry and Intracellular Uptake by Hepatocarcinoma (HepG2) Cells.

    PubMed

    Mondini, Sara; Leonzino, Marianna; Drago, Carmelo; Ferretti, Anna M; Usseglio, Sandro; Maggioni, Daniela; Tornese, Paolo; Chini, Bice; Ponti, Alessandro

    2015-07-07

    Nanoparticles (NPs) have received much attention in recent years for their diverse potential biomedical applications. However, the synthesis of NPs with desired biodistribution and pharmacokinetics is still a major challenge, with NP size and surface chemistry being the main factors determining the behavior of NPs in vivo. Here we report on the surface chemistry and in vitro cellular uptake of magnetic iron oxide NPs coated with zwitterionic dopamine sulfonate (ZDS). ZDS-coated NPs were compared to similar iron oxide NPs coated with PEG-like 2-[2-(2-methoxyethoxy)ethoxy]acetic acid (MEEA) to investigate how surface chemistry affects their in vitro behavior. ZDS-coated NPs had a very dense coating, guaranteeing high colloidal stability in several aqueous media and negligible interaction with proteins. Treatment of HepG2 cells with increasing doses (2.5-100 μg Fe/mL) of ZDS-coated iron oxide NPs had no effect on cell viability and resulted in a low, dose-dependent NP uptake, inferior than most reported data for the internalization of iron oxide NPs by HepG2 cells. MEEA-coated NPs were scarcely stable and formed micrometer-sized aggregates in aqueous media. They decreased cell viability for dose ≥50 μg Fe/mL, and were more efficiently internalized than ZDS-coated NPs. In conclusion, our data indicate that the ZDS layer prevented both aggregation and sedimentation of iron oxide NPs and formed a biocompatible coating that did not display any biocorona effect. The very low cellular uptake of ZDS-coated iron NPs can be useful to achieve highly selective targeting upon specific functionalization.

  11. How Chimeric Antigen Receptor Design Affects Adoptive T Cell Therapy.

    PubMed

    Gacerez, Albert T; Arellano, Benjamine; Sentman, Charles L

    2016-12-01

    Chimeric antigen receptor (CAR) T cells have been developed to treat tumors and have shown great success against B cell malignancies. Exploiting modular designs and swappable domains, CARs can target an array of cell surface antigens and, upon receptor-ligand interactions, direct signaling cascades, thereby driving T cell effector functions. CARs have been designed using receptors, ligands, or scFv binding domains. Different regions of a CAR have each been found to play a role in determining the overall efficacy of CAR T cells. Therefore, this review provides an overview of CAR construction and common designs. Each CAR region is discussed in the context of its importance to a CAR's function. Additionally, the review explores how various engineering strategies have been applied to CAR T cells in order to regulate CAR T cell function and activity. J. Cell. Physiol. 231: 2590-2598, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. Surface code—biophysical signals for apoptotic cell clearance

    NASA Astrophysics Data System (ADS)

    Biermann, Mona; Maueröder, Christian; Brauner, Jan M.; Chaurio, Ricardo; Janko, Christina; Herrmann, Martin; Muñoz, Luis E.

    2013-12-01

    Apoptotic cell death and the clearance of dying cells play an important and physiological role in embryonic development and normal tissue turnover. In contrast to necrosis, apoptosis proceeds in an anti-inflammatory manner. It is orchestrated by the timed release and/or exposure of so-called ‘find-me’, ‘eat me’ and ‘tolerate me’ signals. Mononuclear phagocytes are attracted by various ‘find-me’ signals, including proteins, nucleotides, and phospholipids released by the dying cell, whereas the involvement of granulocytes is prevented via ‘stay away’ signals. The exposure of anionic phospholipids like phosphatidylserine (PS) by apoptotic cells on the outer leaflet of the plasma membrane is one of the main ‘eat me’ signals. PS is recognized by a number of innate receptors as well as by soluble bridging molecules on the surface of phagocytes. Importantly, phagocytes are able to discriminate between viable and apoptotic cells both exposing PS. Due to cytoskeleton remodeling PS has a higher lateral mobility on the surfaces of apoptotic cells thereby promoting receptor clustering on the phagocyte. PS not only plays an important role in the engulfment process, but also acts as ‘tolerate me’ signal inducing the release of anti-inflammatory cytokines by phagocytes. An efficient and fast clearance of apoptotic cells is required to prevent secondary necrosis and leakage of intracellular danger signals into the surrounding tissue. Failure or prolongation of the clearance process leads to the release of intracellular antigens into the periphery provoking inflammation and development of systemic inflammatory autoimmune disease like systemic lupus erythematosus. Here we review the current findings concerning apoptosis-inducing pathways, important players of apoptotic cell recognition and clearance as well as the role of membrane remodeling in the engulfment of apoptotic cells by phagocytes.

  13. Microbial cell budgets of an Arctic glacier surface quantified using flow cytometry.

    PubMed

    Irvine-Fynn, T D L; Edwards, A; Newton, S; Langford, H; Rassner, S M; Telling, J; Anesio, A M; Hodson, A J

    2012-11-01

    Uncertainty surrounds estimates of microbial cell and organic detritus fluxes from glacier surfaces. Here, we present the first enumeration of biological particles draining from a supraglacial catchment, on Midtre Lovénbreen (Svalbard) over 36 days. A stream cell flux of 1.08 × 10(7)  cells m(-2)  h(-1) was found, with strong inverse, non-linear associations between water discharge and biological particle concentrations. Over the study period, a significant decrease in cell-like particles exhibiting 530 nm autofluorescence was noted. The observed total fluvial export of ~7.5 × 10(14) cells equates to 15.1-72.7 g C, and a large proportion of these cells were small (< 0.5 μm in diameter). Differences between the observed fluvial export and inputs from ice-melt and aeolian deposition were marked: results indicate an apparent storage rate of 8.83 × 10(7)  cells m(-2)  h(-1). Analysis of surface ice cores revealed cell concentrations comparable to previous studies (6 × 10(4)  cells ml(-1)) but, critically, showed no variation with depth in the uppermost 1 m. The physical retention and growth of particulates at glacier surfaces has two implications: to contribute to ice mass thinning through feedbacks altering surface albedo, and to potentially seed recently deglaciated terrain with cells, genes and labile organic matter. This highlights the merit of further study into glacier surface hydraulics and biological processes. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  14. Monitoring cell culture media degradation using surface enhanced Raman scattering (SERS) spectroscopy.

    PubMed

    Calvet, Amandine; Ryder, Alan G

    2014-08-20

    The quality of the cell culture media used in biopharmaceutical manufacturing is a crucial factor affecting bioprocess performance and the quality of the final product. Due to their complex composition these media are inherently unstable, and significant compositional variations can occur particularly when in the prepared liquid state. For example photo-degradation of cell culture media can have adverse effects on cell viability and thus process performance. There is therefore, from quality control, quality assurance and process management view points, an urgent demand for the development of rapid and inexpensive tools for the stability monitoring of these complex mixtures. Spectroscopic methods, based on fluorescence or Raman measurements, have now become viable alternatives to more time-consuming and expensive (on a unit analysis cost) chromatographic and/or mass spectrometry based methods for routine analysis of media. Here we demonstrate the application of surface enhanced Raman scattering (SERS) spectroscopy for the simple, fast, analysis of cell culture media degradation. Once stringent reproducibility controls are implemented, chemometric data analysis methods can then be used to rapidly monitor the compositional changes in chemically defined media. SERS shows clearly that even when media are stored at low temperature (2-8°C) and in the dark, significant chemical changes occur, particularly with regard to cysteine/cystine concentration. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Use of upscaled elevation and surface roughness data in two-dimensional surface water models

    USGS Publications Warehouse

    Hughes, J.D.; Decker, J.D.; Langevin, C.D.

    2011-01-01

    In this paper, we present an approach that uses a combination of cell-block- and cell-face-averaging of high-resolution cell elevation and roughness data to upscale hydraulic parameters and accurately simulate surface water flow in relatively low-resolution numerical models. The method developed allows channelized features that preferentially connect large-scale grid cells at cell interfaces to be represented in models where these features are significantly smaller than the selected grid size. The developed upscaling approach has been implemented in a two-dimensional finite difference model that solves a diffusive wave approximation of the depth-integrated shallow surface water equations using preconditioned Newton–Krylov methods. Computational results are presented to show the effectiveness of the mixed cell-block and cell-face averaging upscaling approach in maintaining model accuracy, reducing model run-times, and how decreased grid resolution affects errors. Application examples demonstrate that sub-grid roughness coefficient variations have a larger effect on simulated error than sub-grid elevation variations.

  16. Surface grafting of a thermoplastic polyurethane with methacrylic acid by previous plasma surface activation and by ultraviolet irradiation to reduce cell adhesion.

    PubMed

    Alves, P; Pinto, S; Kaiser, Jean-Pierre; Bruinink, Arie; de Sousa, Hermínio C; Gil, M H

    2011-02-01

    The material performance, in a biological environment, is mainly mediated by its surface properties and by the combination of chemical, physical, biological, and mechanical properties required, for a specific application. In this study, the surface of a thermoplastic polyurethane (TPU) material (Elastollan(®)1180A50) was activated either by plasma or by ultra-violet (UV) irradiation. After surface activation, methacrylic acid (MAA) was linked to the surface of TPU in order to improve its reactivity and to reduce cell adhesion. Grafted surfaces were evaluated by X-ray photoelectron spectroscopy (XPS), by atomic force microscopy (AFM) and by contact angle measurements. Blood compatibility studies and cell adhesion tests with human bone marrow cells (HBMC) were also performed. If was found that UV grafting method led to better results than the plasma activation method, since cell adhesion was reduced when methacrylic acid was grafted to the TPU surface by UV. Copyright © 2010 Elsevier B.V. All rights reserved.

  17. MMSET deregulation affects cell cycle progression and adhesion regulons in t(4;14) myeloma plasma cells

    PubMed Central

    Brito, Jose L.R.; Walker, Brian; Jenner, Matthew; Dickens, Nicholas J.; Brown, Nicola J.M.; Ross, Fiona M.; Avramidou, Athanasia; Irving, Julie A.E.; Gonzalez, David; Davies, Faith E.; Morgan, Gareth J.

    2009-01-01

    Background The recurrent immunoglobulin translocation, t(4;14)(p16;q32) occurs in 15% of multiple myeloma patients and is associated with poor prognosis, through an unknown mechanism. The t(4;14) up-regulates fibroblast growth factor receptor 3 (FGFR3) and multiple myeloma SET domain (MMSET) genes. The involvement of MMSET in the pathogenesis of t(4;14) multiple myeloma and the mechanism or genes deregulated by MMSET upregulation are still unclear. Design and Methods The expression of MMSET was analyzed using a novel antibody. The involvement of MMSET in t(4;14) myelomagenesis was assessed by small interfering RNA mediated knockdown combined with several biological assays. In addition, the differential gene expression of MMSET-induced knockdown was analyzed with expression microarrays. MMSET gene targets in primary patient material was analyzed by expression microarrays. Results We found that MMSET isoforms are expressed in multiple myeloma cell lines, being exclusively up-regulated in t(4;14)-positive cells. Suppression of MMSET expression affected cell proliferation by both decreasing cell viability and cell cycle progression of cells with the t(4;14) translocation. These findings were associated with reduced expression of genes involved in the regulation of cell cycle progression (e.g. CCND2, CCNG1, BRCA1, AURKA and CHEK1), apoptosis (CASP1, CASP4 and FOXO3A) and cell adhesion (ADAM9 and DSG2). Furthermore, we identified genes involved in the latter processes that were differentially expressed in t(4;14) multiple myeloma patient samples. Conclusions In conclusion, dysregulation of MMSET affects the expression of several genes involved in the regulation of cell cycle progression, cell adhesion and survival. PMID:19059936

  18. Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.

    PubMed

    Arpornmaeklong, Premjit; Pripatnanont, Prisana; Chookiatsiri, Chonticha; Tangtrakulwanich, Boonsin

    This study aimed to investigate the effects of titanium surface topography and simvastatin on growth and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in estrogen-deprived (ED) cell culture. Human BMSCs were seeded on cell culture plates, smooth-surface titanium (Ti) disks, and sandblasted with large grits and acid etched (SLA)-surface Ti disks; and subsequently cultured in regular (fetal bovine serum [FBS]), ED, and ED-with 100 nM simvastatin (ED-SIM) culture media for 14 to 21 days. Live/dead cell staining, scanning electron microscope examination, and cell viability assay were performed to determine cell attachment, morphology, and growth. Expression levels of osteoblast-associated genes, Runx2 and bone sialoprotein and levels of alkaline phosphatase (ALP) activity, calcium content, and osteocalcin in culture media were measured to determine osteoblastic differentiation. Expression levels of bone morphogenetic protein-2 (BMP-2) were investigated to examine stimulating effects of simvastatin (n = 4 to 5, mean ± SD). In vitro mineralization was verified by calcein staining. Human BMSCs exhibited different attachment and shapes on smooth and SLA titanium surfaces. Estrogen-deprived cell culture decreased cell attachment and growth, particularly on the SLA titanium surface, but cells were able to grow to reach confluence on day 21 in the ED-osteogenic (OS) culture medium. Promoting effects of the SLA titanium surface in ED-OS were significantly decreased. Simvastatin significantly increased osteogenic differentiation of human BMSCs on the SLA titanium surface in the ED-OS medium, and the promoting effects of simvastatin corresponded with the increasing of BMP-2 gene expression on the SLA titanium surface in ED-OS-SIM culture medium. The ED cell culture model provided a well-defined platform for investigating the effects of hormones and growth factors on cells and titanium surface interaction. Titanium, the SLA surface, and simvastatin

  19. CXCR3 surface expression in human airway epithelial cells: cell cycle dependence and effect on cell proliferation.

    PubMed

    Aksoy, Mark O; Yang, Yi; Ji, Rong; Reddy, P J; Shahabuddin, Syed; Litvin, Judith; Rogers, Thomas J; Kelsen, Steven G

    2006-05-01

    We recently demonstrated that human bronchial epithelial cells (HBEC) constitutively express the CXC chemokine receptor CXCR3, which when activated, induces directed cell migration. The present study in HBEC examined the relative expression of the CXCR3 splice variants CXCR3-A and -B, cell cycle dependence of CXCR3 expression, and the effects of the CXCR3 ligand, the interferon-gamma-inducible CXC chemokine I-TAC/CXCL11, on DNA synthesis and cell proliferation. Both CXCR3-A and -B mRNA, assessed by real-time RT-PCR, were expressed in normal HBEC (NHBEC) and the HBEC line 16-HBE. However, CXCR3-B mRNA was 39- and 6-fold greater than CXCR3-A mRNA in NHBEC and 16-HBE, respectively. Although most HBEC (>80%) assessed by flow cytometry and immunofluorescence microscopy contained intracellular CXCR3, only a minority (<40%) expressed it on the cell surface. In this latter subset of cells, most (>75%) were in the S + G(2)/M phases of the cell cycle. Stimulation of CXCR3 with I-TAC enhanced thymidine incorporation and cell proliferation and increased p38 and ERK1/2 phosphorylation. These data indicate that 1) human airway epithelial cells primarily express CXCR3-B mRNA, 2) surface expression of CXCR3 is largely confined to the S + G(2)/M phases of the cell cycle, and 3) activation of CXCR3 induces DNA synthesis, cell proliferation, and activation of MAPK pathways. We speculate that activation of CXCR3 exerts a mitogenic effect in HBEC, which may be important during airway mucosal injury in obstructive airway diseases such as asthma and chronic obstructive pulmonary disease.

  20. Behaviour of human endothelial cells on surface modified NiTi alloy.

    PubMed

    Plant, Stuart D; Grant, David M; Leach, Lopa

    2005-09-01

    Intravascular stents are being designed which utilise the shape memory properties of NiTi alloy. Despite the clinical advantages afforded by these stents their application has been limited by concerns about the large nickel ion content of the alloy. In this study, the surface chemistry of NiTi alloy was modified by mechanical polishing and oxidising heat treatments and subsequently characterised using X-ray photon spectroscopy (XPS). The effect of these surfaces on monolayer formation and barrier integrity of human umbilical vein endothelial cells (HUVEC) was then assessed by confocal imaging of the adherens junctional molecule VE-cadherin, perijunctional actin and permeability to 42kDa dextrans. Dichlorofluoroscein assays were used to measure oxidative stress in the cells. XPS analysis of NiTi revealed its surface to be dominated by TiO(2). However, where oxidation had occurred after mechanical polishing or post polishing heat treatments at 300 and 400 degrees C in air, a significant amount of metallic nickel or nickel oxide species (10.5 and 18.5 at%) remained on the surface. Exposure of HUVECs to these surfaces resulted in increased oxidative stress within the cells, loss of VE-cadherin and F-actin and significantly increased paracellular permeability. These pathological phenomena were not found in cells grown on NiTi which had undergone heat treatment at 600 degrees C. At this temperature thickening of the TiO(2) layer had occurred due to diffusion of titanium ions from the bulk of the alloy, displacing nickel ions to sub-surface areas. This resulted in a significant reduction in nickel ions detectable on the sample surface (4.8 at%). This study proposes that the integrity of human endothelial monolayers on NiTi is dependent upon the surface chemistry of the alloy and that this can be manipulated, using simple oxidising heat treatments.

  1. Chronic alcohol exposure affects the cell components involved in membrane traffic in neuronal dendrites.

    PubMed

    Romero, Ana M; Renau-Piqueras, Jaime; Marín, M Pilar; Esteban-Pretel, Guillermo

    2015-01-01

    The specific traffic of the membrane components in neurons is a major requirement to establish and maintain neuronal domains-the axonal and the somatodendritic domains-and their polarized morphology. Unlike axons, dendrites contain membranous organelles, which are involved in the secretory pathway, including the endoplasmic reticulum, the Golgi apparatus and post-Golgi apparatus carriers, the cytoskeleton, and plasma membrane. A variety of molecules and factors are also involved in this process. Previous studies have shown that chronic alcohol exposure negatively affects several of these cell components, such as the Golgi apparatus or cytoskeleton in neurons. Yet very little information is available on the possible effects of this exposure on the remaining cell elements involved in intracellular trafficking in neurons, particularly in dendrites. By qualitative and quantitative electron microscopy, immunofluorescence and immunoblotting, we herein show that chronic exposure to moderate levels (30 mM) of ethanol in cultured neurons reduces the volume and surface density of the rough endoplasmic reticulum, and increases the levels of GRP78, a chaperone involved in endoplasmic reticulum stress. Ethanol also significantly diminishes the proportion of neurons that show an extension of Golgi into dendrites and dendritic Golgi outposts, a structure present exclusively in longer, thicker apical dendrites. Both Golgi apparatus types were also fragmented into a large number of cells. We also investigated the effect of alcohol on the levels of microtubule-based motor proteins KIF5, KIF17, KIFC2, dynein, and myosin IIb, responsible for transporting different cargoes in dendrites. Of these, alcohol differently affects several of them by lowering dynein and raising KIF5, KIFC2, and myosin IIb. These results, together with other previously published ones, suggest that practically all the protein trafficking steps in dendrites are altered to a greater or lesser extent by chronic

  2. Potential Role of Induced Pluripotent Stem Cells (IPSCs) for Cell-Based Therapy of the Ocular Surface

    PubMed Central

    Casaroli-Marano, Ricardo P.; Nieto-Nicolau, Núria; Martínez-Conesa, Eva M.; Edel, Michael; Álvarez-Palomo, Ana B.

    2015-01-01

    The integrity and normal function of the corneal epithelium are crucial for maintaining the cornea’s transparency and vision. The existence of a cell population with progenitor characteristics in the limbus maintains a dynamic of constant epithelial repair and renewal. Currently, cell-based therapies for bio replacement—cultured limbal epithelial transplantation (CLET) and cultured oral mucosal epithelial transplantation (COMET)—present very encouraging clinical results for treating limbal stem cell deficiency (LSCD) and restoring vision. Another emerging therapeutic approach consists of obtaining and implementing human progenitor cells of different origins in association with tissue engineering methods. The development of cell-based therapies using stem cells, such as human adult mesenchymal or induced pluripotent stem cells (IPSCs), represent a significant breakthrough in the treatment of certain eye diseases, offering a more rational, less invasive, and better physiological treatment option in regenerative medicine for the ocular surface. This review will focus on the main concepts of cell-based therapies for the ocular surface and the future use of IPSCs to treat LSCD. PMID:26239129

  3. Atrial natriuretic peptide degradation by CPA47 cells: evidence for a divalent cation-independent cell-surface proteolytic activity.

    PubMed

    Frost, S J; Chen, Y M; Whitson, P A

    1992-11-23

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88% of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41% of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  4. Atrial natriuretic peptide degradation by CPA47 cells - Evidence for a divalent cation-independent cell-surface proteolytic activity

    NASA Technical Reports Server (NTRS)

    Frost, S. J.; Chen, Y. M.; Whitson, P. A.

    1992-01-01

    Atrial natriuretic peptide (ANP) is rapidly cleared and degraded in vivo. Nonguanylate-cyclase receptors (C-ANPR) and a metalloproteinase, neutral endopeptidase (EC 3.4.24.11) (NEP 24.11), are thought to be responsible for its metabolism. We investigated the mechanisms of ANP degradation by an endothelial-derived cell line, CPA47. CPA47 cells degraded 88 percent of 125I-ANP after 1 h at 37 degrees C as determined by HPLC. Medium preconditioned by these cells degraded 41 percent of the 125I-ANP, and this activity was inhibited by a divalent cation chelator, EDTA. Furthermore, a cell-surface proteolytic activity degraded 125I-ANP in the presence of EDTA when receptor-mediated endocytosis was inhibited either by low temperature (4 degrees C) or by hyperosmolarity at 37 degrees C. The metalloproteinase, NEP 24.11, is unlikely to be the cell-surface peptidase because 125I-ANP is degraded by CPA47 cells at 4 degrees C in the presence of 5 mM EDTA. These data indicate that CPA47 cells can degrade ANP by a novel divalent cation-independent cell-surface proteolytic activity.

  5. Recent advances in yeast cell-surface display technologies for waste biorefineries.

    PubMed

    Liu, Zhuo; Ho, Shih-Hsin; Hasunuma, Tomohisa; Chang, Jo-Shu; Ren, Nan-Qi; Kondo, Akihiko

    2016-09-01

    Waste biorefinery aims to maximize the output of value-added products from various artificial/agricultural wastes by using integrated bioprocesses. To make waste biorefinery economically feasible, it is thus necessary to develop a low-cost, environment-friendly technique to perform simultaneous biodegradation and bioconversion of waste materials. Cell-surface display engineering is a novel, cost-effective technique that can auto-immobilize proteins on the cell exterior of microorganisms, and has been applied for use with waste biofinery. Through tethering different enzymes (e.g., cellulase, lipase, and protease) or metal-binding peptides on cell surfaces, various yeast strains can effectively produce biofuels and biochemicals from sugar/protein-rich waste materials, catalyze waste oils into biodiesels, or retrieve heavy metals from wastewater. This review critically summarizes recent applications of yeast cell-surface display on various types of waste biorefineries, highlighting its potential and future challenges with regard to commercializing this technology. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Variation of solar cell sensitivity and solar radiation on tilted surfaces

    NASA Technical Reports Server (NTRS)

    Klucher, T. M.

    1978-01-01

    An empirical study was performed (1) to evaluate the validity of various insolation models used to compute solar radiation incident on tilted surfaces from global data measured on horizontal surfaces and (2) to determine the variation of solar cell sensitivity to solar radiation over a wide range of atmospheric condition. Evaluation of the insolation data indicates that the isotropic sky model of Liu and Jordan underestimates the amount of solar radiation falling on tilted surfaces by as much as 10%. An anisotropic-clear-sky model proposed by Temps and Coulson was also evaluated and found to be deficient under cloudy conditions. A new model, formulated herein, reduced the deviations between measured and predicted insolation to less than 3%. Evaluation of solar cell sensitivity data indicates small change (2-3%) in sensitivity from winter to summer for tilted cells. The feasibility of using such global data as a means for calibrating terrestrial solar cells as done by Treble is discussed.

  7. Activation of Cell Surface Bound 20S Proteasome Inhibits Vascular Cell Growth and Arteriogenesis

    PubMed Central

    Ito, Wulf D.; Lund, Natalie; Zhang, Ziyang; Buck, Friedrich; Lellek, Heinrich; Horst, Andrea; Machens, Hans-Günther; Schunkert, Heribert; Schaper, Wolfgang; Meinertz, Thomas

    2015-01-01

    Arteriogenesis is an inflammatory process associated with rapid cellular changes involving vascular resident endothelial progenitor cells (VR-EPCs). Extracellular cell surface bound 20S proteasome has been implicated to play an important role in inflammatory processes. In our search for antigens initially regulated during collateral growth mAb CTA 157-2 was generated against membrane fractions of growing collateral vessels. CTA 157-2 stained endothelium of growing collateral vessels and the cell surface of VR-EPCs. CTA 157-2 bound a protein complex (760 kDa) that was identified as 26 kDa α7 and 21 kDa β3 subunit of 20S proteasome in mass spectrometry. Furthermore we demonstrated specific staining of 20S proteasome after immunoprecipitation of VR-EPC membrane extract with CTA 157-2 sepharose beads. Functionally, CTA 157-2 enhanced concentration dependently AMC (7-amino-4-methylcoumarin) cleavage from LLVY (N-Succinyl-Leu-Leu-Val-Tyr) by recombinant 20S proteasome as well as proteasomal activity in VR-EPC extracts. Proliferation of VR-EPCs (BrdU incorporation) was reduced by CTA 157-2. Infusion of the antibody into the collateral circulation reduced number of collateral arteries, collateral proliferation, and collateral conductance in vivo. In conclusion our results indicate that extracellular cell surface bound 20S proteasome influences VR-EPC function in vitro and collateral growth in vivo. PMID:26146628

  8. Effect of Atmospheric Plasma Treatment to Titanium Surface on Initial Osteoblast-Like Cell Spreading. .

    PubMed

    Kim, In-Hye; Son, Jun-Sik; Kwon, Tae-Yub; Kim, Kyo-Han

    2015-01-01

    Plasma treatments are becoming a popular method for modifying the characteristics of a range of substrate surfaces. Atmospheric pressure plasma is cost-efficient, safe and simple compared to high-pressure plasma. This study examined the effects of atmospheric pressure plasma to a titanium (Ti) surface on osteoblast-like cell (osteoblast) spreading and cellular networks. The characteristics of the Ti surface before and after the atmospheric plasma treatment were analyzed by X-ray photoemission spectroscopy (XPS), scanning electron microscopy (SEM), contact angle measurements, and an optical 3D profiling system. The morphology of osteoblasts attached to the Ti surfaces was observed by SEM and confocal laser scanning microscopy. The atmospheric pressure plasma made the Ti surfaces more hydrophilic. The osteoblasts that adhered to the untreated surface were round and spherical, whereas the cells covered a larger surface area on the plasma-treated surface. The plasma-treated Ti surface showed enhanced cell spreading and migration with more developed cellular networks. In conclusion, an atmospheric plasma treatment is a potential surface modifying method that can enhance the initial the cell affinity at the early stages in vitro.

  9. Guard cells elongate: relationship of volume and surface area during stomatal movement.

    PubMed

    Meckel, Tobias; Gall, Lars; Semrau, Stefan; Homann, Ulrike; Thiel, Gerhard

    2007-02-01

    Stomata in the epidermis of photosynthetically active plant organs are formed by pairs of guard cells, which create a pore, to facilitate CO2 and water exchange with the environment. To control this gas exchange, guard cells actively change their volume and, consequently, surface area to alter the aperture of the stomatal pore. Due to the limited elasticity of the plasma membrane, such changes in surface area require an exocytic addition or endocytic retrieval of membrane during stomatal movement. Using confocal microscopic data, we have reconstructed detailed three-dimensional models of open and closed stomata to precisely quantify the necessary area to be exo- and endocytosed by the guard cells. Images were obtained under a strong emphasis on a precise calibration of the method and by avoiding unphysiological osmotical imbalance, and hence osmocytosis. The data reveal that guard cells of Vicia faba L., whose aperture increases by 111.89+/-22.39%, increase in volume and surface area by 24.82+/-6.26% and 14.99+/-2.62%, respectively. In addition, the precise volume to surface area relationship allows quantitative modeling of the three-dimensional changes. While the major volume change is caused by a slight increase in the cross section of the cells, an elongation of the guard cells achieves the main aperture change.

  10. Intentional formation of a protein corona on nanoparticles: Serum concentration affects protein corona mass, surface charge, and nanoparticle-cell interaction.

    PubMed

    Gräfe, Christine; Weidner, Andreas; Lühe, Moritz V D; Bergemann, Christian; Schacher, Felix H; Clement, Joachim H; Dutz, Silvio

    2016-06-01

    The protein corona, which immediately is formed after contact of nanoparticles and biological systems, plays a crucial role for the biological fate of nanoparticles. In the here presented study we describe a strategy to control the amount of corona proteins which bind on particle surface and the impact of such a protein corona on particle-cell interactions. For corona formation, polyethyleneimine (PEI) coated magnetic nanoparticles (MNP) were incubated in a medium consisting of fetal calf serum (FCS) and cell culture medium. To modulate the amount of proteins bind to particles, the composition of the incubation medium was varied with regard to the FCS content. The protein corona mass was estimated and the size distribution of the participating proteins was determined by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Additionally, the zeta potential of incubated particles was measured. Human blood-brain barrier-representing cell line HBMEC was used for in vitro incubation experiments. To investigate the consequences of the FCS dependent protein corona formation on the interaction of MNP and cells flow cytometry and laser scanning microscopy were used. Zeta potential as well as SDS-PAGE clearly reveal an increase in the amount of corona proteins on MNP with increasing amount of FCS in incubation medium. For MNP incubated with lower FCS concentrations especially medium-sized proteins of molecular weights between 30kDa and 100kDa could be found within the protein corona, whereas for MNP incubated within higher FCS concentrations the fraction of corona proteins of 30kDa and less increased. The presence of the protein corona reduces the interaction of PEI-coated MNP with HBMEC cells within a 30min-incubation. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Galectin-3 modulates the polarized surface delivery of β1-integrin in epithelial cells.

    PubMed

    Hönig, Ellena; Ringer, Karina; Dewes, Jenny; von Mach, Tobias; Kamm, Natalia; Kreitzer, Geri; Jacob, Ralf

    2018-05-10

    Epithelial cells require a precise intracellular transport and sorting machinery in order to establish and maintain their polarized architecture. This machinery includes beta-galactoside binding galectins for glycoprotein targeting to the apical membrane. Galectin-3 sorts cargo destined for the apical plasma membrane into vesicular carriers. After delivery of cargo to the apical milieu, galectin-3 recycles back into sorting organelles. We analyzed the role of galectin-3 in the polarized distribution of β1-integrin in MDCK cells. Integrins are located primarily at the basolateral domain of epithelial cells. We demonstrate that a minor pool of β1-integrin interacts with galectin-3 at the apical plasma membrane. Knockdown of galectin-3 decreases apical delivery of β1-integrin. This loss is restored by supplementation with recombinant galectin-3 and galectin-3 overexpression. Our data suggest that galectin-3 targets newly synthesized β1-integrin to the apical membrane and promotes apical delivery of β1-integrin internalized from the basolateral membrane. In parallel, galectin-3 knockout results in a reduction in cell proliferation and an impairment in proper cyst development. Our results suggest that galectin-3 modulates the surface distribution of β1-integrin and affects the morphogenesis of polarized cells. © 2018. Published by The Company of Biologists Ltd.

  12. DEVELOPMENT OF A MODEL TO INVESTIGATE RED BLOOD CELL SURFACE CHARACTERISTICS AFTER CRYOPRESERVATION.

    PubMed

    Gordiyenko, O I; Anikieieva, M O; Rozanova, S L; Kovalenko, S Ye; Kovalenkol, I F; Gordiyenko, E O

    2015-01-01

    Maintaining cell surface properties after freezing and thawing, characterized in particular by the surface potential and associated with it cell ability to intercellular adhesion, could be used as a characteristic of successful cryopreservation. This study was conducted to research applying different erythrocytes freezing modes and analyses the regimes cryopreservation effect on the cell surface charge and adhesion to microorganisms. Human erythrocytes frozen by three modes. In order to determine adhesion index was used dried bacterial cells of S. thermophilus. The surface charge of erythrocytes was evaluated using Alcian blue cationic dye. The results showed the significant decrease in the lactobacillus adhesion to erythrocytes frozen glycerol and 1,2-propanediol. After erythrocytes were freezen with glycerol and 1,2-propanediol, the cationic dye binding to erythrocytes significantly reduced. AB binding to erythrocytes frozen with PEG-1500 does not differ from control data. Erythrocytes frozen with PEG-1500 mantained surface properties after thawing better, compared to erythrocytes cryopreserved by other methods.

  13. Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming.

    PubMed

    No, Jin-Gu; Choi, Mi-Kyung; Kwon, Dae-Jin; Yoo, Jae Gyu; Yang, Byoung-Chul; Park, Jin-Ki; Kim, Dong-Hoon

    2015-01-01

    Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot(TM) reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.

  14. Response of MG63 osteoblast-like cells onto polycarbonate membrane surfaces with different micropore sizes.

    PubMed

    Lee, Sang Jin; Choi, Jin San; Park, Ki Suk; Khang, Gilson; Lee, Young Moo; Lee, Hai Bang

    2004-08-01

    Response of different types of cells on materials is important for the applications of tissue engineering and regenerative medicine. It is recognized that the behavior of the cell adhesion, proliferation, and differentiation on materials depends largely on surface characteristics such as wettability, chemistry, charge, rigidity, and roughness. In this study, we examined the behavior of MG63 osteoblast-like cells cultured on a polycarbonate (PC) membrane surfaces with different micropore sizes (0.2-8.0 microm in diameter). Cell adhesion and proliferation to the PC membrane surfaces were determined by cell counting and MTT assay. The effect of surface micropore on the MG63 cells was evaluated by cell morphology, protein content, and alkaline phosphatase (ALP) specific activity. It seems that the cell adhesion and proliferation were progressively inhibited as the PC membranes had micropores with increasing size, probably due to surface discontinuities produced by track-etched pores. Increasing micropore size of the PC membrane results in improved protein synthesis and ALP specific activity in isolated cells. There was a statistically significant difference (P<0.05) between different micropore sizes. The MG63 cells also maintained their phenotype under conditions that support a round cell shape. RT-PCR analysis further confirmed the osteogenic phenotype of the MG63 cells onto the PC membranes with different micropore sizes. In results, as micropore size is getting larger, cell number is reduced and cell differentiation and matrix production is increased. This study demonstrated that the surface topography plays an important role for phenotypic expression of the MG63 osteoblast-like cells.

  15. On physical changes on surface of human cervical epithelial cells during cancer transformations

    NASA Astrophysics Data System (ADS)

    Sokolov, Igor; Dokukin, Maxim; Guz, Nataliia; Woodworth, Craig

    2013-03-01

    Physical changes of the cell surface of cells during transformation from normal to cancerous state are rather poorly studied. Here we describe our recent studies of such changes done on human cervical epithelial cells during their transformation from normal through infected with human papillomavirus type-16 (HPV-16), immortalized (precancerous), to cancerous cells. The changes were studied with the help of atomic force microscopy (AFM) and through the measurement of physical adhesion of fluorescent silica beads to the cell surface. Based on the adhesion experiments, we clearly see the difference in nonspecific adhesion which occurs at the stage of immortalization of cells, precancerous cells. The analysis done with the help of AFM shows that the difference observed comes presumably from the alteration of the cellular ``brush,'' a layer that surrounds cells and which consists of mostly microvilli, microridges, and glycocalyx. Further AFM analysis reveals the emergence of fractal scaling behavior on the surface of cells when normal cells turn into cancerous. The possible causes and potential significance of these observations will be discussed.

  16. Effects of surface active agents on DNAPL migration and distribution in saturated porous media.

    PubMed

    Cheng, Zhou; Gao, Bin; Xu, Hongxia; Sun, Yuanyuan; Shi, Xiaoqing; Wu, Jichun

    2016-11-15

    Dissolved surface active agents such as surfactant and natural organic matter can affect the distribution and fate of dense nonaqueous liquids (DNAPLs) in soil and groundwater systems. This work investigated how two common groundwater surface active agents, humic acid (HA) and Tween 80, affected tetrachloroethylene (PCE) migration and source zone architecture in saturated porous media under environmentally relevant conditions. Batch experiments were first conducted to measure the contact angles and interfacial tensions (IFT) between PCE and quartz surface in water containing different amount of surface active agents. Results showed that the contact angle increased and IFT decreased with concentration of surface active agent increasing, and Tween 80 was much more effective than HA. Five 2-D flow cell experiments were then conducted. Correspondingly, Tween 80 showed strong effects on the migration and distribution of PCE in the porous media due to its ability to change the medium wettability from water-wet into intermediate/NAPL-wet. The downward migration velocities of the PCE in three Tween 80 cells were slower than those in the other two cells. In addition, the final saturation of the PCE in the cells containing surface active agents was higher than that in the water-only cell. Results from this work indicate that the presence of surface active agents in groundwater may strongly affect the fate and distribution of DNAPL through altering porous medium wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Effect of surface modification on protein retention and cell proliferation under strain.

    PubMed

    Dunkers, J P; Lee, H-J; Matos, M A; Pakstis, L M; Taboas, J M; Hudson, S D; Cicerone, M T

    2011-07-01

    When culturing cells on flexible surfaces, it is important to consider extracellular matrix treatments that will remain on the surface under mechanical strain. Here we investigate differences in laminin deposited on oxidized polydimethylsiloxane (PDMS) with plasma treatment (plasma-only) vs. plasma and aminopropyltrimethoxysilane treatment (silane-linked). We use specular X-ray reflectivity (SXR), transmission electron microscopy (TEM), and immunofluorescence to probe the quantity and uniformity of laminin. The surface coverage of laminin is approximately 45% for the plasma-only and 50% for the silane-linked treatment as determined by SXR. TEM and immunofluorescence reveal additional islands of laminin aggregates on the plasma-only PDMS compared with the relatively smooth and uniform silane-linked laminin surface. We also examine laminin retention under strain and vascular smooth muscle cell viability and proliferation under static and strain conditions. Equibiaxial stretching of the PDMS surfaces shows greatly improved retention of the silane-linked laminin over plasma-only. There are significantly more cells on the silane-linked surface after 4 days of equibiaxial strain. Published by Elsevier Ltd.

  18. Analysis of the interaction of an electron beam with back surface field solar cells

    NASA Technical Reports Server (NTRS)

    Von Roos, O.; Luke, K. L.

    1983-01-01

    In this paper the short circuit current Isc induced by the electron beam of a scanning electron microscope in a back surface field solar cell will be determined theoretically. It will be shown that, in a configuration used previously for solar cells with an ohmic back surface, the Isc gives a convenient means for estimating the back surface recombination velocities and thus the quality of back surface field cells. Numerical data will be presented applicable to a point source model for the electron-hole pair generation.

  19. Three-dimensional manipulation of single cells using surface acoustic waves

    PubMed Central

    Guo, Feng; Mao, Zhangming; Chen, Yuchao; Xie, Zhiwei; Lata, James P.; Li, Peng; Ren, Liqiang; Liu, Jiayang; Yang, Jian; Dao, Ming; Suresh, Subra; Huang, Tony Jun

    2016-01-01

    The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving “acoustic tweezers” in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner. PMID:26811444

  20. Three-dimensional manipulation of single cells using surface acoustic waves.

    PubMed

    Guo, Feng; Mao, Zhangming; Chen, Yuchao; Xie, Zhiwei; Lata, James P; Li, Peng; Ren, Liqiang; Liu, Jiayang; Yang, Jian; Dao, Ming; Suresh, Subra; Huang, Tony Jun

    2016-02-09

    The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving "acoustic tweezers" in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner.

  1. Plant cell division is specifically affected by nitrotyrosine

    PubMed Central

    Jovanović, Aleksandra M.; Durst, Steffen; Nick, Peter

    2010-01-01

    Virtually all eukaryotic α-tubulins harbour a C-terminal tyrosine that can be reversibly removed and religated, catalysed by a specific tubulin–tyrosine carboxypeptidase (TTC) and a specific tubulin–tyrosine ligase (TTL), respectively. The biological function of this post-translational modification has remained enigmatic. 3-nitro-L-tyrosine (nitrotyrosine, NO2Tyr), can be incorporated into detyrosinated α-tubulin instead of tyrosine, producing irreversibly nitrotyrosinated α-tubulin. To gain insight into the possible function of detyrosination, the effect of NO2Tyr has been assessed in two plant model organisms (rice and tobacco). NO2Tyr causes a specific, sensitive, and dose-dependent inhibition of cell division that becomes detectable from 1 h after treatment and which is not observed with non-nitrosylated tyrosine. These effects are most pronounced in cycling tobacco BY-2 cells, where the inhibition of cell division is accompanied by a stimulation of cell length, and a misorientation of cross walls. NO2Tyr reduces the abundance of the detyrosinated form of α-tubulin whereas the tyrosinated α-tubulin is not affected. These findings are discussed with respect to a model where NO2Tyr is accepted as substrate by TTL and subsequently blocks TTC activity. The irreversibly tyrosinated α-tubulin impairs microtubular functions that are relevant to cell division in general, and cell wall deposition in particular. PMID:20018903

  2. Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

    PubMed Central

    2011-01-01

    Background Several naturally occurring cationic antimicrobial peptides (CAPs), including bovine lactoferricin (LfcinB), display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS) on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS) on their cytotoxic activity. Methods Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides' affinity for HS and CS were also investigated. Results The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. Conclusions In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS. PMID:21453492

  3. Surfaceome and Proteosurfaceome in Parietal Monoderm Bacteria: Focus on Protein Cell-Surface Display

    PubMed Central

    Desvaux, Mickaël; Candela, Thomas; Serror, Pascale

    2018-01-01

    The cell envelope of parietal monoderm bacteria (archetypal Gram-positive bacteria) is formed of a cytoplasmic membrane (CM) and a cell wall (CW). While the CM is composed of phospholipids, the CW is composed at least of peptidoglycan (PG) covalently linked to other biopolymers, such as teichoic acids, polysaccharides, and/or polyglutamate. Considering the CW is a porous structure with low selective permeability contrary to the CM, the bacterial cell surface hugs the molecular figure of the CW components as a well of the external side of the CM. While the surfaceome corresponds to the totality of the molecules found at the bacterial cell surface, the proteinaceous complement of the surfaceome is the proteosurfaceome. Once translocated across the CM, secreted proteins can either be released in the extracellular milieu or exposed at the cell surface by associating to the CM or the CW. Following the gene ontology (GO) for cellular components, cell-surface proteins at the CM can either be integral (GO: 0031226), i.e., the integral membrane proteins, or anchored to the membrane (GO: 0046658), i.e., the lipoproteins. At the CW (GO: 0009275), cell-surface proteins can be covalently bound, i.e., the LPXTG-proteins, or bound through weak interactions to the PG or wall polysaccharides, i.e., the cell wall binding proteins. Besides monopolypeptides, some proteins can associate to each other to form supramolecular protein structures of high molecular weight, namely the S-layer, pili, flagella, and cellulosomes. After reviewing the cell envelope components and the different molecular mechanisms involved in protein attachment to the cell envelope, perspectives in investigating the proteosurfaceome in parietal monoderm bacteria are further discussed. PMID:29491848

  4. Display of a β-mannanase and a chitosanase on the cell surface of Lactobacillus plantarum towards the development of whole-cell biocatalysts.

    PubMed

    Nguyen, Hoang-Minh; Mathiesen, Geir; Stelzer, Elena Maria; Pham, Mai Lan; Kuczkowska, Katarzyna; Mackenzie, Alasdair; Agger, Jane W; Eijsink, Vincent G H; Yamabhai, Montarop; Peterbauer, Clemens K; Haltrich, Dietmar; Nguyen, Thu-Ha

    2016-10-04

    Lactobacillus plantarum is considered as a potential cell factory because of its GRAS (generally recognized as safe) status and long history of use in food applications. Its possible applications include in situ delivery of proteins to a host, based on its ability to persist at mucosal surfaces of the human intestine, and the production of food-related enzymes. By displaying different enzymes on the surface of L. plantarum cells these could be used as whole-cell biocatalysts for the production of oligosaccharides. In this present study, we aimed to express and display a mannanase and a chitosanase on the cell surface of L. plantarum. ManB, a mannanase from Bacillus licheniformis DSM13, and CsnA, a chitosanase from Bacillus subtilis ATCC 23857 were fused to different anchoring motifs of L. plantarum for covalent attachment to the cell surface, either via an N-terminal lipoprotein anchor (Lp_1261) or a C-terminal cell wall anchor (Lp_2578), and the resulting fusion proteins were expressed in L. plantarum WCFS1. The localization of the recombinant proteins on the bacterial cell surface was confirmed by flow cytometry and immunofluorescence microscopy. The highest mannanase and chitosanase activities obtained for displaying L. plantarum cells were 890 U and 1360 U g dry cell weight, respectively. In reactions with chitosan and galactomannans, L. plantarum CsnA- and ManB-displaying cells produced chito- and manno-oligosaccharides, respectively, as analyzed by high performance anion exchange chromatography (HPAEC) and mass spectrometry (MS). Surface-displayed ManB is able to break down galactomannan (LBG) into smaller manno-oligosaccharides, which can support growth of L. plantarum. This study shows that mannanolytic and chitinolytic enzymes can be anchored to the cell surface of L. plantarum in active forms. L. plantarum chitosanase- and mannanase-displaying cells should be of interest for the production of potentially 'prebiotic' oligosaccharides. This approach

  5. In Azospirillum brasilense, mutations in flmA or flmB genes affect polar flagellum assembly, surface polysaccharides, and attachment to maize roots.

    PubMed

    Rossi, Fernando Ariel; Medeot, Daniela Beatriz; Liaudat, Juan Pablo; Pistorio, Mariano; Jofré, Edgardo

    2016-09-01

    Azospirillum brasilense is a soil bacterium capable of promoting plant growth. Several surface components were previously reported to be involved in the attachment of A. brasilense to root plants. Among these components are the exopolysaccharide (EPS), lipopolysaccharide (LPS) and the polar flagellum. Flagellin from polar flagellum is glycosylated and it was suggested that genes involved in such a posttranslational modification are the same ones involved in the biosynthesis of sugars present in the O-antigen of the LPS. In this work, we report on the characterization of two homologs present in A. brasilense Cd, to the well characterized flagellin modification genes, flmA and flmB, from Aeromonas caviae. We show that mutations in either flmA or flmB genes of A. brasilense resulted in non-motile cells due to alterations in the polar flagellum assembly. Moreover, these mutations also affected the capability of A. brasilense cells to adsorb to maize roots and to produce LPS and EPS. By generating a mutant containing the polar flagellum affected in their rotation, we show the importance of the bacterial motility for the early colonization of maize roots. Copyright © 2016 Elsevier GmbH. All rights reserved.

  6. Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically

    PubMed Central

    Peter, Beatrix; Farkas, Eniko; Forgacs, Eniko; Saftics, Andras; Kovacs, Boglarka; Kurunczi, Sandor; Szekacs, Inna; Csampai, Antal; Bosze, Szilvia; Horvath, Robert

    2017-01-01

    The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity. PMID:28186133

  7. Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically.

    PubMed

    Peter, Beatrix; Farkas, Eniko; Forgacs, Eniko; Saftics, Andras; Kovacs, Boglarka; Kurunczi, Sandor; Szekacs, Inna; Csampai, Antal; Bosze, Szilvia; Horvath, Robert

    2017-02-10

    The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity.

  8. Green tea polyphenol tailors cell adhesivity of RGD displaying surfaces: multicomponent models monitored optically

    NASA Astrophysics Data System (ADS)

    Peter, Beatrix; Farkas, Eniko; Forgacs, Eniko; Saftics, Andras; Kovacs, Boglarka; Kurunczi, Sandor; Szekacs, Inna; Csampai, Antal; Bosze, Szilvia; Horvath, Robert

    2017-02-01

    The interaction of the anti-adhesive coating, poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its Arg-Gly-Asp (RGD) functionalized form, PLL-g-PEG-RGD, with the green tea polyphenol, epigallocatechin-gallate (EGCg) was in situ monitored. After, the kinetics of cellular adhesion on the EGCg exposed coatings were recorded in real-time. The employed plate-based waveguide biosensor is applicable to monitor small molecule binding and sensitive to sub-nanometer scale changes in cell membrane position and cell mass distribution; while detecting the signals of thousands of adhering cells. The combination of this remarkable sensitivity and throughput opens up new avenues in testing complicated models of cell-surface interactions. The systematic studies revealed that, despite the reported excellent antifouling properties of the coatings, EGCg strongly interacted with them, and affected their cell adhesivity in a concentration dependent manner. Moreover, the differences between the effects of the fresh and oxidized EGCg solutions were first demonstrated. Using a semiempirical quantumchemical method we showed that EGCg binds to the PEG chains of PLL-g-PEG-RGD and effectively blocks the RGD sites by hydrogen bonds. The calculations supported the experimental finding that the binding is stronger for the oxidative products. Our work lead to a new model of polyphenol action on cell adhesion ligand accessibility and matrix rigidity.

  9. Micromechanical and surface adhesive properties of single saccharomyces cerevisiae cells

    NASA Astrophysics Data System (ADS)

    Farzi, Bahman; Cetinkaya, Cetin

    2017-09-01

    The adhesion and mechanical properties of a biological cell (e.g. cell membrane elasticity and adhesiveness) are often strong indicators for the state of its health. Many existing techniques for determining mechanical properties of cells require direct physical contact with a single cell or a group of cells. Physical contact with the cell can trigger complex mechanotransduction mechanisms, leading to cellular responses, and consequently interfering with measurement accuracy. In the current work, based on ultrasonic excitation and interferometric (optical) motion detection, a non-contact method for characterizing the adhesion and mechanical properties of single cells is presented. It is experimentally demonstrated that the rocking (rigid body) motion and internal vibrational resonance frequencies of a single saccharomyces cerevisiae (SC) (baker’s yeast) cell can be acquired with the current approach, and the Young’s modulus and surface tension of the cell membrane as well as surface adhesion energy can be extracted from the values of these acquired resonance frequencies. The detected resonance frequency ranges for single SC cells include a rocking (rigid body) frequency of 330  ±  70 kHz and two breathing resonance frequencies of 1.53  ±  0.12 and 2.02  ±  0.31 MHz. Based on these values, the average work-of-adhesion of SC cells on a silicon substrate in aqueous medium is extracted, for the first time, as WASC-Si=16.2+/- 3.8 mJ {{m}-2} . Similarly, the surface tension and the Young’s modulus of the SC cell wall are predicted as {{σ }SC}=0.16+/- 0.02 N {{m}-1} and {{E}SC}= 9.20  ±  2.80 MPa, respectively. These results are compared to those reported in the literature by utilizing various methods, and good agreements are found. The current approach eliminates the measurement inaccuracies associated with the physical contact. Exciting and detecting cell dynamics at micro-second time-scales is significantly faster than the

  10. Affinity adsorption of cells to surfaces and strategies for cell detachment.

    PubMed

    Hubble, John

    2007-01-01

    The use of bio-specific interactions for the separation and recovery of bio-molecules is now widely established and in many cases the technique has successfully crossed the divide between bench and process scale operation. Although the major specificity advantage of affinity-based separations also applies to systems intended for cell fractionation, developments in this area have been slower. Many of the problems encountered result from attempts to take techniques developed for molecular systems and, with only minor modification to the conditions used, apply them for the separation of cells. This approach tends to ignore or at least trivialise the problems, which arise from the heterogeneous nature of a cell suspension and the multivalent nature of the cell/surface interaction. To develop viable separation processes on a larger scale, effective contacting strategies are required in separators that also allow detachment or recovery protocols that overcome the enhanced binding strength generated by multivalent interactions. The effects of interaction valency on interaction strength needs to be assessed and approaches developed to allow effective detachment and recovery of adsorbed cells without compromising cell viability. This article considers the influence of operating conditions on cell attachment and the extent to which multivalent interactions determine the strength of cell binding and subsequent detachment.

  11. Targeting Host Cell Surface Nucleolin for RSV Therapy: Challenges and Opportunities.

    PubMed

    Mastrangelo, Peter; Norris, Michael J; Duan, Wenming; Barrett, Edward G; Moraes, Theo J; Hegele, Richard G

    2017-09-19

    Nucleolin (NCL) has been reported as a cellular receptor for the human respiratory syncytial virus (RSV). We studied the effects of re-purposing AS1411, an anti-cancer compound that binds cell surface NCL, as a possible novel strategy for RSV therapy in vitro and in vivo. AS1411 was administered to RSV-infected cultures of non-polarized (HEp-2) and polarized (MDCK) epithelial cells and to virus-infected mice and cotton rats. Results of in vitro experiments showed that AS1411, used in micromolar concentrations, was associated with decreases in the number of virus-positive cells. Intranasal administration of AS1411 (50 mg/kg) to RSV-infected mice and cotton rats was associated with partial reductions in lung viral titers, decreased virus-associated airway inflammation, and decreased IL-4/IFN-γ ratios when compared to untreated, infected animals. In conclusion, our findings indicate that therapeutic use of AS1411 has modest effects on RSV replication and host response. While the results underscore the challenges of targeting cell surface NCL as a potential novel strategy for RSV therapy, they also highlight the potential of cell surface NCL as a therapeutic target.

  12. Basigin-2 Is a Cell Surface Receptor for Soluble Basigin Ligand*S⃞

    PubMed Central

    Belton, Robert J.; Chen, Li; Mesquita, Fernando S.; Nowak, Romana A.

    2008-01-01

    The metastatic spread of a tumor is dependent upon the ability of the tumor to stimulate surrounding stromal cells to express enzymes required for tissue remodeling. The immunoglobulin superfamily protein basigin (EMMPRIN/CD147) is a cell surface glycoprotein expressed by tumor cells that stimulates matrix metalloproteinase and vascular endothelial growth factor expression in stromal cells. The ability of basigin to stimulate expression of molecules involved in tissue remodeling and angiogenesis makes basigin a potential target for the development of strategies to block metastasis. However, the identity of the cell surface receptor for basigin remains controversial. The goal of this study was to determine the identity of the receptor for basigin. Using a novel recombinant basigin protein (rBSG) corresponding to the extracellular domain of basigin, it was demonstrated that the native, nonglycosylated rBSG protein forms dimers in solution. Furthermore, rBSG binds to the surface of uterine fibroblasts, activates the ERK1/2 signaling pathway, and induces expression of matrix metalloproteinases 1, 2, and 3. Proteins that interact with rBSG were isolated using a biotin label transfer technique and sequenced by matrix-assisted laser desorption ionization tandem mass spectrophotometry. The results demonstrate that rBSG interacts with basigin expressed on the surface of fibroblasts and is subsequently internalized. During internalization, rBSG associates with a novel form of human basigin (basigin-3). It was concluded that cell surface basigin functions as a membrane receptor for soluble basigin and this homophilic interaction is not dependent upon glycosylation of the basigin ligand. PMID:18434307

  13. Versatile aptasensor for electrochemical quantification of cell surface glycan and naked-eye tracking glycolytic inhibition in living cells.

    PubMed

    Zhang, Jing-Jing; Cheng, Fang-Fang; Zheng, Ting-Ting; Zhu, Jun-Jie

    2017-03-15

    Quantifying the glycan expression status on cell surfaces is of vital importance for insight into the glycan function in biological processes and related diseases. Here we developed a versatile aptasensor for electrochemical quantification of cell surface glycan by taking advantage of the cell-specific aptamer, and the lectin-functionalized gold nanoparticles acting as both a glycan recognition unit and a signal amplification probe. To construct the aptasensor, amine-functionalized mucin 1 protein (MUC1) aptamer was first covalently conjugated to carboxylated-magnetic beads (MBs) using the succinimide coupling (EDC-NHS) method. On the basis of the specific recognition between aptamer and MUC1 protein that overexpressed on the surface of MCF-7 cells, the aptamer conjugated MBs showed a predominant capability for cell capture with high selectivity. Moreover, a lectin-based nanoprobe was designed by noncovalent assembly of concanavalin A (ConA) on gold nanoparticles (AuNPs). This nanoprobe incorporated the abilities of both the specific carbohydrate recognition and the signal amplification based on the gold-promoted reduction of silver ions. By coupling with electrochemical stripping analysis, the proposed sandwich-type cytosensor showed an excellent analytical performance for the ultrasensitive detection of MCF-7 cells and quantification of cell surface glycan. More importantly, taking advantage of Con A-gold nanoprobe catalyzed silver enhancement, the proposed method was further used for naked-eye tracking glycolytic inhibition in living cells. This aptasensor holds great promise as a new point-of-care diagnostic tool for analyzing glycan expression on living cells and further helps cancer diagnosis and treatment. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. CD22 is a recycling receptor that can shuttle cargo between the cell surface and endosomal compartments of B cells.

    PubMed

    O'Reilly, Mary K; Tian, Hua; Paulson, James C

    2011-02-01

    CD22 is a member of the sialic acid-binding Ig-like lectin (Siglec) family that is known to be a regulator of B cell signaling. Its B cell-specific expression makes it an attractive target for immunotoxin-mediated B cell depletion therapy for the treatment of B cell lymphomas and autoimmune diseases. Although CD22 is well documented to be an endocytic receptor, it is believed that after internalization, it is targeted for degradation. We show in this study that CD22 is instead constitutively recycled to the cell surface. We also find that glycan ligand-based cargo is released from CD22 and accumulates intracellularly as CD22 recycles between the cell surface and endosomal compartments. In contrast, Abs to CD22 do not accumulate but remain bound to CD22 and recycle to the cell surface. The results have implications for development of agents that target CD22 as an endocytic receptor for delivery of cytotoxic cargo to B cells.

  15. Simple surface engineering of polydimethylsiloxane with polydopamine for stabilized mesenchymal stem cell adhesion and multipotency

    PubMed Central

    Chuah, Yon Jin; Koh, Yi Ting; Lim, Kaiyang; Menon, Nishanth V.; Wu, Yingnan; Kang, Yuejun

    2015-01-01

    Polydimethylsiloxane (PDMS) has been extensively exploited to study stem cell physiology in the field of mechanobiology and microfluidic chips due to their transparency, low cost and ease of fabrication. However, its intrinsic high hydrophobicity renders a surface incompatible for prolonged cell adhesion and proliferation. Plasma-treated or protein-coated PDMS shows some improvement but these strategies are often short-lived with either cell aggregates formation or cell sheet dissociation. Recently, chemical functionalization of PDMS surfaces has proved to be able to stabilize long-term culture but the chemicals and procedures involved are not user- and eco-friendly. Herein, we aim to tailor greener and biocompatible PDMS surfaces by developing a one-step bio-inspired polydopamine coating strategy to stabilize long-term bone marrow stromal cell culture on PDMS substrates. Characterization of the polydopamine-coated PDMS surfaces has revealed changes in surface wettability and presence of hydroxyl and secondary amines as compared to uncoated surfaces. These changes in PDMS surface profile contribute to the stability in BMSCs adhesion, proliferation and multipotency. This simple methodology can significantly enhance the biocompatibility of PDMS-based microfluidic devices for long-term cell analysis or mechanobiological studies. PMID:26647719

  16. The Biological Function of the Prion Protein: A Cell Surface Scaffold of Signaling Modules.

    PubMed

    Linden, Rafael

    2017-01-01

    The prion glycoprotein (PrP C ) is mostly located at the cell surface, tethered to the plasma membrane through a glycosyl-phosphatydil inositol (GPI) anchor. Misfolding of PrP C is associated with the transmissible spongiform encephalopathies (TSEs), whereas its normal conformer serves as a receptor for oligomers of the β-amyloid peptide, which play a major role in the pathogenesis of Alzheimer's Disease (AD). PrP C is highly expressed in both the nervous and immune systems, as well as in other organs, but its functions are controversial. Extensive experimental work disclosed multiple physiological roles of PrP C at the molecular, cellular and systemic levels, affecting the homeostasis of copper, neuroprotection, stem cell renewal and memory mechanisms, among others. Often each such process has been heralded as the bona fide function of PrP C , despite restricted attention paid to a selected phenotypic trait, associated with either modulation of gene expression or to the engagement of PrP C with a single ligand. In contrast, the GPI-anchored prion protein was shown to bind several extracellular and transmembrane ligands, which are required to endow that protein with the ability to play various roles in transmembrane signal transduction. In addition, differing sets of those ligands are available in cell type- and context-dependent scenarios. To account for such properties, we proposed that PrP C serves as a dynamic platform for the assembly of signaling modules at the cell surface, with widespread consequences for both physiology and behavior. The current review advances the hypothesis that the biological function of the prion protein is that of a cell surface scaffold protein, based on the striking similarities of its functional properties with those of scaffold proteins involved in the organization of intracellular signal transduction pathways. Those properties are: the ability to recruit spatially restricted sets of binding molecules involved in specific

  17. Factors affecting the development of somatic cell nuclear transfer embryos in Cattle.

    PubMed

    Akagi, Satoshi; Matsukawa, Kazutsugu; Takahashi, Seiya

    2014-01-01

    Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.

  18. Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

    PubMed Central

    AKAGI, Satoshi; MATSUKAWA, Kazutsugu; TAKAHASHI, Seiya

    2014-01-01

    Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle. PMID:25341701

  19. Polydopamine-mediated surface modification of scaffold materials for human neural stem cell engineering.

    PubMed

    Yang, Kisuk; Lee, Jung Seung; Kim, Jin; Lee, Yu Bin; Shin, Heungsoo; Um, Soong Ho; Kim, Jeong Beom; Park, Kook In; Lee, Haeshin; Cho, Seung-Woo

    2012-10-01

    Surface modification of tissue engineering scaffolds and substrates is required for improving the efficacy of stem cell therapy by generating physicochemical stimulation promoting proliferation and differentiation of stem cells. However, typical surface modification methods including chemical conjugation or physical absorption have several limitations such as multistep, complicated procedures, surface denaturation, batch-to-batch inconsistencies, and low surface conjugation efficiency. In this study, we report a mussel-inspired, biomimetic approach to surface modification for efficient and reliable manipulation of human neural stem cell (NSC) differentiation and proliferation. Our study demonstrates that polydopamine coating facilitates highly efficient, simple immobilization of neurotrophic growth factors and adhesion peptides onto polymer substrates. The growth factor or peptide-immobilized substrates greatly enhance differentiation and proliferation of human NSCs (human fetal brain-derived NSCs and human induced pluripotent stem cell-derived NSCs) at a level comparable or greater than currently available animal-derived coating materials (Matrigel) with safety issues. Therefore, polydopamine-mediated surface modification can provide a versatile platform technology for developing chemically defined, safe, functional substrates and scaffolds for therapeutic applications of human NSCs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Surface receptor Toso controls B cell-mediated regulation of T cell immunity.

    PubMed

    Yu, Jinbo; Duong, Vu Huy Hoang; Westphal, Katrin; Westphal, Andreas; Suwandi, Abdulhadi; Grassl, Guntram A; Brand, Korbinian; Chan, Andrew C; Föger, Niko; Lee, Kyeong-Hee

    2018-05-01

    The immune system is tightly controlled by regulatory processes that allow for the elimination of invading pathogens, while limiting immunopathological damage to the host. In the present study, we found that conditional deletion of the cell surface receptor Toso on B cells unexpectedly resulted in impaired proinflammatory T cell responses, which led to impaired immune protection in an acute viral infection model and was associated with reduced immunopathological tissue damage in a chronic inflammatory context. Toso exhibited its B cell-inherent immunoregulatory function by negatively controlling the pool of IL-10-competent B1 and B2 B cells, which were characterized by a high degree of self-reactivity and were shown to mediate immunosuppressive activity on inflammatory T cell responses in vivo. Our results indicate that Toso is involved in the differentiation/maintenance of regulatory B cells by fine-tuning B cell receptor activation thresholds. Furthermore, we showed that during influenza A-induced pulmonary inflammation, the application of Toso-specific antibodies selectively induced IL-10-competent B cells at the site of inflammation and resulted in decreased proinflammatory cytokine production by lung T cells. These findings suggest that Toso may serve as a novel therapeutic target to dampen pathogenic T cell responses via the modulation of IL-10-competent regulatory B cells.

  1. Surface functionalization of nanobiomaterials for application in stem cell culture, tissue engineering, and regenerative medicine.

    PubMed

    Rana, Deepti; Ramasamy, Keerthana; Leena, Maria; Jiménez, Constanza; Campos, Javier; Ibarra, Paula; Haidar, Ziyad S; Ramalingam, Murugan

    2016-05-01

    Stem cell-based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial-based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell-based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554-567, 2016. © 2016 American Institute of Chemical Engineers.

  2. Human mesenchymal stem cell behavior on femtosecond laser-textured Ti-6Al-4V surfaces.

    PubMed

    Cunha, Alexandre; Zouani, Omar Farouk; Plawinski, Laurent; Botelho do Rego, Ana Maria; Almeida, Amélia; Vilar, Rui; Durrieu, Marie-Christine

    2015-01-01

    The aim of the present work was to investigate ultrafast laser surface texturing as a surface treatment of Ti-6Al-4V alloy dental and orthopedic implants to improve osteoblastic commitment of human mesenchymal stem cells (hMSCs). Surface texturing was carried out by direct writing with an Yb:KYW chirped-pulse regenerative amplification laser system with a central wavelength of 1030 nm and a pulse duration of 500 fs. The surface topography and chemical composition were investigated by scanning electron microscopy and x-ray photoelectron spectroscopy, respectively. Three types of surface textures with potential interest to improve implant osseointegration can be produced by this method: laser-induced periodic surface structures (LIPSSs); nanopillars (NPs); and microcolumns covered with LIPSSs, forming a bimodal roughness distribution. The potential of the laser treatment in improving hMSC differentiation was assessed by in vitro study of hMSCs spreading, adhesion, elongation and differentiation using epifluorescence microscopy at different times after cell seeding, after specific stainings and immunostainings. Cell area and focal adhesion area were lower on the laser-textured surfaces than on a polished reference surface. Obviously, the laser-textured surfaces have an impact on cell shape. Osteoblastic commitment was observed independently of the surface topography after 2 weeks of cell seeding. When the cells were cultured (after 4 weeks of seeding) in osteogenic medium, LIPSS- and NP- textured surfaces enhanced matrix mineralization and bone-like nodule formation as compared with polished and microcolumn-textured surfaces. The present work shows that surface nanotextures consisting of LIPSSs and NPs can, potentially, improve hMSC differentiation into an osteoblastic lineage.

  3. Evaluation of Encapsulant Adhesion to Surface Metallization of Photovoltaic Cells: Preprint

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tracy, Jared; Dauskardt, Reinhold; Bosco, Nick

    Delamination of encapsulant materials from PV cell surfaces often appears to originate at regions with metallization. Using a fracture mechanics based metrology, the adhesion of EVA encapsulant to screen printed silver metallization was evaluated. At room temperature, the fracture energy, Gc [J/m2], of the EVA/silver interface (952 J/m2) was ~70% lower than that of the EVA/AR coating (>2900 J/m2) and ~60% lower than that of the EVA to the surface of cell (2265 J/m2). After only 300 hours of damp heat aging, the adhesion energy of the silver interface dropped to and plateaued at ~50-60 J/m2, while that of themore » EVA/AR coating and EVA/cell remained mostly unchanged. Elemental surface analysis showed that the EVA separates from the silver in a purely adhesive manner, indicating that bonds at the interface were likely displaced in the presence of humidity and elevated temperature, and may explain the propensity for delamination to occur at metallized surfaces in the field.« less

  4. Burn-injury affects gut-associated lymphoid tissues derived CD4+ T cells.

    PubMed

    Fazal, Nadeem; Shelip, Alla; Alzahrani, Alhusain J

    2013-01-01

    After scald burn-injury, the intestinal immune system responds to maintain immune balance. In this regard CD4+T cells in Gut-Associated Lymphoid Tissues (GALT), like mesenteric lymph nodes (MLN) and Peyer's patches (PP) respond to avoid immune suppression following major injury such as burn. Therefore, we hypothesized that the gut CD4+T cells become dysfunctional and turn the immune homeostasis towards depression of CD4+ T cell-mediated adaptive immune responses. In the current study we show down regulation of mucosal CD4+ T cell proliferation, IL-2 production and cell surface marker expression of mucosal CD4+ T cells moving towards suppressive-type. Acute burn-injury lead to up-regulation of regulatory marker (CD25+), down regulation of adhesion (CD62L, CD11a) and homing receptor (CD49d) expression, and up-regulation of negative co-stimulatory (CTLA-4) molecule. Moreover, CD4+CD25+ T cells of intestinal origin showed resistance to spontaneous as well as induced apoptosis that may contribute to suppression of effector CD4+ T cells. Furthermore, gut CD4+CD25+ T cells obtained from burn-injured animals were able to down-regulate naïve CD4+ T cell proliferation following adoptive transfer of burn-injured CD4+CD25+ T cells into sham control animals, without any significant effect on cell surface activation markers. Together, these data demonstrate that the intestinal CD4+ T cells evolve a strategy to promote suppressive CD4+ T cell effector responses, as evidenced by enhanced CD4+CD25+ T cells, up-regulated CTLA-4 expression, reduced IL-2 production, tendency towards diminished apoptosis of suppressive CD4+ T cells, and thus lose their natural ability to regulate immune homeostasis following acute burn-injury and prevent immune paralysis.

  5. Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells

    PubMed Central

    Ortega, Fabian E.; Rengarajan, Michelle; Chavez, Natalie; Radhakrishnan, Prathima; Gloerich, Martijn; Bianchini, Julie; Siemers, Kathleen; Luckett, William S.; Lauer, Peter; Nelson, W. James; Theriot, Julie A.

    2017-01-01

    The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting αE-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell–cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin–mediated coupling of the bacterium to F-actin is not required. PMID:28877987

  6. Ions, metabolites, and cells: Water as a reporter of surface conditions during bacterial growth.

    PubMed

    Jarisz, Tasha A; Lane, Sarah; Gozdzialski, Lea; Hore, Dennis K

    2018-06-14

    Surface-specific nonlinear vibrational spectroscopy, combined with bulk solution measurements and imaging, is used to study the surface conditions during the growth of E. coli. As a result of the silica high surface charge density, the water structure at the silica-aqueous interface is known to be especially sensitive to pH and ionic strength, and surface concentration profiles develop that can be appreciably different from the bulk solution conditions. We illustrate that, in the presence of growing cells, a unique surface micro-environment is established as a result of metabolites accumulating on the silica surface. Even in the subsequent absence of the cells, this surface layer works to reduce the interfacial ionic strength as revealed by the enhanced signal from surface water molecules. In the presence of growing cells, an additional boost in surface water signal is attributed to a local pH that is higher than that of the bulk solution.

  7. Ions, metabolites, and cells: Water as a reporter of surface conditions during bacterial growth

    NASA Astrophysics Data System (ADS)

    Jarisz, Tasha A.; Lane, Sarah; Gozdzialski, Lea; Hore, Dennis K.

    2018-06-01

    Surface-specific nonlinear vibrational spectroscopy, combined with bulk solution measurements and imaging, is used to study the surface conditions during the growth of E. coli. As a result of the silica high surface charge density, the water structure at the silica-aqueous interface is known to be especially sensitive to pH and ionic strength, and surface concentration profiles develop that can be appreciably different from the bulk solution conditions. We illustrate that, in the presence of growing cells, a unique surface micro-environment is established as a result of metabolites accumulating on the silica surface. Even in the subsequent absence of the cells, this surface layer works to reduce the interfacial ionic strength as revealed by the enhanced signal from surface water molecules. In the presence of growing cells, an additional boost in surface water signal is attributed to a local pH that is higher than that of the bulk solution.

  8. Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

    NASA Astrophysics Data System (ADS)

    Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

    2017-02-01

    Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells.

  9. Intestinal lamina propria dendritic cells maintain T cell homeostasis but do not affect commensalism

    PubMed Central

    Welty, Nathan E.; Staley, Christopher; Ghilardi, Nico; Sadowsky, Michael J.; Igyártó, Botond Z.

    2013-01-01

    Dendritic cells (DCs) in the intestinal lamina propria (LP) are composed of two CD103+ subsets that differ in CD11b expression. We report here that Langerin is expressed by human LP DCs and that transgenic human langerin drives expression in CD103+CD11b+ LP DCs in mice. This subset was ablated in huLangerin-DTA mice, resulting in reduced LP Th17 cells without affecting Th1 or T reg cells. Notably, cognate DC–T cell interactions were not required for Th17 development, as this response was intact in huLangerin-Cre I-Aβfl/fl mice. In contrast, responses to intestinal infection or flagellin administration were unaffected by the absence of CD103+CD11b+ DCs. huLangerin-DTA x BatF3−/− mice lacked both CD103+ LP DC subsets, resulting in defective gut homing and fewer LP T reg cells. Despite these defects in LP DCs and resident T cells, we did not observe alterations of intestinal microbial communities. Thus, CD103+ LP DC subsets control T cell homeostasis through both nonredundant and overlapping mechanisms. PMID:24019552

  10. The interaction between LYVE-1 with hyaluronan on the cell surface may play a role in the diversity of adhesion to cancer cells.

    PubMed

    Du, Yan; Liu, Hua; He, Yiqing; Liu, Yiwen; Yang, Cuixia; Zhou, Muqing; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

    2013-01-01

    Hyaluronan (HA), a simple disaccharide unit, can polymerize and is considered a primary component of the extracellular matrix, which has a wide range of biological functions. In recent years, HA was found on the surface of tumor cells. According to previous reports, differing HA content on the cell surface of tumor cells is closely related to lymph node metastases, but the mechanisms mediating this process remained unclear. This research intended to study the surface content of HA on tumor cells and analyze cell adhesive changes caused by the interaction between HA and its lymphatic endothelial receptor (LYVE-1). We screened and observed high HA content on HS-578T breast cells and low HA content on MCF-7 breast cells through particle exclusion, immunofluorescence and flow cytometry experiments. The expression of LYVE-1, the lymph-vessel specific HA receptor, was consistent with our previous report and enhanced the adhesion of HA(high)-HS-578T cells to COS-7(LYVE-1(+)) through HA in cell static adhesion and dynamic parallel plate flow chamber experiments. MCF-7 breast cells contain little HA on the surface; however, our results showed little adhesion difference between MCF-7 cells and COS-7(LYVE-1(+)) and COS-7(LYVE-1(-)) cells. Similar results were observed concerning the adhesion of HS-578T cells or MCF-7 cells to SVEC4-10 cells. Furthermore, we observed for the first time that the cell surface HA content of high transfer tumor cells was rich, and we visualized the cross-linking of HA cable structures, which may activate LYVE-1 on lymphatic endothelial cells, promoting tumor adhesion. In summary, high-low cell surface HA content of tumor cells through the interaction with LYVE-1 leads to adhesion differences.

  11. Inactivation of the srtA Gene Affects Localization of Surface Proteins and Decreases Adhesion of Streptococcus pneumoniae to Human Pharyngeal Cells In Vitro

    PubMed Central

    Kharat, Arun S.; Tomasz, Alexander

    2003-01-01

    Inactivation of sortase gene srtA in Streptococcus pneumoniae strain R6 caused the release of β-galactosidase and neuraminidase A (NanA) from the cell wall into the surrounding medium. Both of these surface proteins contain the LPXTG motif in the C-terminal domain. Complementation with plasmid-borne srtA reversed protein release. Deletion of murM, a gene involved in the branching of pneumococcal peptidoglycan, also caused partial release of β-galactosidase, suggesting preferential attachment of the protein to branched muropeptides in the cell wall. Inactivation of srtA caused decreased adherence to human pharyngeal cells in vitro but had no effect on the virulence of a capsular type III strain of S. pneumoniae in the mouse intraperitoneal model. The observations suggest that—as in other gram-positive bacteria—sortase-dependent display of proteins occurs in S. pneumoniae and that some of these proteins may be involved in colonization of the human host. PMID:12704150

  12. Regulation of Kv2.1 K+ conductance by cell surface channel density

    PubMed Central

    Fox, Philip D.; Loftus, Rob J.; Tamkun, Michael M.

    2013-01-01

    The Kv2.1 voltage-gated K+ channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are non-conducting. Using TIRF microscopy the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared to K+ channel conductance measured by whole-cell voltage-clamp of the same cell. This approach indicated that as channel density increases non-clustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the non-conducting state with 17% conducting K+ at higher surface densities. The non-conducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immuno-fluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared to the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 days, respectively. Together these data indicate that the non-conducting state depends primarily on surface density as opposed to cluster location and that this non-conducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K+ conductance further supports a non-conducting role for Kv2.1 in excitable tissues. PMID:23325261

  13. Physical-mechanical image of the cell surface on the base of AFM data in contact mode

    NASA Astrophysics Data System (ADS)

    Starodubtseva, M. N.; Starodubtsev, I. E.; Yegorenkov, N. I.; Kuzhel, N. S.; Konstantinova, E. E.; Chizhik, S. A.

    2017-10-01

    Physical and mechanical properties of the cell surface are well-known markers of a cell state. The complex of the parameters characterizing the cell surface properties, such as the elastic modulus (E), the parameters of adhesive (Fa), and friction (Ff) forces can be measured using atomic force microscope (AFM) in a contact mode and form namely the physical-mechanical image of the cell surface that is a fundamental element of the cell mechanical phenotype. The paper aims at forming the physical-mechanical images of the surface of two types of glutaraldehyde-fixed cancerous cells (human epithelial cells of larynx carcinoma, HEp-2c cells, and breast adenocarcinoma, MCF-7 cells) based on the data obtained by AFM in air and revealing the basic difference between them. The average values of friction, elastic and adhesive forces, and the roughness of lateral force maps, as well as dependence of the fractal dimension of lateral force maps on Z-scale factor have been studied. We have revealed that the response of microscale areas of the HEp-2c cell surface having numerous microvilli to external mechanical forces is less expressed and more homogeneous in comparison with the response of MCF-7 cell surface.

  14. Functional Anchoring Lipids for Drug Delivery Carrier Fabrication and Cell Surface Re-Engineering Applications

    NASA Astrophysics Data System (ADS)

    Vabbilisetty, Pratima

    For decades, lipid vesicular bodies such as liposomes have been widely used and explored as biomimetic models of cell membranes and as drug/gene delivery carrier systems. Similarly, micellar iron oxide nanoparticles have also been investigated as potential MRI agents as well as drug delivery carrier systems. Cell surface carbohydrate-protein interactions allow them to serve as markers for recognition of many molecular and cellular activities thereby, are exploited as attractive molecules for surface modification of nanocarrier systems with purpose for tissues specific targeting and biocompatibility. In addition, the cell lipid membrane serves as an important platform for occurrence of many biological processes that are governed and guided by cell surface receptors. Introduction of chemoselective functional groups, via bio-orthogonal conjugation strategies, at the cell surface facilitates many cellular modifications and paves path for novel and potential biomedical applications. Anchoring lipids are needed for liposome surface functionalization with ligands of interest and play important roles in ligand grafting density, liposomes stability and biological activity. On the other hand, anchoring lipids are also needed for cell surface re-engineering by lipid fusion approach and have high impact for ligand insertion efficiency and biological activity. Overall, in this dissertation study, functional anchoring lipids for glyco-functionalized carrier systems and for efficient cell surface re-engineering applications were systematically investigated, respectively. Firstly, investigation of the synthesis of glyco-functionalized liposome systems based on phosphatidylethonalamine (PE) and cholesterol (Chol) anchoring lipids, prepared by post chemically selective functionalization via Staudinger ligation were carried out. The effect of anchor lipids on the stability, encapsulation and releasing capacity of the glycosylated liposomes were investigated by dynamic light

  15. Droplet Microarray Based on Patterned Superhydrophobic Surfaces Prevents Stem Cell Differentiation and Enables High-Throughput Stem Cell Screening.

    PubMed

    Tronser, Tina; Popova, Anna A; Jaggy, Mona; Bastmeyer, Martin; Levkin, Pavel A

    2017-12-01

    Over the past decades, stem cells have attracted growing interest in fundamental biological and biomedical research as well as in regenerative medicine, due to their unique ability to self-renew and differentiate into various cell types. Long-term maintenance of the self-renewal ability and inhibition of spontaneous differentiation, however, still remain challenging and are not fully understood. Uncontrolled spontaneous differentiation of stem cells makes high-throughput screening of stem cells also difficult. This further hinders investigation of the underlying mechanisms of stem cell differentiation and the factors that might affect it. In this work, a dual functionality of nanoporous superhydrophobic-hydrophilic micropatterns is demonstrated in their ability to inhibit differentiation of mouse embryonic stem cells (mESCs) and at the same time enable formation of arrays of microdroplets (droplet microarray) via the effect of discontinuous dewetting. Such combination makes high-throughput screening of undifferentiated mouse embryonic stem cells possible. The droplet microarray is used to investigate the development, differentiation, and maintenance of stemness of mESC, revealing the dependence of stem cell behavior on droplet volume in nano- and microliter scale. The inhibition of spontaneous differentiation of mESCs cultured on the droplet microarray for up to 72 h is observed. In addition, up to fourfold increased cell growth rate of mESCs cultured on our platform has been observed. The difference in the behavior of mESCs is attributed to the porosity and roughness of the polymer surface. This work demonstrates that the droplet microarray possesses the potential for the screening of mESCs under conditions of prolonged inhibition of stem cells' spontaneous differentiation. Such a platform can be useful for applications in the field of stem cell research, pharmacological testing of drug efficacy and toxicity, biomedical research as well as in the field of

  16. Identification and characterization of Vibrio cholerae surface proteins by radioiodination.

    PubMed Central

    Richardson, K; Parker, C D

    1985-01-01

    Whole cells and isolated outer membrane from Vibrio cholerae (Classical, Inaba) were radiolabeled with Iodogen or Iodo-beads as catalyst. Radiolabeling of whole cells was shown to be surface specific by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis of whole cells and cell fractions. Surface-labeled whole cells regularly showed 16 distinguishable protein species, of which nine were found in radiolabeled outer membrane preparations obtained by a lithium chloride-lithium acetate procedure. Eight of these proteins were found in outer membranes prepared by sucrose density gradient centrifugation and Triton X-100 extraction of radiolabeled whole cells. The mobility of several proteins was shown to be affected by temperature, and the major protein species exposed on the cell surface was shown to consist of at least two different peptides. Images PMID:3980099

  17. Altered Calcium Dynamics in Cardiac Cells Grown on Silane-Modified Surfaces

    PubMed Central

    Ravenscroft-Chang, Melissa S.; Stohlman, Jayna; Molnar, Peter; Natarajan, Anupama; Canavan, Heather E.; Teliska, Maggie; Stancescu, Maria; Krauthamer, Victor; Hickman, J.J.

    2013-01-01

    Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca2+ because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca2+ imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca2+ concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca2+ changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca2+ dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems. PMID:19828193

  18. Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

    NASA Astrophysics Data System (ADS)

    Mikulska, Anna; Filipowska, Joanna; Osyczka, Anna; Nowakowska, Maria; Szczubiałka, Krzysztof

    2014-12-01

    Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2

  19. Osteoinductive activity of insulin-functionalized cell culture surfaces obtained using diazonium chemistry

    PubMed Central

    Mikulska, Anna; Filipowska, Joanna; Osyczka, Anna M.; Nowakowska, Maria; Szczubiałka, Krzysztof

    2015-01-01

    Polymeric surfaces suitable for cell culture (DR/Pec) were constructed from diazoresin (DR) and pectin (Pec) in a form of ultrathin films using the layer-by-layer (LbL) technique. The surfaces were functionalized with insulin using diazonium chemistry. Such functionalized surfaces were used to culture human mesenchymal stem cells (hMSCs) to assess their suitability for bone tissue engineering and regeneration. The activity of insulin immobilized on the surfaces (DR/Pec/Ins) was compared to that of insulin dissolved in the culture medium. Human MSC grown on insulin-immobilized DR/Pec surfaces displayed increased proliferation and higher osteogenic activity. The latter was determined by means of alkaline phosphatase (ALP) activity, which increases at early stages of osteoblasts differentiation. Insulin dissolved in the culture medium did not stimulate cell proliferation and its osteogenic activity was significantly lower. Addition of recombinant human bone morphogenetic protein 2 (rhBMP-2) to the culture medium further increased ALP activity in hMSCs indicating additive osteogenic action of immobilized insulin and rhBMP-2. PMID:25629028

  20. DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

    PubMed

    Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

    2006-01-01

    Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.

  1. SERS imaging of cell-surface biomolecules metabolically labeled with bioorthogonal Raman reporters.

    PubMed

    Xiao, Ming; Lin, Liang; Li, Zefan; Liu, Jie; Hong, Senlian; Li, Yaya; Zheng, Meiling; Duan, Xuanming; Chen, Xing

    2014-08-01

    Live imaging of biomolecules with high specificity and sensitivity as well as minimal perturbation is essential for studying cellular processes. Here, we report the development of a bioorthogonal surface-enhanced Raman scattering (SERS) imaging approach that exploits small Raman reporters for visualizing cell-surface biomolecules. The cells were cultured and imaged by SERS microscopy on arrays of Raman-enhancing nanoparticles coated on silicon wafers or glass slides. The Raman reporters including azides, alkynes, and carbondeuterium bonds are small in size and spectroscopically bioorthogonal (background-free). We demonstrated that various cell-surface biomolecules including proteins, glycans, and lipids were metabolically incorporated with the corresponding precursors bearing a Raman reporter and visualized by SERS microscopy. The coupling of SERS microscopy with bioorthogonal Raman reporters expands the capabilities of live-cell microscopy beyond the modalities of fluorescence and label-free imaging. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Generating Chondromimetic Mesenchymal Stem Cell Spheroids by Regulating Media Composition and Surface Coating.

    PubMed

    Sridharan, BanuPriya; Laflin, Amy D; Detamore, Michael S

    2018-04-01

    Spheroids of mesenchymal stem cells (MSCs) in cartilage tissue engineering have been shown to enhance regenerative potential owing to their 3D structure. In this study, we explored the possibility of priming spheroids under different media to replace the use of inductive surface coatings for chondrogenic differentiation. Rat bone marrow-derived MSCs were organized into cell spheroids by the hanging drop technique and subsequently cultured on hyaluronic acid (HA) coated or non-coated well plates under different cell media conditions. Endpoint analysis included cell viability, DNA and Glycosaminoglycan (GAG) and collagen content, gene expression and immunohistochemistry. For chondrogenic applications, MSC spheroids derived on non-coated surfaces outperformed the spheroids derived from HA-coated surfaces in matrix synthesis and collagen II gene expression. Spheroids on non-coated surfaces gave rise to the highest collagen and GAG when primed with medium containing insulin-like growth factor (IGF) for 1 week during spheroid formation. Spheroids that were grown in chondroinductive raw material-inclusive media such as aggrecan or chondroitin sulfate exhibited the highest Collagen II gene expression in the non-coated surface at 1 week. Media priming by growth factors and raw materials might be a more predictive influencer of chondrogenesis compared to inductive-surfaces. Such tailored bioactivity of the stem cell spheroids in the stage of the spheroid formation may give rise to a platform technology that may eventually produce spheroids capable of chondrogenesis achieved by mere media manipulation, skipping the need for additional culture on a modified surface, that paves the way for cost-effective technologies.

  3. ALDH isozymes downregulation affects cell growth, cell motility and gene expression in lung cancer cells.

    PubMed

    Moreb, Jan S; Baker, Henry V; Chang, Lung-Ji; Amaya, Maria; Lopez, M Cecilia; Ostmark, Blanca; Chou, Wayne

    2008-11-24

    Aldehyde dehydrogenase isozymes ALDH1A1 and ALDH3A1 are highly expressed in non small cell lung cancer. Neither the mechanisms nor the biologic significance for such over expression have been studied. We have employed oligonucleotide microarrays to analyze changes in gene profiles in A549 lung cancer cell line in which ALDH activity was reduced by up to 95% using lentiviral mediated expression of siRNA against both isozymes (Lenti 1+3). Stringent analysis methods were used to identify gene expression patterns that are specific to the knock down of ALDH activity and significantly different in comparison to wild type A549 cells (WT) or cells similarly transduced with green fluorescent protein (GFP) siRNA. We confirmed significant and specific down regulation of ALDH1A1 and ALDH3A1 in Lenti 1+3 cells and in comparison to 12 other ALDH genes detected. The results of the microarray analysis were validated by real time RT-PCR on RNA obtained from Lenti 1+3 or WT cells treated with ALDH activity inhibitors. Detailed functional analysis was performed on 101 genes that were significantly different (P < 0.001) and their expression changed by > or = 2 folds in the Lenti 1+3 group versus the control groups. There were 75 down regulated and 26 up regulated genes. Protein binding, organ development, signal transduction, transcription, lipid metabolism, and cell migration and adhesion were among the most affected pathways. These molecular effects of the ALDH knock-down are associated with in vitro functional changes in the proliferation and motility of these cells and demonstrate the significance of ALDH enzymes in cell homeostasis with a potentially significant impact on the treatment of lung cancer.

  4. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32)

    PubMed Central

    2011-01-01

    Background Elevated numbers of regulatory T cells (Tregs) have been implicated in certain cancers. Depletion of Tregs has been shown to increase anti-tumor immunity. Tregs also play a critical role in the suppression of autoimmune responses. The study of Tregs has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated Tregs. However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of Tregs expressing LRRC32. Results Using naturally-occurring freshly isolated Tregs, we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ Tregs are distinct from LRRC32- Tregs with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ Tregs are more potent suppressors than LRRC32- Tregs. Conclusions A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent Treg populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of Tregs and the refinement of immunotherapeutic strategies aimed at targeting these cells. PMID:21615933

  5. Signal peptide cleavage is essential for surface expression of a regulatory T cell surface protein, leucine rich repeat containing 32 (LRRC32).

    PubMed

    Chan, Derek V; Somani, Ally-Khan; Young, Andrew B; Massari, Jessica V; Ohtola, Jennifer; Sugiyama, Hideaki; Garaczi, Edina; Babineau, Denise; Cooper, Kevin D; McCormick, Thomas S

    2011-05-26

    Elevated numbers of regulatory T cells (T(regs)) have been implicated in certain cancers. Depletion of T(regs) has been shown to increase anti-tumor immunity. T(regs) also play a critical role in the suppression of autoimmune responses. The study of T(regs) has been hampered by a lack of adequate surface markers. Leucine Rich Repeat Containing 32 (LRRC32), also known as Glycoprotein A Repetitions Predominant (GARP), has been postulated as a novel surface marker of activated T(regs). However, there is limited information regarding the processing of LRRC32 or the regulatory phenotype and functional activity of T(regs) expressing LRRC32. Using naturally-occurring freshly isolated T(regs), we demonstrate that low levels of LRRC32 are present intracellularly prior to activation and that freshly isolated LRRC32+ T(regs) are distinct from LRRC32- T(regs) with respect to the expression of surface CD62L. Using LRRC32 transfectants of HEK cells, we demonstrate that the N-terminus of LRRC32 is cleaved prior to expression of the protein at the cell surface. Furthermore, we demonstrate using a construct containing a deleted putative signal peptide region that the presence of a signal peptide region is critical to cell surface expression of LRRC32. Finally, mixed lymphocyte assays demonstrate that LRRC32+ T(regs) are more potent suppressors than LRRC32- T(regs). A cleaved signal peptide site in LRRC32 is necessary for surface localization of native LRRC32 following activation of naturally-occurring freshly-isolated regulatory T cells. LRRC32 expression appears to alter the surface expression of activation markers of T cells such as CD62L. LRRC32 surface expression may be useful as a marker that selects for more potent T(reg) populations. In summary, understanding the processing and expression of LRRC32 may provide insight into the mechanism of action of T(regs) and the refinement of immunotherapeutic strategies aimed at targeting these cells.

  6. Physical labeling of papillomavirus-infected, immortal, and cancerous cervical epithelial cells reveal surface changes at immortal stage.

    PubMed

    Swaminathan Iyer, K; Gaikwad, R M; Woodworth, C D; Volkov, D O; Sokolov, Igor

    2012-06-01

    A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p < 0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, premalignant cells.

  7. Physical Labeling of Papillomavirus-Infected, Immortal, and Cancerous Cervical Epithelial Cells Reveal Surface Changes at Immortal Stage

    PubMed Central

    Iyer, K. Swaminathan; Gaikwad, R. M.; Woodworth, C. D.; Volkov, D. O.

    2013-01-01

    A significant change of surface features of malignant cervical epithelial cells compared to normal cells has been previously reported. Here, we are studying the question at which progressive stage leading to cervical cancer the surface alteration happens. A non-traditional method to identify malignant cervical epithelial cells in vitro, which is based on physical (in contrast to specific biochemical) labelling of cells with fluorescent silica micron-size beads, is used here to examine cells at progressive stages leading to cervical cancer which include normal epithelial cells, cells infected with human papillomavirus type-16 (HPV-16), cells immortalized by HPV-16, and carcinoma cells. The study shows a statistically significant (at p <0.01) difference between both immortal and cancer cells and a group consisting of normal and infected. There is no significant difference between normal and infected cells. Immortal cells demonstrate the signal which is closer to cancer cells than to either normal or infected cells. This implies that the cell surface, surface cellular brush changes substantially when cells become immortal. Physical labeling of the cell surface represents a substantial departure from the traditional biochemical labeling methods. The results presented show the potential significance of physical properties of the cell surface for development of clinical methods for early detection of cervical cancer, even at the stage of immortalized, pre-malignant cells. PMID:22351422

  8. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy†

    PubMed Central

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-soo; Torelli, Marco D.; Hamers, Robert J.; Murhpy, Catherine J.; Orr, Galya

    2015-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate eficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells. PMID:24816810

  9. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localizationmore » patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.« less

  10. Antimicrobial design of titanium surface that kill sessile bacteria but support stem cells adhesion

    NASA Astrophysics Data System (ADS)

    Zhu, Chen; Bao, Ni-Rong; Chen, Shuo; Zhao, Jian-Ning

    2016-12-01

    Implant-related bacterial infection is one of the most severe postoperative complications in orthopedic or dental surgery. In this context, from the perspective of surface modification, increasing efforts have been made to enhance the antibacterial capability of titanium surface. In this work, a hierarchical hybrid surface architecture was firstly constructed on titanium surface by two-step strategy of acid etching and H2O2 aging. Then silver nanoparticles were firmly immobilized on the hierarchical surface by ion implantation, showing no detectable release of silver ions from surface. The designed titanium surface showed good bioactivity. More importantly, this elaborately designed titanium surface can effectively inactivate the adherent S. aureus on surface by virtue of a contact-killing mode. Meanwhile, the designed titanium surface can significantly facilitate the initial adhesion and spreading behaviors of bone marrow mesenchymal stem cells (MSCs) on titanium. The results suggested that, the elaborately designed titanium surface might own a cell-favoring ability that can help mammalian cells win the initial adhesion race against bacteria. We hope the present study can provide a new insight for the better understanding and designing of antimicrobial titanium surface, and pave the way to satisfying clinical requirements.

  11. Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

    PubMed Central

    Zhang, Li; Liang, Shuli; Zhou, Xinying; Jin, Zi; Jiang, Fengchun; Han, Shuangyan; Zheng, Suiping

    2013-01-01

    Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro. In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by β-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface. PMID:23835174

  12. Genetic modification of mesenchymal stem cells to express a single-chain antibody against EGFRvIII on the cell surface.

    PubMed

    Balyasnikova, Irina V; Franco-Gou, Rosa; Mathis, J Michael; Lesniak, Maciej S

    2010-06-01

    Human adult mesenchymal stem cells (hMSCs) are under active investigation as cellular carriers for gene therapy. hMSCs possess natural tropism toward tumours; however, the targeting of hMSCs to specific cell populations within tumours is unexplored. In the case of glioblastoma multiforme (GBM), at least half of the tumours express EGFRvIII on the cell surface, an ideal target for antibody-mediated gene/drug delivery. In this study, we investigated the feasibility of genetically modifying hMSCs to express a single-chain antibody (scFv) to EGFRvIII on their surfaces. Nucleofection was used to transfect hMSCs with cDNA encoding scFv EGFRvIII fused with PDGFR or human B7-1 transmembrane domains. The expression of scFv EGFRvIII on the cell surface was assessed by FACS. A stable population of scFv EGFRvIII-expressing hMSCs was selected, based on antibiotic resistance, and enriched using FACS. We found that nucleofection allows the efficient expression of scFv EGFRvIII on the cell surface of hMSCs. hMSCs transfected with the construct encoding scFv EGFRvIII as a fusion with PDGFRtm showed scFv EGFRvIII expression in up to 86% of cells. Most importantly, human MSCs expressing scFv against EGFRvIII demonstrated enhanced binding to U87-EGFRvIII cells in vitro and significantly increased retention in human U87-EGFRvIII-expressing tumours in vivo. In summary, we provide the first conclusive evidence of genetic modification of hMSCs with a single-chain antibody against an antigen expressed on the surface of tumour cells, thereby opening up a new venue for enhanced delivery of gene therapy applications in the context of malignant brain cancer. Copyright 2009 John Wiley & Sons, Ltd.

  13. Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction.

    PubMed

    Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

    2014-05-01

    Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent.

  14. Isthmin targets cell-surface GRP78 and triggers apoptosis via induction of mitochondrial dysfunction

    PubMed Central

    Chen, M; Zhang, Y; Yu, V C; Chong, Y-S; Yoshioka, T; Ge, R

    2014-01-01

    Isthmin (ISM) is a secreted 60-kDa protein that potently induces endothelial cell (EC) apoptosis. It suppresses tumor growth and angiogenesis in mice when stably overexpressed in cancer cells. Although αvβ5 integrin serves as a low-affinity receptor for ISM, the mechanism by which ISM mediates antiangiogenesis and apoptosis in ECs remain to be fully resolved. In this work, we report the identification of cell-surface glucose-regulated protein 78 kDa (GRP78) as a high-affinity receptor for ISM (Kd=8.6 nM). We demonstrated that ISM-GRP78 interaction triggers apoptosis not only in activated ECs but also in cancer cells expressing high level of cell-surface GRP78. Normal cells and benign tumor cells tend to express low level of cell-surface GRP78 and are resistant to ISM-induced apoptosis. Upon binding to GRP78, ISM is internalized into ECs through clathrin-dependent endocytosis that is essential for its proapoptotic activity. Once inside the cell, ISM co-targets with GRP78 to mitochondria where it interacts with ADP/ATP carriers on the inner membrane and blocks ATP transport from mitochondria to cytosol, thereby causing apoptosis. Hence, ISM is a novel proapoptotic ligand that targets cell-surface GRP78 to trigger apoptosis by inducing mitochondrial dysfunction. The restricted and high-level expression of cell-surface GRP78 on cancer cells and cancer ECs make them uniquely susceptible to ISM-targeted apoptosis. Indeed, systemic delivery of recombinant ISM potently suppressed subcutaneous 4T1 breast carcinoma and B16 melanoma growth in mice by eliciting apoptosis selectively in the cancer cells and cancer ECs. Together, this work reveals a novel ISM-GRP78 apoptosis pathway and demonstrates the potential of ISM as a cancer-specific and dual-targeting anticancer agent. PMID:24464222

  15. Adhesion molecules affected by treatment of lung cancer cells with epidermal growth factor.

    PubMed

    Fonseca, Fernando L A; Azzalis, Ligia A; Feder, David; Nogoceke, Everson; Junqueira, Virginia B C; Valenti, Vitor E; de Abreu, Luiz Carlos

    2011-10-01

    Lung cancer is one of the leading causes of death in the world. Some tumor events are attributed to an important group of molecules (cadherins and integrins). We evaluated the interactions of cell adhesion molecules in cell lines from lung cancer. Two lung cancer cell lines were nonmetastatic (H358 and H441) and two were metastatic (H1299 and H292). All cell lines were treated with epidermal growth factor (EGF), and Western blot analysis was performed to assess the interactions between these proteins. The bronchoalveolar cells H358 showed the three analyzed proteins: E-cadherin, β-catenin, and p120 catenin. The adenocarcinoma cells H441 did not present p120 catenin, and carcinoma cells did not show E-cadherin (H1299) or p120 catenin (H292). FAK (pTyr925) was dephosphorylated in adenocarcinoma cells H441, absent in carcinoma cells H1299, and upregulated in the other carcinoma cells H292. p130Cas showed no difference when the cell lines were treated with EGF for 30 min; it was absent in the metastatic carcinoma cells H1299. Paxillin was dephosphorylated in adenocarcinoma cells H441 and also absent in other metastatic carcinoma cells H292. Vinculin showed the same results, and talin was downregulated in adenocarcinoma cells H441 when the cells were treated with EGF. Rap1 was downregulated and PYK2 was upregulated in the same cell line. Our data help to comprehend the mechanism involved in cell migration to the blood and metastasis generation. In conclusion, the expression patterns of cell-cell adhesion were not affected by EGF treatment but it affected cell-extracellular matrix adhesion.

  16. Factors affecting the potassium concentration at the mucosal surface of the proximal and the distal colon of guinea pig.

    PubMed Central

    Kück-Biere, U; von Engelhardt, W

    1990-01-01

    K+ concentrations were measured in vitro with K+ sensitive microelectrodes in the microclimate at the luminal cell surface of the colon of guinea pigs. The serosal K+ concentration was mostly 5.4 mmol/1, the mucosal K+ concentrations were changed (0, 5, 50, or 70 mmol/l). Under control conditions K+ concentrations in the microclimate of the proximal colon were also low (6-9 mmol/l) and rather independent from K+ concentrations in the bulk luminal solution. In the distal colon K+ concentrations in the microclimate increased from 3.7 mmol/l when no K+ was in the luminal solution, up to 22 mmol/l when the mucosal K+ concentrations was 70 mmol/l. Attempts to decrease K+ conductance of the apical membrane with Ba++, to impair K+ transport with ouabain and to increase the paracellular shunt with deoxycholic acid did not affect K+ concentrations in the microclimate of the proximal colon but decreased K+ concentrations in the distal colon. When valinomycin or triaminopyrimidine were added to the mucosal solution at high K+ concentrations in the luminal solutions the K+ concentration in the microclimate was raised. At low luminal K+ concentrations valinomycin had no effect, triaminopyrimidine significantly diminished K+ concentrations at the cell surface. Regional differences in paracellular shunt conductance and in the preepithelial diffusion barrier are thought to be responsible for the observed differences between the proximal and the distal colon. Obviously, however, further unknown mechanisms have to be involved. PMID:2108077

  17. In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

    PubMed

    Uhl, P B; Szober, C M; Amann, B; Alge-Priglinger, C; Ueffing, M; Hauck, S M; Deeg, C A

    2014-09-23

    Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Isolation of pancreatic progenitor cells with the surface marker of hematopoietic stem cells.

    PubMed

    Ma, Fengxia; Chen, Fang; Chi, Ying; Yang, Shaoguang; Lu, Shihong; Han, Zhongchao

    2012-01-01

    To isolate pancreatic progenitor cells with the surface markers of hematopoietic stem cells, the expression of stem cell antigen (Sca-1) and c-Kit and the coexpression of them with pancreatic duodenal homeobox-1 (PDX-1), neurogenin 3 (Ngn3), and insulin were examined in murine embryonic pancreas. Then different pancreatic cell subpopulations were isolated by magnet-activated cell sorting. Isolated cells were cultured overnight in hanging drops. When cells formed spheres, they were laid on floating filters at the air/medium interface. With this new culture system, pancreatic progenitor cells were induced to differentiate to endocrine and exocrine cells. It was shown that c-Kit and Sca-1 were expressed differently in embryonic pancreas at 12.5, 15.5, and 17.5 days of gestation. The expression of c-Kit and Sca-1 was the highest at 15.5 days of gestation. c-Kit rather than Sca-1 coexpressed with PDX-1, Ngn3, and insulin. Cells differentiated from c-Kit-positive cells contained more insulin-producing cells and secreted more insulin in response to glucose stimulation than that from c-Kit-negative cells. These results suggested that c-Kit could be used to isolate pancreatic progenitor cells and our new culture system permitted pancreatic progenitor cells to differentiate to mature endocrine cells.

  19. Release of complement regulatory proteins from ocular surface cells in infections.

    PubMed

    Cocuzzi, E; Guidubaldi, J; Bardenstein, D S; Chen, R; Jacobs, M R; Medof, E M

    2000-11-01

    The decay accelerating factor (DAF or CD55) and the membrane inhibitor of reactive lysis (MIRL or CD59), two complement regulatory proteins that protect self cells from autologous complement-mediated injury, are attached to corneal and cqonjunctival epithelial cells by glycosylphos-phatidylinositol (GPI) anchors. We sought to 1) determine the frequency with which bacteria recovered from patients with infections of the eye elaborate factors that can remove these surface proteins from ocular cells, 2) determine the spectrum of bacteria from other sites that have similar effects, and 3) establish the time interval required for reconstitution of the two regulators. Culture supernatants of 18 ocular isolates [P. aeruginosa (n = 3), S. marcescens (n = 1), S. epidermidis (n = 9), and S. aureus (n = 5)], and > 100 other clinical specimens isolated in the hospital's microbiology laboratory [P. mirabilis (n = 1), S. aureus (n = 65), S. epidermidis (n = 24), B. cereus (n = 12), H. influenzae (n = 15), and Enterobacter sp. (n = 21)] were incubated at 37 degrees C for various times with conjunctival epithelial cells, conjunctival fibroblasts or HeLa cells and the release of DAF and CD59 proteins from the surfaces of the cells analyzed by 2-site immunoradiometric assays and by Western blotting. The kinetics of recovery of DAF and CD59 expression on the cells was measured by flow cytometry. DAF and/or CD59 release from the cell monolayers varied from < 5% to > 99% at as much as a 1:81 dilution of the supernatant from some bacteria. On conjunctival epithelial cells, more than 8 hr was required for 44% recovery of DAF expression and for 50% recovery of CD59 expression. Bacteria produce phospholipases and/or other enzymes which can efficiently remove DAF and CD59 from ocular cell surfaces. This phenomenon may correlate with their in vivo pathogenicity.

  20. TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

    PubMed

    Oida, Takatoku; Weiner, Howard L

    2010-11-24

    It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

  1. The active translation of MHCII mRNA during dendritic cells maturation supplies new molecules to the cell surface pool.

    PubMed

    Malanga, Donatella; Barba, Pasquale; Harris, Paul E; Maffei, Antonella; Del Pozzo, Giovanna

    2007-04-01

    The transition of human dendritic cells (DCs) from the immature to the mature phenotype is characterized by an increased density of MHC class II (MHCII) molecules on the plasma membrane, a key requirement of their competence as professional antigen presenting cells (APCs). MHCII molecules on the cell surface derive from newly synthesized as well as from preexisting proteins. So far, all the studies done on DCs during maturation, to establish the relative contribution of newly synthesized MHCII molecules to the cell surface pool did not produced a clear, unified scenario. We report that, in human DCs stimulated ex vivo with LPS, the changes in the RNA accumulation specific for at least two MHCII genes (HLA-DRA and HLA-DQA1) due to transcriptional upregulation, is associated with the active translation at high rate of these transcripts. Our finding reveals that, across the 24h of the maturation process in human DCs, newly synthesized MHCII proteins are supplied to the APCs cell surface pool.

  2. Surface nanoporosity has a greater influence on osteogenic and bacterial cell adhesion than crystallinity and wettability

    NASA Astrophysics Data System (ADS)

    Rodriguez-Contreras, Alejandra; Guadarrama Bello, Dainelys; Nanci, Antonio

    2018-07-01

    There has been much emphasis on the influence of crystallinity and wettability for modulating cell activity, particularly for bone biomaterials. In this context, we have generated titanium oxide layers with similar mesoporous topography and surface roughness but with amorphous or crystalline oxide layers and differential wettability. We then investigated their influence on the behavior of MC3T3 osteoblastic and bacterial cells. There was no difference in cell adhesion, spreading and growth on amorphous and crystalline surfaces. The number of focal adhesions was similar, however, cells on the amorphous surface exhibited a higher frequency of mature adhesions. The crystallinity of the surface layers also had no bearing on bacterial adhesion. While it cannot be excluded that surface crystallinity, roughness and wettability contribute to some degree to determining cell behavior, our data suggest that physical characteristics of surfaces represent the major determinant.

  3. A simplified model for dynamics of cell rolling and cell-surface adhesion

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cimrák, Ivan, E-mail: ivan.cimrak@fri.uniza.sk

    2015-03-10

    We propose a three dimensional model for the adhesion and rolling of biological cells on surfaces. We study cells moving in shear flow above a wall to which they can adhere via specific receptor-ligand bonds based on receptors from selectin as well as integrin family. The computational fluid dynamics are governed by the lattice-Boltzmann method. The movement and the deformation of the cells is described by the immersed boundary method. Both methods are fully coupled by implementing a two-way fluid-structure interaction. The adhesion mechanism is modelled by adhesive bonds including stochastic rules for their creation and rupture. We explore amore » simplified model with dissociation rate independent of the length of the bonds. We demonstrate that this model is able to resemble the mesoscopic properties, such as velocity of rolling cells.« less

  4. Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

    PubMed

    Walz, Jenna A; Mace, Charles R

    2018-06-05

    Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

  5. Endocrine disrupting chemicals affect the adipogenic differentiation of mesenchymal stem cells in distinct ontogenetic windows

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Biemann, Ronald, E-mail: ronald.biemann@medizin.uni-halle.de; Navarrete Santos, Anne; Navarrete Santos, Alexander

    Highlights: Black-Right-Pointing-Pointer Endocrine disrupting chemicals affect adipogenesis in mesenchymal stem cells (MSC). Black-Right-Pointing-Pointer The adipogenic impact depends strongly on the window of exposure. Black-Right-Pointing-Pointer Bisphenol A reduces the potential of MSC to differentiate into adipocytes. Black-Right-Pointing-Pointer DEHP and TBT trigger the adipogenic differentiation of mesenchymal stem cells. Black-Right-Pointing-Pointer BPA, DEHP and TBT did not affect adipogenesis in embryonic stem cells. -- Abstract: Endocrine disrupting chemicals (EDC) like bisphenol A (BPA), bis(2-ethylhexyl)phthalate (DEHP) and tributyltin (TBT) are ubiquitously present in the environment and in human tissues. They bind to nuclear hormone receptors and affect cellular and developmental processes. In this study,more » we show that BPA, DEHP and TBT affect the adipogenic differentiation of murine mesenchymal stem cells (MSC, C3H/10T1/2) in a concentration-, stage- and compound-specific manner. C3H/10T1/2 cells and embryonic stem cells (CGR8) were exposed to BPA, DEHP or TBT at different stages of cell determination and differentiation (undifferentiated growth, adipogenic induction and terminal adipogenic differentiation). The final amount of differentiated adipocytes, cellular triglyceride content and mRNA expression of adipogenic marker genes (adiponectin, FABP4, PPAR{gamma}2, LPL) were quantified and compared with corresponding unexposed cells. BPA (10 {mu}M) decreased subsequent adipogenic differentiation of MSC, when cells were exposed during undifferentiated growth. In contrast, DEHP (100 {mu}M) during the hormonal induction period, and TBT (100 nM) in all investigated stages, enhanced adipogenesis. Importantly, exposure of undifferentiated murine embryonic stem cells did not show any effect of the investigated EDC on subsequent adipogenic differentiation.« less

  6. Multilayer Choline Phosphate Molecule Modified Surface with Enhanced Cell Adhesion but Resistance to Protein Adsorption.

    PubMed

    Chen, Xingyu; Yang, Ming; Liu, Botao; Li, Zhiqiang; Tan, Hong; Li, Jianshu

    2017-08-22

    Choline phosphate (CP), which is a new zwitterionic molecule, and has the reverse order of phosphate choline (PC) and could bind to the cell membrane though the unique CP-PC interaction. Here we modified a glass surface with multilayer CP molecules using surface-initiated atom-transfer radical polymerization (SI-ATRP) and the ring-opening method. Polymeric brushes of (dimethylamino)ethyl methacrylate (DMAEMA) were synthesized by SI-ATRP from the glass surface. Then the grafted PDMAEMA brushes were used to introduce CP groups to fabricate the multilayer CP molecule modified surface. The protein adsorption experiment and cell culture test were used to evaluate the biocompatibility of the modified surfaces by using human umbilical veinendothelial cells (HUVECs). The protein adsorption results demonstrated that the multilayer CP molecule decorated surface could prevent the adsorption of fibrinogen and serum protein. The adhesion and proliferation of cells were improved significantly on the multilayer CP molecule modified surface. Therefore, the biocompatibility of the material surface could be improved by the modified multilayer CP molecule, which exhibits great potential for biomedical applications, e.g., scaffolds in tissue engineering.

  7. Relaxin affects cell organization and early and late stages of spermatogenesis in a coculture of rat testicular cells.

    PubMed

    Pimenta, M T; Francisco, R A R; Silva, R P; Porto, C S; Lazari, M F M

    2015-07-01

    Relaxin and its receptor RXFP1 are co-expressed in Sertoli cells, and relaxin can stimulate proliferation of Sertoli cells. In this study, we investigated a role of relaxin in spermatogenesis, using a short-term culture of testicular cells of the rat that allowed differentiation of spermatogonia to spermatids. Sertoli, germ, and peritubular myoid cells were the predominant cell types in the culture. Sertoli and germ cells expressed RXFP1. Cultures were incubated without (control) or with 0.5% fetal bovine serum (FBS) or 100 ng/mL H2 relaxin (RLN) for 2 days. Cell organization, number, and differentiation were analyzed after 2 (D2), 5 (D5) or 8 (D8) days of culturing. Although the proportion of germ cells decayed from D2 to D5, the relative contribution of HC, 1C, 2C, and 4C germ cell populations remained constant in the control group during the whole culture. RLN did not affect the proportion of germ cell populations compared with control, but increased gene and/or protein expression of the undifferentiated and differentiated spermatogonia markers PLZF and c-KIT, and of the post-meiotic marker Odf2 in D5. RLN favored organization of cells in tubule-like structures, the arrangement of myoid cells around the tubules, arrangement of c-KIT-positive spermatogonia at the basal region of the tubules, and expression of the cell junction protein β-catenin close to the plasma membrane region. Knockdown of relaxin with small interfering RNA (siRNA) reduced expression of β-catenin at the cell junctions, and shifted its expression to the nucleus. We propose that relaxin may affect spermatogenesis by modulating spermatogonial self renewal and favoring cell contact. © 2015 American Society of Andrology and European Academy of Andrology.

  8. Analysis of high-throughput screening reveals the effect of surface topographies on cellular morphology.

    PubMed

    Hulsman, Marc; Hulshof, Frits; Unadkat, Hemant; Papenburg, Bernke J; Stamatialis, Dimitrios F; Truckenmüller, Roman; van Blitterswijk, Clemens; de Boer, Jan; Reinders, Marcel J T

    2015-03-01

    Surface topographies of materials considerably impact cellular behavior as they have been shown to affect cell growth, provide cell guidance, and even induce cell differentiation. Consequently, for successful application in tissue engineering, the contact interface of biomaterials needs to be optimized to induce the required cell behavior. However, a rational design of biomaterial surfaces is severely hampered because knowledge is lacking on the underlying biological mechanisms. Therefore, we previously developed a high-throughput screening device (TopoChip) that measures cell responses to large libraries of parameterized topographical material surfaces. Here, we introduce a computational analysis of high-throughput materiome data to capture the relationship between the surface topographies of materials and cellular morphology. We apply robust statistical techniques to find surface topographies that best promote a certain specified cellular response. By augmenting surface screening with data-driven modeling, we determine which properties of the surface topographies influence the morphological properties of the cells. With this information, we build models that predict the cellular response to surface topographies that have not yet been measured. We analyze cellular morphology on 2176 surfaces, and find that the surface topography significantly affects various cellular properties, including the roundness and size of the nucleus, as well as the perimeter and orientation of the cells. Our learned models capture and accurately predict these relationships and reveal a spectrum of topographies that induce various levels of cellular morphologies. Taken together, this novel approach of high-throughput screening of materials and subsequent analysis opens up possibilities for a rational design of biomaterial surfaces. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  9. Emergence of an apical epithelial cell surface in vivo

    PubMed Central

    Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B.

    2016-01-01

    Epithelial sheets are crucial components of all metazoan animals, enclosing organs and protecting the animal from its environment. Epithelial homeostasis poses unique challenges, as addition of new cells and loss of old cells must be achieved without disrupting the fluid-tight barrier and apicobasal polarity of the epithelium. Several studies have identified cell biological mechanisms underlying extrusion of cells from epithelia, but far less is known of the converse mechanism by which new cells are added. Here, we combine molecular, pharmacological and laser-dissection experiments with theoretical modelling to characterize forces driving emergence of an apical surface as single nascent cells are added to a vertebrate epithelium in vivo. We find that this process involves the interplay between cell-autonomous actin-generated pushing forces in the emerging cell and mechanical properties of neighboring cells. Our findings define the forces driving this cell behavior, contributing to a more comprehensive understanding of epithelial homeostasis. PMID:26766441

  10. Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics.

    PubMed

    Marín, Elvira; Haesaert, Annelies; Padilla, Laura; Adán, Jaume; Hernáez, María L; Monteoliva, Lucía; Gil, Concha

    2018-01-01

    Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis . These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV.

  11. Unraveling Gardnerella vaginalis Surface Proteins Using Cell Shaving Proteomics

    PubMed Central

    Marín, Elvira; Haesaert, Annelies; Padilla, Laura; Adán, Jaume; Hernáez, María L.; Monteoliva, Lucía; Gil, Concha

    2018-01-01

    Gardnerella vaginalis is one of the main etiologic agents of bacterial vaginosis (BV). This infection is responsible for a wide range of public health costs and is associated with several adverse outcomes during pregnancy. Improving our understanding of G. vaginalis protein cell surface will assist in BV diagnosis. This study represents the first proteomic approach that has analyzed the exposed proteins on G. vaginalis cell surface using a shaving approach. The 261 G. vaginalis proteins identified using this approach were analyzed with bioinformatic tools to detect characteristic motifs from surface-exposed proteins, such as signal peptides (36 proteins), lipobox domains (17 proteins), LPXTG motifs (5 proteins) and transmembrane alpha-helices (66 proteins). One third of the identified proteins were found to have at least one typical motif of surface-exposed proteins. Furthermore, the subcellular location was examined using two predictors (PSORT and Gpos-mPLoc). These bioinformatic tools classified 17% of the identified proteins as surface-associated proteins. Interestingly, we identified 13 members of the ATP-binding cassette (ABC) superfamily, which were mainly involved in the translocation of various substrates across membranes. To validate the location of the G. vaginalis surface-exposed proteins, an immunofluorescence assay with antibodies against Escherichia coli GroEL was performed to reveal the extracellular location of the moonlighting GroEL. In addition, monoclonal antibodies (mAb) against G. vaginalis Cna protein were produced and used to validate the location of Cna on the surface of the G. vaginalis. These high affinity anti-Cna mAb represent a useful tool for the study of this pathogenic microorganism and the BV. PMID:29867878

  12. Cell Surface Expression of Biologically Active Influenza C Virus HEF Glycoprotein Expressed from cDNA

    PubMed Central

    Pekosz, Andrew; Lamb, Robert A.

    1999-01-01

    The hemagglutinin, esterase, and fusion (HEF) glycoprotein of influenza C virus possesses receptor binding, receptor destroying, and membrane fusion activities. The HEF cDNAs from influenza C/Ann Arbor/1/50 (HEF-AA) and influenza C/Taylor/1223/47 (HEF-Tay) viruses were cloned and expressed, and transport of HEF to the cell surface was monitored by susceptibility to cleavage by exogenous trypsin, indirect immunofluorescence microscopy, and flow cytometry. Previously it has been found in studies with the C/Johannesburg/1/66 strain of influenza C virus (HEF-JHB) that transport of HEF to the cell surface is severely inhibited, and it is thought that the short cytoplasmic tail, Arg-Thr-Lys, is involved in blocking HEF cell surface expression (F. Oeffner, H.-D. Klenk, and G. Herrler, J. Gen. Virol. 80:363–369, 1999). As the cytoplasmic tail amino acid sequences of HEF-AA and HEF-Tay are identical to that of HEF-JHB, the data indicate that cell surface expression of HEF-AA and HEF-Tay is not inhibited by this amino acid sequence. Furthermore, the abundant cell surface transport of HEF-AA and HEF-Tay indicates that their cell surface expression does not require coexpression of another viral protein. The HEF-AA and HEF-Tay HEF glycoproteins bound human erythrocytes, promoted membrane fusion in a low-pH and trypsin-dependent manner, and displayed esterase activity, indicating that the HEF glycoprotein alone mediates all three known functions at the cell surface. PMID:10482635

  13. The Arabidopsis SOS5 Locus Encodes a Putative Cell Surface Adhesion Protein and Is Required for Normal Cell Expansion

    PubMed Central

    Shi, Huazhong; Kim, YongSig; Guo, Yan; Stevenson, Becky; Zhu, Jian-Kang

    2003-01-01

    Cell surface proteoglycans have been implicated in many aspects of plant growth and development, but genetic evidence supporting their function has been lacking. Here, we report that the Salt Overly Sensitive5 (SOS5) gene encodes a putative cell surface adhesion protein and is required for normal cell expansion. The sos5 mutant was isolated in a screen for Arabidopsis salt-hypersensitive mutants. Under salt stress, the root tips of sos5 mutant plants swell and root growth is arrested. The root-swelling phenotype is caused by abnormal expansion of epidermal, cortical, and endodermal cells. The SOS5 gene was isolated through map-based cloning. The predicted SOS5 protein contains an N-terminal signal sequence for plasma membrane localization, two arabinogalactan protein–like domains, two fasciclin-like domains, and a C-terminal glycosylphosphatidylinositol lipid anchor signal sequence. The presence of fasciclin-like domains, which typically are found in animal cell adhesion proteins, suggests a role for SOS5 in cell-to-cell adhesion in plants. The SOS5 protein was present at the outer surface of the plasma membrane. The cell walls are thinner in the sos5 mutant, and those between neighboring epidermal and cortical cells in sos5 roots appear less organized. SOS5 is expressed ubiquitously in all plant organs and tissues, including guard cells in the leaf. PMID:12509519

  14. Reaction and Aggregation Dynamics of Cell Surface Receptors

    NASA Astrophysics Data System (ADS)

    Wang, Michelle Dong

    This dissertation is composed of both theoretical and experimental studies of cell surface receptor reaction and aggregation. Project I studies the reaction rate enhancement due to surface diffusion of a bulk dissolved ligand with its membrane embedded target, using numerical calculations. The results show that the reaction rate enhancement is determined by ligand surface adsorption and desorption kinetic rates, surface and bulk diffusion coefficients, and geometry. In particular, we demonstrate that the ligand surface adsorption and desorption kinetic rates, rather than their ratio (the equilibrium constant), are important in rate enhancement. The second and third projects are studies of acetylcholine receptor clusters on cultured rat myotubes using fluorescence techniques after labeling the receptors with tetramethylrhodamine -alpha-bungarotoxin. The second project studies when and where the clusters form by making time-lapse movies. The movies are made from overlay of the pseudocolored total internal reflection fluorescence (TIRF) images of the cluster, and the schlieren images of the cell cultures. These movies are the first movies made using TIRF, and they clearly show the cluster formation from the myoblast fusion, the first appearance of clusters, and the eventual disappearance of clusters. The third project studies the fine structural features of individual clusters observed under TIRF. The features were characterized with six parameters by developing a novel fluorescence technique: spatial fluorescence autocorrelation. These parameters were then used to study the feature variations with age, and with treatments of drugs (oligomycin and carbachol). The results show little variation with age. However, drug treatment induced significant changes in some parameters. These changes were different for oligomycin and carbachol, which indicates that the two drugs may eliminate clusters through different mechanisms.

  15. Seasonal Influences on Ground-Surface Water Interactions in an Arsenic-Affected Aquifer in Cambodia

    NASA Astrophysics Data System (ADS)

    Richards, L. A.; Magnone, D.; Van Dongen, B.; Bryant, C.; Boyce, A.; Ballentine, C. J.; Polya, D. A.

    2015-12-01

    Millions of people in South and Southeast Asia consume drinking water daily which contains dangerous levels of arsenic exceeding health-based recommendations [1]. A key control on arsenic mobilization in aquifers in these areas has been controversially identified as the interaction of 'labile' organic matter contained in surface waters with groundwaters and sediments at depth [2-4], which may trigger the release of arsenic from the solid- to aqueous-phase via reductive dissolution of iron-(hyr)oxide minerals [5]. In a field site in Kandal Province, Cambodia, which is an arsenic-affected area typical to others in the region, there are strong seasonal patterns in groundwater flow direction, which are closely related to monsoonal rains [6] and may contribute to arsenic release in this aquifer. The aim of this study is to explore the implications of the high susceptibility of this aquifer system to seasonal changes on potential ground-surface water interactions. The main objectives are to (i) identify key zones where there are likely ground-surface water interactions, (ii) assess the seasonal impact of such interactions and (iii) quantify the influence of interactions using geochemical parameters (such as As, Fe, NO3, NH4, 14C, 3T/3He, δ18O, δ2H). Identifying the zones, magnitude and seasonal influence of ground-surface water interactions elucidates new information regarding potential locations/pathways of arsenic mobilization and/or transport in affected aquifers and may be important for water management strategies in affected areas. This research is supported by NERC (NE/J023833/1) to DP, BvD and CJB and a NERC PhD studentship (NE/L501591/1) to DM. References: [1] World Health Organization, 2008. [2] Charlet & Polya (2006), Elements, 2, 91-96. [3] Harvey et al. (2002), Science, 298, 1602-1606. [4] Lawson et al. (2013), Env. Sci. Technol. 47, 7085 - 7094. [5] Islam et al. (2004), Nature, 430, 68-71. [6] Benner et al. (2008) Appl. Geochem. 23(11), 3072 - 3087.

  16. Sorting of Marburg Virus Surface Protein and Virus Release Take Place at Opposite Surfaces of Infected Polarized Epithelial Cells

    PubMed Central

    Sänger, Christian; Mühlberger, Elke; Ryabchikova, Elena; Kolesnikova, Larissa; Klenk, Hans-Dieter; Becker, Stephan

    2001-01-01

    Marburg virus, a filovirus, causes severe hemorrhagic fever with hitherto poorly understood molecular pathogenesis. We have investigated here the vectorial transport of the surface protein GP of Marburg virus in polarized epithelial cells. To this end, we established an MDCKII cell line that was able to express GP permanently (MDCK-GP). The functional integrity of GP expressed in these cells was analyzed using vesicular stomatitis virus pseudotypes. Further experiments revealed that GP is transported in MDCK-GP cells mainly to the apical membrane and is released exclusively into the culture medium facing the apical membrane. When MDCKII cells were infected with Marburg virus, the majority of GP was also transported to the apical membrane, suggesting that the protein contains an autonomous apical transport signal. Release of infectious progeny virions, however, took place exclusively at the basolateral membrane of the cells. Thus, vectorial budding of Marburg virus is presumably determined by factors other than the surface protein. PMID:11152500

  17. Fibrillar Structure and Charge Determine the Interaction of Polyglutamine Protein Aggregates with the Cell Surface*

    PubMed Central

    Trevino, R. Sean; Lauckner, Jane E.; Sourigues, Yannick; Pearce, Margaret M.; Bousset, Luc; Melki, Ronald; Kopito, Ron R.

    2012-01-01

    The pathogenesis of most neurodegenerative diseases, including transmissible diseases like prion encephalopathy, inherited disorders like Huntington disease, and sporadic diseases like Alzheimer and Parkinson diseases, is intimately linked to the formation of fibrillar protein aggregates. It is becoming increasingly appreciated that prion-like intercellular transmission of protein aggregates can contribute to the stereotypical spread of disease pathology within the brain, but the mechanisms underlying the binding and uptake of protein aggregates by mammalian cells are largely uninvestigated. We have investigated the properties of polyglutamine (polyQ) aggregates that endow them with the ability to bind to mammalian cells in culture and the properties of the cell surface that facilitate such uptake. Binding and internalization of polyQ aggregates are common features of mammalian cells and depend upon both trypsin-sensitive and trypsin-resistant saturable sites on the cell surface, suggesting the involvement of cell surface proteins in this process. polyQ aggregate binding depends upon the presence of a fibrillar amyloid-like structure and does not depend upon electrostatic interaction of fibrils with the cell surface. Sequences in the huntingtin protein that flank the amyloid-forming polyQ tract also influence the extent to which aggregates are able to bind to cell surfaces. PMID:22753412

  18. Wrinkled, wavelength-tunable graphene-based surface topographies for directing cell alignment and morphology

    PubMed Central

    Wang, Zhongying; Tonderys, Daniel; Leggett, Susan E.; Williams, Evelyn Kendall; Kiani, Mehrdad T.; Steinberg, Ruben Spitz; Qiu, Yang; Wong, Ian Y.; Hurt, Robert H.

    2015-01-01

    Textured surfaces with periodic topographical features and long-range order are highly attractive for directing cell-material interactions. They mimic physiological environments more accurately than planar surfaces and can fundamentally alter cell alignment, shape, gene expression, and cellular assembly into superstructures or microtissues. Here we demonstrate for the first time that wrinkled graphene-based surfaces are suitable as textured cell attachment substrates, and that engineered wrinkling can dramatically alter cell alignment and morphology. The wrinkled surfaces are fabricated by graphene oxide wet deposition onto pre-stretched elastomers followed by relaxation and mild thermal treatment to stabilize the films in cell culture medium. Multilayer graphene oxide films form periodic, delaminated buckle textures whose wavelengths and amplitudes can be systematically tuned by variation in the wet deposition process. Human and murine fibroblasts attach to these textured films and remain viable, while developing pronounced alignment and elongation relative to those on planar graphene controls. Compared to lithographic patterning of nanogratings, this method has advantages in the simplicity and scalability of fabrication, as well as the opportunity to couple the use of topographic cues with the unique conductive, adsorptive, or barrier properties of graphene materials for functional biomedical devices. PMID:25848137

  19. Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR.

    PubMed

    Yin, Yancun; Chen, Chen; Chen, Jinliang; Zhan, Renhui; Zhang, Qiang; Xu, Xiaoyan; Li, Defang; Li, Minjing

    2017-07-01

    The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (-)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Surface glycosaminoglycans mediate adherence between HeLa cells and Lactobacillus salivarius Lv72.

    PubMed

    Martín, Rebeca; Martín, Carla; Escobedo, Susana; Suárez, Juan E; Quirós, Luis M

    2013-09-17

    The adhesion of lactobacilli to the vaginal surface is of paramount importance to develop their probiotic functions. For this reason, the role of HeLa cell surface proteoglycans in the attachment of Lactobacillus salivarius Lv72, a mutualistic strain of vaginal origin, was investigated. Incubation of cultures with a variety of glycosaminoglycans (chondroitin sulfate A and C, heparin and heparan sulfate) resulted in marked binding interference. However, no single glycosaminoglycan was able to completely abolish cell binding, the sum of all having an additive effect that suggests cooperation between them and recognition of specific adhesins on the bacterial surface. In contrast, chondroitin sulfate B enhanced cell to cell attachment, showing the relevance of the stereochemistry of the uronic acid and the sulfation pattern on binding. Elimination of the HeLa surface glycosaminoglycans with lyases also resulted in severe adherence impairment. Advantage was taken of the Lactobacillus-glycosaminoglycans interaction to identify an adhesin from the bacterial surface. This protein, identify as a soluble binding protein of an ABC transporter system (OppA) by MALDI-TOF/(MS), was overproduced in Escherichia coli, purified and shown to interfere with L. salivarius Lv72 adhesion to HeLa cells. These data suggest that glycosaminoglycans play a fundamental role in attachment of mutualistic bacteria to the epithelium that lines the cavities where the normal microbiota thrives, OppA being a bacterial adhesin involved in the process.