Development of synthetic media mimicking food soils to study the behaviour of Listeria monocytogenes on stainless steel surfaces.

PubMed

Overney, Anaïs; Chassaing, Danielle; Carpentier, Brigitte; Guillier, Laurent; Firmesse, Olivier

2016-12-05

Listeria monocytogenes is one of the main targets of hygiene procedures in the ready-to-eat food industry due to its ability to persist for months or even years in processing plants, where it can contaminate food during processing. The factors associated with persistence are often those that foster growth, which itself depends on food contamination of surfaces. It is therefore essential to experiment by using food soils or media modelling these soils to understand the behaviour of L. monocytogenes on surfaces of food processing plants. Thus, we set up an experimental plan including three physiological parameters characteristic of the behaviour of cells on surfaces, namely spatial distribution, adhesion forces and the physiological state of sessile L. monocytogenes. These were recorded in two food soils: smoked salmon juice and meat exudate. According to our results, the behaviour of L. monocytogenes on stainless steel surfaces is highly dependent on the food soil used. The presence of viable but non-culturable (VBNC) cells was demonstrated using meat exudate, while all viable cells were recovered using smoked salmon juice. Moreover, on the basis of our criteria and after validation with three strains of L. monocytogenes, we showed that smoked salmon juice can be substituted by a modified culture medium, demonstrating that drawbacks associated with the use of food soils can be overcome. Copyright © 2016 Elsevier B.V. All rights reserved.

Structural Analysis of the Synaptic Protein Neuroligin and Its β-Neurexin Complex: Determinants for Folding and Cell Adhesion

PubMed Central

Fabrichny, Igor P.; Leone, Philippe; Sulzenbacher, Gerlind; Comoletti, Davide; Miller, Meghan T.; Taylor, Palmer; Bourne, Yves; Marchot, Pascale

2009-01-01

SUMMARY The neuroligins are postsynaptic cell adhesion proteins whose associations with presynaptic neurexins participate in synaptogenesis. Mutations in the neuroligin and neurexin genes appear to be associated with autism and mental retardation. The crystal structure of a neuroligin reveals features not found in its catalytically active relatives, such as the fully hydrophobic interface forming the functional neuroligin dimer; the conformations of surface loops surrounding the vestigial active center; the location of determinants that are critical for folding and processing; and the absence of a macromolecular dipole and presence of an electronegative, hydrophilic surface for neurexin binding. The structure of a β-neurexin-neuroligin complex reveals the precise orientation of the bound neurexin and, despite a limited resolution, provides substantial information on the Ca2+-dependent interactions network involved in trans-synaptic neurexin-neuroligin association. These structures exemplify how an α/β-hydrolase fold varies in surface topography to confer adhesion properties and provide templates for analyzing abnormal processing or recognition events associated with autism. PMID:18093521

Biofilms associated with poultry processing equipment.

PubMed

Lindsay, D; Geornaras, I; von Holy, A

1996-01-01

Aerobic and Gram-negative bacteria were enumerated on non-metallic surfaces and stainless steel test pieces attached to equipment surfaces by swabbing and a mechanical dislodging procedure, respectively, in a South African grade B poultry processing plant. Changes in bacterial numbers were also monitored over time on metal test pieces. The highest bacterial counts were obtained from non-metallic surfaces such as rubber fingered pluckers and plastic defeathering curtains which exceeded the highest counts found on the metal surfaces by at least 1 log CFU cm-2. Gram-negative bacterial counts on all non-metallic surface types were at least 2 log CFU cm-2 lower than corresponding aerobic plate counts. On metal surfaces, the highest microbial numbers were obtained after 14 days exposure, with aerobic plate counts ranging from 3.57 log CFU cm-2 to 5.13 log CFU cm-2, and Gram-negative counts from 0.70 log CFU cm-2 to 3.31 log CFU cm-2. Scanning electron microscopy confirmed the presence of bacterial cells on non-metallic and metallic surfaces associated with poultry processing. Rubber 'fingers', plastic curtains, conveyor belt material and stainless steel test surfaces placed on the scald tank overflow and several chutes revealed extensive and often confluent bacterial biofilms. Extracellular polymeric substances, but few bacterial cells were visible on test pieces placed on evisceration equipment, spinchiller blades and the spinchiller outlet.

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion

PubMed Central

Knuchel, Sarah; Anderle, Pascale; Werfelli, Patricia; Diamantis, Eva; Rüegg, Curzio

2015-01-01

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion. PMID:25973543

Fibroblast surface-associated FGF-2 promotes contact-dependent colorectal cancer cell migration and invasion through FGFR-SRC signaling and integrin αvβ5-mediated adhesion.

PubMed

Knuchel, Sarah; Anderle, Pascale; Werfelli, Patricia; Diamantis, Eva; Rüegg, Curzio

2015-06-10

Carcinoma-associated fibroblasts were reported to promote colorectal cancer (CRC) invasion by secreting motility factors and extracellular matrix processing enzymes. Less is known whether fibroblasts may induce CRC cancer cell motility by contact-dependent mechanisms. To address this question we characterized the interaction between fibroblasts and SW620 and HT29 colorectal cancer cells in 2D and 3D co-culture models in vitro. Here we show that fibroblasts induce contact-dependent cancer cell elongation, motility and invasiveness independently of deposited matrix or secreted factors. These effects depend on fibroblast cell surface-associated fibroblast growth factor (FGF) -2. Inhibition of FGF-2 or FGF receptors (FGFRs) signaling abolishes these effects. FGFRs activate SRC in cancer cells and inhibition or silencing of SRC in cancer cells, but not in fibroblasts, prevents fibroblasts-mediated effects. Using an RGD-based integrin antagonist and function-blocking antibodies we demonstrate that cancer cell adhesion to fibroblasts requires integrin αvβ5. Taken together, these results demonstrate that fibroblasts induce cell-contact-dependent colorectal cancer cell migration and invasion under 2D and 3D conditions in vitro through fibroblast cell surface-associated FGF-2, FGF receptor-mediated SRC activation and αvβ5 integrin-dependent cancer cell adhesion to fibroblasts. The FGF-2-FGFRs-SRC-αvβ5 integrin loop might be explored as candidate therapeutic target to block colorectal cancer invasion.

The influence of surface properties of plasma-etched polydimethylsiloxane (PDMS) on cell growth and morphology.

PubMed

Pennisi, Cristian P; Zachar, Vladimir; Gurevich, Leonid; Patriciu, Andrei; Struijk, Johannes J

2010-01-01

Polydimethylsiloxane (PDMS) or silicone rubber is a widely used implant material. Approaches to promote tissue integration to PDMS are desirable to avoid clinical problems associated with sliding and friction between tissue and implant. Plasma-etching is a useful way to control cell behavior on PDMS without additional coatings. In this work, different plasma processing conditions were used to modify the surface properties of PDMS substrates. Surface nanotopography and wettability were measured to study their effect on in vitro growth and morphology of fibroblasts. While fluorinated plasma treatments produced nanorough hydrophobic and superhydrophobic surfaces that had negative or little influences on cellular behavior, water vapor/oxygen plasma produced smooth hydrophillic surfaces that enhanced cell growth.

Expression of TNF-alpha and immunohistochemical distribution of hepatic macrophage surface markers in carbon tetrachloride-induced chronic liver injury in rats.

PubMed

Orfila, C; Lepert, J C; Alric, L; Carrera, G; Beraud, M; Vinel, J P; Pipy, B

1999-10-01

In liver injury induced by carbon tetrachloride, secondary hepatic injury occurs from inflammatory processes originating from products released by activated Kupffer cells, which play a central role in hepatic inflammation. The purpose of our study was to demonstrate, in rats, the relationships between a function of the hepatic macrophages, TNF-alpha production and the state of activation of these cells, characterized by their phenotype, in the different phases of the process and development of fibrosis in a carbon tetrachloride-induced cirrhosis model. The immunohistochemical localization of proinflammatory cytokine TNF-alpha and surface surface makers (ED1 and ED2) was studied in hepatitis and cirrhosis in response to 3 and 9 weeks ingestion of carbon tetrachloride. After carbon tetrachloride ingestion, accompanying the increased necrosis, immunohistochemical analysis of liver tissue sections demonstrated the significantly increased number of cells expressing ED1, ED2 and TNF-alpha, compared to normal. The number of cells expressing the surface phenotypic markers of liver macrophages increased and this change was concomitantly associated with an increased cellular expression of TNF-alpha. Local macrophage proliferation and influx of newly recruited blood monocytes resulted in an increase of the macrophage population. The populational changes involved difference in functional activity and enhanced TNF-alpha expression. This cytokine expressed in the carbon tetrachloride-induced inflammatory process is associated with the development of fibrosis and may contribute to disease severity.

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.

Synergistic responses of superficial chemistry and micro topography of titanium created by wire-type electric discharge machining.

PubMed

Kataoka, Yu; Tamaki, Yukimichi; Miyazaki, Takashi

2011-01-01

Wire-type electric discharge machining has been applied to the manufacture of endosseous titanium implants as this computer associated technique allows extremely accurate complex sample shaping with an optimal micro textured surface during the processing. Since the titanium oxide layer is sensitively altered by each processing, the authors hypothesized that this technique also up-regulates biological responses through the synergistic effects of the superficial chemistry and micro topography. To evaluate the respective in vitro cellular responses on the superficial chemistry and micro topography of titanium surface processed by wire-type electric discharge, we used titanium-coated epoxy resin replica of the surface. An oxide layer on the titanium surface processed by wire-type electric discharge activated the initial responses of osteoblastic cells through an integrin-mediated mechanism. Since the mRNA expression of ALP on those replicas was up-regulated compared to smooth titanium samples, the micro topography of a titanium surface processed by wire-type electric discharge promotes the osteogenic potential of cells. The synergistic response of the superficial chemistry and micro topography of titanium processed by wire-type electric discharge was demonstrated in this study.

Ectodomain shedding of Limbic System-Associated Membrane Protein (LSAMP) by ADAM Metallopeptidases promotes neurite outgrowth in DRG neurons.

PubMed

Sanz, Ricardo L; Ferraro, Gino B; Girouard, Marie-Pier; Fournier, Alyson E

2017-08-11

IgLONs are members of the immunoglobulin superfamily of cell adhesion proteins implicated in the process of neuronal outgrowth, cell adhesion and subdomain target recognition. IgLONs form homophilic and heterophilic complexes on the cell surface that repress or promote growth depending on the neuronal population, the developmental stage and surface repertoire of IgLON family members. In the present study, we identified a metalloproteinase-dependent mechanism necessary to promote growth in embryonic dorsal root ganglion cells (DRGs). Treatment of embryonic DRG neurons with pan-metalloproteinase inhibitors, tissue inhibitor of metalloproteinase-3, or an inhibitor of ADAM Metallopeptidase Domain 10 (ADAM10) reduces outgrowth from DRG neurons indicating that metalloproteinase activity is important for outgrowth. The IgLON family members Neurotrimin (NTM) and Limbic System-Associated Membrane Protein (LSAMP) were identified as ADAM10 substrates that are shed from the cell surface of DRG neurons. Overexpression of LSAMP and NTM suppresses outgrowth from DRG neurons. Furthermore, LSAMP loss of function decreases the outgrowth sensitivity to an ADAM10 inhibitor. Together our findings support a role for ADAM-dependent shedding of cell surface LSAMP in promoting outgrowth from DRG neurons.

In vitro osteoinduction of human mesenchymal stem cells in biomimetic surface modified titanium alloy implants.

PubMed

Santander, Sonia; Alcaine, Clara; Lyahyai, Jaber; Pérez, Maria Angeles; Rodellar, Clementina; Doblaré, Manuel; Ochoa, Ignacio

2012-01-01

Interaction between cells and implant surface is crucial for clinical success. This interaction and the associated surface treatment are essential for achieving a fast osseointegration process. Several studies of different topographical or chemical surface modifications have been proposed previously in literature. The Biomimetic Advanced Surface (BAS) topography is a combination of a shot blasting and anodizing procedure. Macroroughness, microporosity of titanium oxide and Calcium/Phosphate ion deposition is obtained. Human mesenchymal stem cells (hMCSs) response in vitro to this treatment has been evaluated. The results obtained show an improved adhesion capacity and a higher proliferation rate when hMSCs are cultured on treated surfaces. This biomimetic modification of the titanium surface induces the expression of osteblastic differentiation markers (RUNX2 and Osteopontin) in the absence of any externally provided differentiation factor. As a main conclusion, our biomimetic surface modification could lead to a substantial improvement in osteoinduction in titanium alloy implants.

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

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.

Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus

PubMed Central

Wolfisberg, Raphael; Kempf, Christoph

2016-01-01

ABSTRACT Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. IMPORTANCE In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle. PMID:27009963

Late Maturation Steps Preceding Selective Nuclear Export and Egress of Progeny Parvovirus.

PubMed

Wolfisberg, Raphael; Kempf, Christoph; Ros, Carlos

2016-06-01

Although the mechanism is not well understood, growing evidence indicates that the nonenveloped parvovirus minute virus of mice (MVM) may actively egress before passive release through cell lysis. We have dissected the late maturation steps of the intranuclear progeny with the aims of confirming the existence of active prelytic egress and identifying critical capsid rearrangements required to initiate the process. By performing anion-exchange chromatography (AEX), we separated intranuclear progeny particles by their net surface charges. Apart from empty capsids (EC), two distinct populations of full capsids (FC) arose in the nuclei of infected cells. The earliest population of FC to appear was infectious but, like EC, could not be actively exported from the nucleus. Further maturation of this early population, involving the phosphorylation of surface residues, gave rise to a second, late population with nuclear export potential. While capsid surface phosphorylation was strictly associated with nuclear export capacity, mutational analysis revealed that the phosphoserine-rich N terminus of VP2 (N-VP2) was dispensable, although it contributed to passive release. The reverse situation was observed for the incoming particles, which were dephosphorylated in the endosomes. Our results confirm the existence of active prelytic egress and reveal a late phosphorylation event occurring in the nucleus as a selective factor for initiating the process. In general, the process of egress of enveloped viruses is active and involves host cell membranes. However, the release of nonenveloped viruses seems to rely more on cell lysis. At least for some nonenveloped viruses, an active process before passive release by cell lysis has been reported, although the underlying mechanism remains poorly understood. By using the nonenveloped model parvovirus minute virus of mice, we could confirm the existence of an active process of nuclear export and further characterize the associated capsid maturation steps. Following DNA packaging in the nucleus, capsids required further modifications, involving the phosphorylation of surface residues, to acquire nuclear export potential. Inversely, those surface residues were dephosphorylated on entering capsids. These spatially controlled phosphorylation-dephosphorylation events concurred with the nuclear export-import potential required to complete the infectious cycle. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Emergence of fractal geometry on the surface of human cervical epithelial cells during progression towards cancer

NASA Astrophysics Data System (ADS)

Dokukin, M. E.; Guz, N. V.; Woodworth, C. D.; Sokolov, I.

2015-03-01

Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation.

Emerging of fractal geometry on surface of human cervical epithelial cells during progression towards cancer

PubMed Central

Dokukin, M. E.; Guz, N. V.; Woodworth, C.D.; Sokolov, I.

2015-01-01

Despite considerable advances in understanding the molecular nature of cancer, many biophysical aspects of malignant development are still unclear. Here we study physical alterations of the surface of human cervical epithelial cells during stepwise in vitro development of cancer (from normal to immortal (premalignant), to malignant). We use atomic force microscopy to demonstrate that development of cancer is associated with emergence of simple fractal geometry on the cell surface. Contrary to the previously expected correlation between cancer and fractals, we find that fractal geometry occurs only at a limited period of development when immortal cells become cancerous; further cancer progression demonstrates deviation from fractal. Because of the connection between fractal behaviour and chaos (or far from equilibrium behaviour), these results suggest that chaotic behaviour coincides with the cancer transformation of the immortalization stage of cancer development, whereas further cancer progression recovers determinism of processes responsible for cell surface formation. PMID:25844044

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.

Extracellular heat shock protein HSP90{beta} secreted by MG63 osteosarcoma cells inhibits activation of latent TGF-{beta}1

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

Suzuki, Shigeki; Kulkarni, Ashok B., E-mail: ak40m@nih.gov

2010-07-30

Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex, which consists of latency-associated peptide (LAP) and the mature ligand. The release of the mature ligand from LAP usually occurs through conformational change of the latent complex and is therefore considered to be the first step in the activation of the TGF-{beta} signaling pathway. So far, factors such as heat, pH changes, and proteolytic cleavage are reportedly involved in this activation process, but the precise molecular mechanism is still far from clear. Identification and characterization of the cell surface proteins that bind to LAP are important to our understandingmore » of the latent TGF-{beta} activation process. In this study, we have identified heat shock protein 90 {beta} (HSP90{beta}) from the cell surface of the MG63 osteosarcoma cell line as a LAP binding protein. We have also found that MG63 cells secrete HSP90{beta} into extracellular space which inhibits the activation of latent TGF-{beta}1, and that there is a subsequent decrease in cell proliferation. TGF-{beta}1-mediated stimulation of MG63 cells resulted in the increased cell surface expression of HSP90{beta}. Thus, extracellular HSP90{beta} is a negative regulator for the activation of latent TGF-{beta}1 modulating TGF-{beta} signaling in the extracellular domain. -- Research highlights: {yields} Transforming growth factor-beta 1 (TGF-{beta}1) is secreted as a latent complex. {yields} This complex consists of latency-associated peptide (LAP) and the mature ligand. {yields} The release of the mature ligand from LAP is the first step in TGF-{beta} activation. {yields} We identified for the first time a novel mechanism for this activation process. {yields} Heat shock protein 90 {beta} is discovered as a negative regulator for this process.« less

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

Differential requirement for cathepsin D for processing of the full length and C-terminal fragment of the malaria antigen MSP1.

PubMed

Tulone, Calogero; Sponaas, Anne-Marit; Raiber, Eun-Ang; Tabor, Alethea B; Langhorne, Jean; Chain, Benny M

2011-01-01

Merozoite Surface Protein 1 is expressed on the surface of malaria merozoites and is important for invasion of the malaria parasite into erythrocytes. MSP1-specific CD4 T cell responses and antibody can confer protective immunity in experimental models of malaria. In this study we explore the contributions of cathepsins D and E, two aspartic proteinases previously implicated in antigen processing, to generating MSP1 CD4 T-cell epitopes for presentation. The absence of cathepsin D, a late endosome/lysosomal enzyme, is associated with a reduced presentation of MSP1 both following in vitro processing of the epitope MSP1 from infected erythrocytes by bone marrow-derived dendritic cells, and following in vivo processing by splenic CD11c+ dendritic cells. By contrast, processing and presentation of the soluble recombinant protein fragment of MSP1 is unaffected by the absence of cathepsin D, but is inhibited when both cathepsin D and E are absent. The role of different proteinases in generating the CD4 T cell repertoire, therefore, depends on the context in which an antigen is introduced to the immune system.

Differential Requirement for Cathepsin D for Processing of the Full Length and C-Terminal Fragment of the Malaria Antigen MSP1

PubMed Central

Raiber, Eun-Ang; Tabor, Alethea B.; Langhorne, Jean; Chain, Benny M.

2011-01-01

Merozoite Surface Protein 1 is expressed on the surface of malaria merozoites and is important for invasion of the malaria parasite into erythrocytes. MSP1-specific CD4 T cell responses and antibody can confer protective immunity in experimental models of malaria. In this study we explore the contributions of cathepsins D and E, two aspartic proteinases previously implicated in antigen processing, to generating MSP1 CD4 T-cell epitopes for presentation. The absence of cathepsin D, a late endosome/lysosomal enzyme, is associated with a reduced presentation of MSP1 both following in vitro processing of the epitope MSP1 from infected erythrocytes by bone marrow-derived dendritic cells, and following in vivo processing by splenic CD11c+ dendritic cells. By contrast, processing and presentation of the soluble recombinant protein fragment of MSP1 is unaffected by the absence of cathepsin D, but is inhibited when both cathepsin D and E are absent. The role of different proteinases in generating the CD4 T cell repertoire, therefore, depends on the context in which an antigen is introduced to the immune system. PMID:22053177

Protein phosphatase 2A mediates resensitization of the neurokinin 1 receptor

PubMed Central

Murphy, Jane E.; Roosterman, Dirk; Cottrell, Graeme S.; Padilla, Benjamin E.; Feld, Micha; Brand, Eva; Cedron, Wendy J.; Steinhoff, Martin

2011-01-01

Activated G protein-coupled receptors (GPCRs) are phosphorylated and interact with β-arrestins, which mediate desensitization and endocytosis. Endothelin-converting enzyme-1 (ECE-1) degrades neuropeptides in endosomes and can promote recycling. Although endocytosis, dephosphorylation, and recycling are accepted mechanisms of receptor resensitization, a large proportion of desensitized receptors can remain at the cell surface. We investigated whether reactivation of noninternalized, desensitized (phosphorylated) receptors mediates resensitization of the substance P (SP) neurokinin 1 receptor (NK1R). Herein, we report a novel mechanism of resensitization by which protein phosphatase 2A (PP2A) is recruited to dephosphorylate noninternalized NK1R. A desensitizing concentration of SP reduced cell-surface SP binding sites by only 25%, and SP-induced Ca2+ signals were fully resensitized before cell-surface binding sites started to recover, suggesting resensitization of cell-surface-retained NK1R. SP induced association of β-arrestin1 and PP2A with noninternalized NK1R. β-Arrestin1 small interfering RNA knockdown prevented SP-induced association of cell-surface NK1R with PP2A, indicating that β-arrestin1 mediates this interaction. ECE-1 inhibition, by trapping β-arrestin1 in endosomes, also impeded SP-induced association of cell-surface NK1R with PP2A. Resensitization of NK1R signaling required both PP2A and ECE-1 activity. Thus, after stimulation with SP, PP2A interacts with noninternalized NK1R and mediates resensitization. PP2A interaction with NK1R requires β-arrestin1. ECE-1 promotes this process by releasing β-arrestin1 from NK1R in endosomes. These findings represent a novel mechanism of PP2A- and ECE-1-dependent resensitization of GPCRs. PMID:21795521

Environmental Stress-mediated EPS Production Shape Microbial Activity on Various Hydrated Rough Surfaces

NASA Astrophysics Data System (ADS)

Wang, G.; Liu, L.; Chen, G.

2016-12-01

The complex environmental physical and chemical processes and interplay with the associating biological responses are keys to understanding the environmental microbiology ensconced in environmental remediation, water quality control, food safety, nutrient cycling, and etc., yet remain poorly understood. Using experimental micromodels, we study how environmental conditions (e.g., hydration fluctuation, nutrient limitation, pH variation, etc.) affect microbial extracellular polymeric substances (EPS) production and their configuration within various hydrated surfaces, and impacts on microbial motility, surface attachment, aggregation, and other bioremediation activities. To elucidate the potential mechanisms underlying the complex bio-physicochemical processes, we developed an individual-based and spatio-temporally resolved modeling platform that explicitly considers microscale aqueous-phase configuration and nutrient transport/diffusion and associated biophysical processes affecting individual microbial cell life history. We quantitatively explore the effects of the above microscale environmental processes on bio-physicochemical interactions affecting microbial growth, motility, surface attachment and aggregation, and shaping population interactions and functions. Simulation scenarios of microbial induced pollutant (e.g., roxarsone) biotransformation on various hydrated rough surfaces will also be present.

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.

Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1.

PubMed

Maher, Christina M; Thomas, Jeffrey D; Haas, Derick A; Longen, Charles G; Oyer, Halley M; Tong, Jane Y; Kim, Felix J

2018-02-01

Emerging evidence suggests that Sigma1 ( SIGMAR1 , also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)-associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress-associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy. Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg Mol Cancer Res; 16(2); 243-55. ©2017 AACR . ©2017 American Association for Cancer Research.

Enhanced osteogenic differentiation of MC3T3-E1 cells on grid-topographic surface and evidence for involvement of YAP mediator.

PubMed

Zhang, Yingying; Gong, He; Sun, Yan; Huang, Yan; Fan, Yubo

2016-05-01

Numerous studies have shown that surface topography can promote cell-substrate associations and deeply influence cell fate. The intracellular mechanism or how micro- or nano-patterned extracellular signal is ultimately linked to activity of nuclear transcription factors remains unknown. It has been reported that Yes-associated protein (YAP) can respond to extracellular matrix microenvironment signals, thus regulates stem cell differentiation process. We propose that YAP may play a role in mediating the topography induced cell differentiation. To this end, we fabricated polydimethylsiloxane (PDMS) micropatterns with grid topology (GT) (3 μm pattern width, 2 μm pattern interval length, 7 μm pattern height); nonpatterned PDMS substrates were used as the planar controls. The MC3T3-E1 cells were then cultured on these surfaces, respectively, in osteogenic inducing medium. Cell differentiation in terms of osteogenesis related gene expression, protein levels, alkaline phosphatase activity and extracellular matrix mineralization was assessed. It was shown that the cells on GT surfaces had stronger osteogenesis capacity. In addition, expression level of YAP was increased when MC3T3-E1 cells grew on GT substrates, which was similar to the levels of osteogenic differentiation markers. It was also shown that YAP knockdown attenuated GT substrates-induced MC3T3-E1 differentiation, which reduced the osteogenic differentiation effect of the GT substrates. Collectively, our findings indicate that GT substrates-induced MC3T3-E1 differentiation may be associated with YAP. This paper provides new target points for transcriptional mechanism research of microenvironment induced cell differentiation and a useful approach to obtain more biofunctionalization scaffolds for tissue engineering. © 2016 Wiley Periodicals, Inc.

Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.

PubMed

Cowles, Kimberly N; Gitai, Zemer

2010-06-01

Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.

Immunity to Intracellular Salmonella Depends on Surface-associated Antigens

PubMed Central

Claudi, Beatrice; Mazé, Alain; Schemmer, Anne K.; Kirchhoff, Dennis; Schmidt, Alexander; Burton, Neil; Bumann, Dirk

2012-01-01

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens. PMID:23093937

SRC-like adaptor protein regulates B cell development and function.

PubMed

Dragone, Leonard L; Myers, Margaret D; White, Carmen; Sosinowski, Tomasz; Weiss, Arthur

2006-01-01

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

PubMed Central

Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

2012-01-01

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

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

Purvina, Maija; Hoste, Astrid; Rossignol, Jean-Michel

Highlights: Black-Right-Pointing-Pointer P20, precursor of the HBeAg, interacts with the cellular protein gC1qR. Black-Right-Pointing-Pointer HBeAg and P20 bind to T cell surface and inhibit mitogen-induced T cell division. Black-Right-Pointing-Pointer HBeAg and P20 inhibition of T cell proliferation is gC1qR and IL-1RAcP-independent. -- Abstract: The hepatitis B virus (HBV) Precore protein is processed through the secretory pathway directly as HBeAg or with the generation of an intermediate (P20). Precore gene has been shown to be implicated in viral persistence, but the functions of HBeAg and its precursors have not been fully elucidated. We show that the secreted proteins HBeAg and P20more » interact with T cell surface and alter Kit-225 and primary T cells proliferation, a process which may facilitate the establishment of HBV persistence. Our data indicate that the N-terminal end of Precore is important for these inhibitory effects and exclude that they are dependent on the association of HBeAg and P20 with two characterized cell surface ligands, the Interleukin-1 Receptor Accessory Protein and gC1qR (present study).« less

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex.

PubMed

Holt, G D; Swiedler, S J; Freed, J H; Hart, G W

1985-07-01

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.

Non-canonical features of the Golgi apparatus in bipolar epithelial neural stem cells

PubMed Central

Taverna, Elena; Mora-Bermúdez, Felipe; Strzyz, Paulina J.; Florio, Marta; Icha, Jaroslav; Haffner, Christiane; Norden, Caren; Wilsch-Bräuninger, Michaela; Huttner, Wieland B.

2016-01-01

Apical radial glia (aRG), the stem cells in developing neocortex, are unique bipolar epithelial cells, extending an apical process to the ventricle and a basal process to the basal lamina. Here, we report novel features of the Golgi apparatus, a central organelle for cell polarity, in mouse aRGs. The Golgi was confined to the apical process but not associated with apical centrosome(s). In contrast, in aRG-derived, delaminating basal progenitors that lose apical polarity, the Golgi became pericentrosomal. The aRG Golgi underwent evolutionarily conserved, accordion-like compression and extension concomitant with cell cycle-dependent nuclear migration. Importantly, in line with endoplasmic reticulum but not Golgi being present in the aRG basal process, its plasma membrane contained glycans lacking Golgi processing, consistent with direct ER-to-cell surface membrane traffic. Our study reveals hitherto unknown complexity of neural stem cell polarity, differential Golgi contribution to their specific architecture, and fundamental Golgi re-organization upon cell fate change. PMID:26879757

Differential proteomic analysis of a human breast tumor and its matched bone metastasis identifies cell membrane and extracellular proteins associated with bone metastasis.

PubMed

Dumont, Bruno; Castronovo, Vincent; Peulen, Olivier; Blétard, Noëlla; Clézardin, Philippe; Delvenne, Philippe; De Pauw, Edwin A; Turtoi, Andrei; Bellahcène, Akeila

2012-04-06

The classical fate of metastasizing breast cancer cells is to seed and form secondary colonies in bones. The molecules closely associated with these processes are predominantly present at the cell surface and in the extracellular space, establishing the first contacts with the target tissue. In this study, we had the rare opportunity to analyze a bone metastatic lesion and its corresponding breast primary tumor obtained simultaneously from the same patient. Using mass spectrometry, we undertook a proteomic study on cell surface and extracellular protein-enriched material. We provide a repertoire of significantly modulated proteins, some with yet unknown roles in the bone metastatic process as well as proteins notably involved in cancer cell invasiveness and in bone metabolism. The comparison of these clinical data with those previously obtained using a human osteotropic breast cancer cell line highlighted an overlapping group of proteins. Certain differentially expressed proteins are validated in the present study using immunohistochemistry on a retrospective collection of breast tumors and matched bone metastases. Our exclusive set of selected proteins supports the setup of further investigations on both clinical samples and experimental bone metastasis models that will help to reveal the finely coordinated expression of proteins that favor the development of metastases in the bone microenvironment.

Adhesion of Chlamydomonas microalgae to surfaces is switchable by light

NASA Astrophysics Data System (ADS)

Kreis, Christian Titus; Le Blay, Marine; Linne, Christine; Makowski, Marcin Michal; Bäumchen, Oliver

2018-01-01

Microalgae are photoactive microbes that live in liquid-infused environments, such as soil, temporary pools and rocks, where they encounter and colonize a plethora of surfaces. Their photoactivity manifests itself in a variety of processes, including light-directed motility (phototaxis), the growth of microalgal populations, and their photosynthetic machinery. Although microbial responses to light have been widely recognized, any influence of light on cell-surface interactions remains elusive. Here, we reveal that the unspecific adhesion of microalgae to surfaces can be reversibly switched on and off by light. Using a micropipette force spectroscopy technique, we measured in vivo single-cell adhesion forces and show that the microalga's flagella provide light-switchable adhesive contacts with the surface. This light-induced adhesion to surfaces is an active and completely reversible process that occurs on a timescale of seconds. Our results suggest that light-switchable adhesiveness is a natural functionality of microalgae to regulate the transition between the planktonic and the surface-associated state, which yields an adhesive adaptation to optimize the photosynthetic efficiency in conjunction with phototaxis.

Cell density related H2 consumption in relation to anoxic Fe(0) corrosion and precipitation of corrosion products by Shewanella oneidensis MR-1.

PubMed

De Windt, Wim; Boon, Nico; Siciliano, Steven D; Verstraete, Willy

2003-11-01

In the absence of oxygen, a protective H2 film is formed around an Fe(0) surface, inhibiting the electron flow from this surface. Our study of anoxic corrosion of Fe(0) beads revealed that, in the presence of Shewanella oneidensis MR-1, H2 removal and precipitation of Fe mineral particles on the cell surface are determining processes for corrosion. These two biologically mediated processes were governed by cell density. H2 removal by Shewanella oneidensis was detected at cell concentrations of 1.0 x 10(6) live cells ml-1 and higher and H2 was electron donor for denitrification of NO3-. The removal of the protective H2 layer from Fe(0) beads by Shewanella oneidensis, resulted in an increase of Fe release out of the Fe(0) beads from 153 +/- 25 mg l(-1) to 196 +/- 7 mg l-1 after 20 h. When the cell concentration exceeded 1.0 x 10(8) live cells ml-1, precipitation of iron minerals on the cell surface was characteristic for the greatest percentage of MR-1 cells, whereas micrometre-scale iron precipitates not associated with culturable cell biomass significantly decreased in number. Addition of supernatant of a corrosion assay with high cell concentration induced metabolic activity in a corrosion assay with low cell concentration, resulting in increased H2 consumption and Fe release from Fe(0) beads. Homoserine lactone-like molecules were detected in the supernatant by a bio-assay, suggesting the involvement of a quorum-sensing regulatory mechanism.

Subunits of the Pyruvate Dehydrogenase Cluster of Mycoplasma pneumoniae Are Surface-Displayed Proteins that Bind and Activate Human Plasminogen

PubMed Central

Gründel, Anne; Friedrich, Kathleen; Pfeiffer, Melanie; Jacobs, Enno; Dumke, Roger

2015-01-01

The dual role of glycolytic enzymes in cytosol-located metabolic processes and in cell surface-mediated functions with an influence on virulence is described for various micro-organisms. Cell wall-less bacteria of the class Mollicutes including the common human pathogen Mycoplasma pneumoniae possess a reduced genome limiting the repertoire of virulence factors and metabolic pathways. After the initial contact of bacteria with cells of the respiratory epithelium via a specialized complex of adhesins and release of cell-damaging factors, surface-displayed glycolytic enzymes may facilitate the further interaction between host and microbe. In this study, we described detection of the four subunits of pyruvate dehydrogenase complex (PDHA-D) among the cytosolic and membrane-associated proteins of M. pneumoniae. Subunits of PDH were cloned, expressed and purified to produce specific polyclonal guinea pig antisera. Using colony blotting, fractionation of total proteins and immunofluorescence experiments, the surface localization of PDHA-C was demonstrated. All recombinant PDH subunits are able to bind to HeLa cells and human plasminogen. These interactions can be specifically blocked by the corresponding polyclonal antisera. In addition, an influence of ionic interactions on PDHC-binding to plasminogen as well as of lysine residues on the association of PDHA-D with plasminogen was confirmed. The PDHB subunit was shown to activate plasminogen and the PDHB-plasminogen complex induces degradation of human fibrinogen. Hence, our data indicate that the surface-associated PDH subunits might play a role in the pathogenesis of M. pneumoniae infections by interaction with human plasminogen. PMID:25978044

Proteinaceous determinants of surface colonization in bacteria: bacterial adhesion and biofilm formation from a protein secretion perspective

PubMed Central

Chagnot, Caroline; Zorgani, Mohamed A.; Astruc, Thierry; Desvaux, Mickaël

2013-01-01

Bacterial colonization of biotic or abiotic surfaces results from two quite distinct physiological processes, namely bacterial adhesion and biofilm formation. Broadly speaking, a biofilm is defined as the sessile development of microbial cells. Biofilm formation arises following bacterial adhesion but not all single bacterial cells adhering reversibly or irreversibly engage inexorably into a sessile mode of growth. Among molecular determinants promoting bacterial colonization, surface proteins are the most functionally diverse active components. To be present on the bacterial cell surface, though, a protein must be secreted in the first place. Considering the close association of secreted proteins with their cognate secretion systems, the secretome (which refers both to the secretion systems and their protein substrates) is a key concept to apprehend the protein secretion and related physiological functions. The protein secretion systems are here considered in light of the differences in the cell-envelope architecture between diderm-LPS (archetypal Gram-negative), monoderm (archetypal Gram-positive) and diderm-mycolate (archetypal acid-fast) bacteria. Besides, their cognate secreted proteins engaged in the bacterial colonization process are regarded from single protein to supramolecular protein structure as well as the non-classical protein secretion. This state-of-the-art on the complement of the secretome (the secretion systems and their cognate effectors) involved in the surface colonization process in diderm-LPS and monoderm bacteria paves the way for future research directions in the field. PMID:24133488

Expression profile of senescence-associated beta-galactosidase and activation of telomerase in human ovarian surface epithelial cells undergoing immortalization.

PubMed

Litaker, J R; Pan, J; Cheung, Y; Zhang, D K; Liu, Y; Wong, S C; Wan, T S; Tsao, S W

1998-11-01

Senescence is a specific physiological stage of cells characterized by long population doubling time. It accounts for the inability of normal somatic cells to undergo indefinite cell division. As the number of population doublings increase, cell cycle regulatory mechanisms come into play and signal cells to exit the cell cycle and become senescent. Senescence has been implicated in the aging process and may function as a tumor suppressor mechanism in human cells. The ability to measure the degree of cellular senescence is important in understanding the biological processes regulating cell aging and immortalization. Senescent cells exhibit an enzyme termed senescence-associated histochemical staining. Cells immortalized by viral oncogenes often enter a stage of crisis at the early phase of immortalization. The cells at crisis have a long population doubling time. Cells at the crisis stage resemble senescent cells and the expression of SA- beta-Gal may be used to monitor the process of immortalization. In this study the expression profile of SA-beta-Gal was examined in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalization by the human papilloma viral oncogene E6 and E7 (HPV E6 and E7). Our results showed a low percentage (12.0%) of HOSE 6-3 cells expressing SA-beta-Gal activity at the pre-crisis stage. The percentage of HOSE 6-3 cells expressing SA-beta-Gal activity was highest (39.2%) at the crisis stage. When HOSE 6-3 cells achieved immortalized status there was a sharp decrease in cells (1. 3%) expressing SA-beta-Gal activity. In addition, an inverse relationship between the expression of SA-beta-Gal activity and telomerase activity was noted in cells undergoing immortalization. The results confirm that the SA-beta-Gal enzyme is a good marker for monitoring the population of cells undergoing senescence at different stages of immortalization and that telomerase activation is a characteristic feature of post-crisis cells.

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

Early T-cell activation biophysics

PubMed Central

Henry, Nelly; Hivroz, Claire

2009-01-01

The T-cell is one of the main players in the mammalian immune response. It ensures antigen recognition at the surface of antigen-presenting cells in a complex and highly sensitive and specific process, in which the encounter of the T-cell receptor with the agonist peptide associated with the major histocompatibility complex triggers T-cell activation. While signaling pathways have been elucidated in increasing detail, the mechanism of TCR triggering remains highly controversial despite active research published in the past 10 years. In this paper, we present a short overview of pending questions on critical initial events associated with T-cell triggering. In particular, we examine biophysical approaches already in use, as well as future directions. We suggest that the most recent advances in fluorescence super-resolution imaging, coupled with the new classes of genetic fluorescent probes, will play an important role in elucidation of the T-cell triggering mechanism. Beyond this aspect, we predict that exploration of mechanical cues in the triggering process will provide new clues leading to clarification of the entire mechanism. PMID:20514131

Volume labeling with Alexa Fluor dyes and surface functionalization of highly sensitive fluorescent silica (SiO2) nanoparticles

NASA Astrophysics Data System (ADS)

Wang, Wei; Nallathamby, Prakash D.; Foster, Carmen M.; Morrell-Falvey, Jennifer L.; Mortensen, Ninell P.; Doktycz, Mitchel J.; Gu, Baohua; Retterer, Scott T.

2013-10-01

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and a polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or ``free'' surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface-modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems. Electronic supplementary information (ESI) available: Cell culture preparation for dose/response imaging experiments. See DOI: 10.1039/c3nr02639f

On the role of extracellular polymeric substances during early stages of Xylella fastidiosa biofilm formation.

PubMed

Lorite, Gabriela S; de Souza, Alessandra A; Neubauer, Daniel; Mizaikoff, Boris; Kranz, Christine; Cotta, Mônica A

2013-02-01

The structural integrity and protection of bacterial biofilms are intrinsically associated with a matrix of extracellular polymeric substances (EPS) produced by the bacteria cells. However, the role of these substances during biofilm adhesion to a surface remains largely unclear. In this study, the influence of EPS on Xylella fastidiosa biofilm formation was investigated. This bacterium is associated with economically important plant diseases; it presents a slow growth rate and thus allows us to pinpoint more precisely the early stages of cell-surface adhesion. Scanning electron microscopy and atomic force microscopy show evidence of EPS production in such early stages and around individual bacteria cells attached to the substrate surface even a few hours after inoculation. In addition, EPS formation was investigated via attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR). To this end, X. fastidiosa cells were inoculated within an ATR liquid cell assembly. IR-ATR spectra clearly reveal EPS formation already during the early stages of X. fastidiosa biofilm formation, thereby providing supporting evidence for the hypothesis of the relevance of the EPS contribution to the adhesion process. Copyright © 2012 Elsevier B.V. All rights reserved.

Proteins at the Biomaterial Electrolyte Interface

NASA Astrophysics Data System (ADS)

Tengvall, Pentti

2005-03-01

Proteins adsorb rapidly onto solid and polymeric surfaces because the association process is in the vast majority of cases energetically favourable, i.e. exothermic. The most common exceptions to this rule are hydrophilic interfaces with low net charge and high mobility, e.g. immobilized PEGs. Current research in the research area tries to understand and control unwanted and wanted adsorption by studying the adsorption kinetics, protein surface binding specificity, protein exchange at interfaces, and surface protein repulsion mechanisms. In blood plasma model systems humoral cascade reactions such as surface mediated coagulation and immune complement raise considerable interest due to the immediate association to blood compatibility, and in tissue applications the binding between surfaces and membrane receptors in cells and tissues. Thus, the understanding of interfacial events at the protein level is of large importance in applications such as blood and tissue contacting biomaterials, in vitro medical and biological diagnostics, food industry and in marine anti-fouling technology. Well described consequences of adsorption are a lowered system energy, increased system entropy, irreversible binding, conformational changes, specific surface/protein interactions, and in biomedical materials applications surface opsonization followed by cell-surface interactions and a host tissue response. This lecture will deal with some mechanisms known to be of importance for the adsorption processes, such as the influence of surface chemistry and surface energy, the composition of the protein solution, the Vroman effect, and residence time. Examples will be shown from ellipsometric experiments using different model surfaces in single/few protein solutions, and specific attention be given to blood serum and plasma experiments on coagulation and immune complement at interfaces.

Surface Modification of Biodegradable Polymers towards Better Biocompatibility and Lower Thrombogenicity

PubMed Central

Rudolph, Andreas; Teske, Michael; Illner, Sabine; Kiefel, Volker; Sternberg, Katrin; Grabow, Niels; Wree, Andreas; Hovakimyan, Marina

2015-01-01

Purpose Drug-eluting stents (DES) based on permanent polymeric coating matrices have been introduced to overcome the in stent restenosis associated with bare metal stents (BMS). A further step was the development of DES with biodegradable polymeric coatings to address the risk of thrombosis associated with first-generation DES. In this study we evaluate the biocompatibility of biodegradable polymer materials for their potential use as coating matrices for DES or as materials for fully bioabsorbable vascular stents. Materials and Methods Five different polymers, poly(L-lactide) PLLA, poly(D,L-lactide) PDLLA, poly(L-lactide-co-glycolide) P(LLA-co-GA), poly(D,L-lactide-co-glycolide) P(DLLA-co-GA) and poly(L-lactide-co-ε-caprolactone), P(LLA-co-CL) were examined in vitro without and with surface modification. The surface modification of polymers was performed by means of wet-chemical (NaOH and ethylenediamine (EDA)) and plasma-chemical (O2 and NH3) processes. The biocompatibility studies were performed on three different cell types: immortalized mouse fibroblasts (cell line L929), human coronary artery endothelial cells (HCAEC) and human umbilical vein endothelial cells (HUVEC). The biocompatibility was examined quantitatively using in vitro cytotoxicity assay. Cells were investigated immunocytochemically for expression of specific markers, and morphology was visualized using confocal laser scanning (CLSM) and scanning electron (SEM) microscopy. Additionally, polymer surfaces were examined for their thrombogenicity using an established hemocompatibility test. Results Both endothelial cell types exhibited poor viability and adhesion on all five unmodified polymer surfaces. The biocompatibility of the polymers could be influenced positively by surface modifications. In particular, a reproducible effect was observed for NH3-plasma treatment, which enhanced the cell viability, adhesion and morphology on all five polymeric surfaces. Conclusion Surface modification of polymers can provide a useful approach to enhance their biocompatibility. For clinical application, attempts should be made to stabilize the plasma modification and use it for coupling of biomolecules to accelerate the re-endothelialization of stent surfaces in vivo. PMID:26641662

Effects of macro- versus nanoporous silicon substrates on human aortic endothelial cell behavior

PubMed Central

2014-01-01

Human aortic endothelial cells play a key role in the pathogenesis of atherosclerosis, which is a common, progressive, and multifactorial disease that is the clinical endpoint of an inflammatory process and endothelial dysfunction. Study and development of new therapies against cardiovascular disease must be tested in vitro cell models, prior to be evaluated in vivo. To this aim, new cell culture platforms are developed that allow cells to grow and respond to their environment in a realistic manner. In this work, the cell adhesion and morphology of endothelial cells are investigated on functionalized porous silicon substrates with two different pore size configurations: macroporous and nanoporous silicon. Herein, we modified the surfaces of porous silicon substrates by aminopropyl triethoxysilane, and we studied how different pore geometries induced different cellular response in the cell morphology and adhesion. The cell growth over the surface of porous silicon becomes an attractive field, especially for medical applications. Surface properties of the biomaterial are associated with cell adhesion and as well as, with proliferation, migration and differentiation. PMID:25246859

Application of a simple cerebellar model to geologic surface mapping

USGS Publications Warehouse

Hagens, A.; Doveton, J.H.

1991-01-01

Neurophysiological research into the structure and function of the cerebellum has inspired computational models that simulate information processing associated with coordination and motor movement. The cerebellar model arithmetic computer (CMAC) has a design structure which makes it readily applicable as an automated mapping device that "senses" a surface, based on a sample of discrete observations of surface elevation. The model operates as an iterative learning process, where cell weights are continuously modified by feedback to improve surface representation. The storage requirements are substantially less than those of a conventional memory allocation, and the model is extended easily to mapping in multidimensional space, where the memory savings are even greater. ?? 1991.

Advances in pancreatic islet monolayer culture on glass surfaces enable super-resolution microscopy and insights into beta cell ciliogenesis and proliferation

PubMed Central

Phelps, Edward A.; Cianciaruso, Chiara; Santo-Domingo, Jaime; Pasquier, Miriella; Galliverti, Gabriele; Piemonti, Lorenzo; Berishvili, Ekaterine; Burri, Olivier; Wiederkehr, Andreas; Hubbell, Jeffrey A.; Baekkeskov, Steinunn

2017-01-01

A robust and reproducible method for culturing monolayers of adherent and well-spread primary islet cells on glass coverslips is required for detailed imaging studies by super-resolution and live-cell microscopy. Guided by an observation that dispersed islet cells spread and adhere well on glass surfaces in neuronal co-culture and form a monolayer of connected cells, we demonstrate that in the absence of neurons, well-defined surface coatings combined with components of neuronal culture media collectively support robust attachment and growth of primary human or rat islet cells as monolayers on glass surfaces. The islet cell monolayer cultures on glass stably maintain distinct mono-hormonal insulin+, glucagon+, somatostatin+ and PP+ cells and glucose-responsive synchronized calcium signaling as well as expression of the transcription factors Pdx-1 and NKX-6.1 in beta cells. This technical advance enabled detailed observation of sub-cellular processes in primary human and rat beta cells by super-resolution microscopy. The protocol is envisaged to have broad applicability to sophisticated analyses of pancreatic islet cells that reveal new biological insights, as demonstrated by the identification of an in vitro protocol that markedly increases proliferation of primary beta cells and is associated with a reduction in ciliated, ostensibly proliferation-suppressed beta cells. PMID:28401888

Heparan sulfate/heparin oligosaccharides protect stromal cell-derived factor-1 (SDF-1)/CXCL12 against proteolysis induced by CD26/dipeptidyl peptidase IV.

PubMed

Sadir, Rabia; Imberty, Anne; Baleux, Françoise; Lortat-Jacob, Hugues

2004-10-15

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine that is constitutively expressed in most tissues and displayed on the cell surface in association with heparan sulfate (HS). Its numerous biological effects are mediated by a specific G protein-coupled receptor, CXCR4. A number of cells inactivate SDF-1 by specific processing of the N-terminal domain of the chemokine. In particular, CD26/dipeptidyl peptidase IV (DPP IV), a serine protease that co-distributes with CXCR4 at the cell surface, mediates the selective removal of the N-terminal dipeptide of SDF-1. We report here that heparin and HS specifically prevent the processing of SDF-1 by DPP IV expressed by Caco-2 cells. The level of processing increases with the level of differentiation of these cells, which correlates with an increase of DPP IV activity. A mutant SDF-1 that does not interact with HS is readily cleaved by DPP IV, a process that is not inhibited by HS, demonstrating that a productive interaction between HS and SDF-1 is required for the protection to take place. Moreover, we found that protection depends on the degree of polymerization of the HS sulfated S-domains. Finally a structural model of SDF-1, in complex with HS oligosaccharides of defined length, rationalizes the experimental data. The mechanisms by which HS regulates SDF-1 may thus include, in addition to its ability to locally concentrate the chemokine at the cell surface, a control of selective protease cleavage events that directly affect the chemokine activity.

The atlA operon of Streptococcus mutans: role in autolysin maturation and cell surface biogenesis.

PubMed

Ahn, Sang-Joon; Burne, Robert A

2006-10-01

The Smu0630 protein (AtlA) was recently shown to be involved in cell separation, biofilm formation, and autolysis. Here, transcriptional studies revealed that atlA is part of a multigene operon under the control of at least three promoters. The morphology and biofilm-forming capacity of a nonpolar altA mutant could be restored to that of the wild-type strain by adding purified AtlA protein to the medium. A series of truncated derivatives of AtlA revealed that full activity required the C terminus and repeat regions. AtlA was cell associated and readily extractable from with sodium dodecyl sulfate. Of particular interest, the surface protein profile of AtlA-deficient strains was dramatically altered compared to the wild-type strain, as was the nature of the association of the multifunctional adhesin P1 with the cell wall. In addition, AtlA-deficient strains failed to develop competence as effectively as the parental strain. Mutation of thmA, which can be cotranscribed with atlA and encodes a putative pore-forming protein, resulted in a phenotype very similar to that of the AtlA-deficient strain. ThmA was also shown to be required for efficient processing of AtlA to its mature form, and treatment of the thmA mutant strain with full-length AtlA protein did not restore normal cell separation and biofilm formation. The effects of mutating other genes in the operon on cell division, biofilm formation, or AtlA biogenesis were not as profound. This study reveals that AtlA is a surface-associated protein that plays a critical role in the network connecting cell surface biogenesis, biofilm formation, genetic competence, and autolysis.

Mechanics of active surfaces

NASA Astrophysics Data System (ADS)

Salbreux, Guillaume; Jülicher, Frank

2017-09-01

We derive a fully covariant theory of the mechanics of active surfaces. This theory provides a framework for the study of active biological or chemical processes at surfaces, such as the cell cortex, the mechanics of epithelial tissues, or reconstituted active systems on surfaces. We introduce forces and torques acting on a surface, and derive the associated force balance conditions. We show that surfaces with in-plane rotational symmetry can have broken up-down, chiral, or planar-chiral symmetry. We discuss the rate of entropy production in the surface and write linear constitutive relations that satisfy the Onsager relations. We show that the bending modulus, the spontaneous curvature, and the surface tension of a passive surface are renormalized by active terms. Finally, we identify active terms which are not found in a passive theory and discuss examples of shape instabilities that are related to active processes in the surface.

Glycosyltransferase-programmed stereosubstitution (GPS) to create HCELL: engineering a roadmap for cell migration.

PubMed

Sackstein, Robert

2009-07-01

During evolution of the vertebrate cardiovascular system, the vast endothelial surface area associated with branching vascular networks mandated the development of molecular processes to efficiently and specifically recruit circulating sentinel host defense cells and tissue repair cells at localized sites of inflammation/tissue injury. The forces engendered by high-velocity blood flow commensurately required the evolution of specialized cell surface molecules capable of mediating shear-resistant endothelial adhesive interactions, thus literally capturing relevant cells from the blood stream onto the target endothelial surface and permitting subsequent extravasation. The principal effectors of these shear-resistant binding interactions comprise a family of C-type lectins known as 'selectins' that bind discrete sialofucosylated glycans on their respective ligands. This review explains the 'intelligent design' of requisite reagents to convert native CD44 into the sialofucosylated glycoform known as hematopoietic cell E-/L-selectin ligand (HCELL), the most potent E-selectin counter-receptor expressed on human cells, and will describe how ex vivo glycan engineering of HCELL expression may open the 'avenues' for the efficient vascular delivery of cells for a variety of cell therapies.

Insulin promotes cell migration by regulating PSA-NCAM

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

Monzo, Hector J.; Coppieters, Natacha; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cellmore » migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.« less

A glycosylphosphatidylinositol anchor is required for membrane localization but dispensable for cell wall association of chitin deacetylase 2 in Cryptococcus neoformans.

PubMed

Gilbert, Nicole M; Baker, Lorina G; Specht, Charles A; Lodge, Jennifer K

2012-01-01

Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. The surface of a pathogenic microbe is a major interface with its host. In fungi, the outer surface consists of a complex matrix known as the cell wall, which includes polysaccharides, proteins, and other molecules. The mammalian host recognizes many of these surface molecules and mounts appropriate responses to combat the microbial infection. Cryptococcus neoformans is a serious fungal pathogen that kills over 600,000 people annually. It converts most of its chitin, a cell wall polysaccharide, to chitosan, which is necessary for virulence. Chitin deacetylase enzymes have been identified in the cell wall, and our studies were undertaken to understand how the deacetylase is linked to the wall and where it has activity. Our results have implications for the current model of chitosan biosynthesis and further challenge the paradigm of covalent linkages between cell wall proteins and polysaccharides through a lipid modification of the protein.

Insulin promotes cell migration by regulating PSA-NCAM.

PubMed

Monzo, Hector J; Coppieters, Natacha; Park, Thomas I H; Dieriks, Birger V; Faull, Richard L M; Dragunow, Mike; Curtis, Maurice A

2017-06-01

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. Copyright © 2017 Elsevier Inc. All rights reserved.

Nanowall formation by maskless wet-etching on a femtosecond laser irradiated silicon surface

NASA Astrophysics Data System (ADS)

Lee, Siwoo; Jo, Kukhyun; Keum, Hee-sung; Chae, Sangmin; Kim, Yonghyeon; Choi, Jiyeon; Lee, Hyun Hwi; Kim, Hyo Jung

2018-04-01

We found that micro-cells surrounded by nanowalls can be formed by a maskless wet-etching process on Si (100) surfaces possessing Laser Induced Periodic Surface Structure (LIPSS) by femtosecond laser irradiation. The LIPSS process could produce periodic one-dimensional micron scale ripples on a Si surface, which could be developed into micro-cells by a subsequent etching process. The solution etching conditions strongly affected both the micro-cell and nanowall shapes such as the height and the thickness of nanowalls. The tetramethylammonium hydroxide solution created thin nanowalls and the resulting micro-cells with a well-flattened bottom while the KOH solution formed thick walls and incomplete micro-cells. The bottoms of micro-cells surrounded by the nanowalls were considerably flat with a 3.10 nm surface roughness. A pentacene layer was deposited on the micro-cells of a Si surface to evaluate the film properties by grazing incidence wide angle x-ray scattering measurements. The pentacene film on the micro-cell Si surface showed a strong film phase, which was comparable to the film phase grown on the atomically flat Si surface.

ABCG1-mediated generation of extracellular cholesterol microdomains[S

PubMed Central

Freeman, Sebastian R.; Jin, Xueting; Anzinger, Joshua J.; Xu, Qing; Purushothaman, Sonya; Fessler, Michael B.; Addadi, Lia; Kruth, Howard S.

2014-01-01

Previous studies have demonstrated that the ATP-binding cassette transporters (ABC)A1 and ABCG1 function in many aspects of cholesterol efflux from macrophages. In this current study, we continued our investigation of extracellular cholesterol microdomains that form during enrichment of macrophages with cholesterol. Human monocyte-derived macrophages and mouse bone marrow-derived macrophages, differentiated with macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulation factor (GM-CSF), were incubated with acetylated LDL (AcLDL) to allow for cholesterol enrichment and processing. We utilized an anti-cholesterol microdomain monoclonal antibody to reveal pools of unesterified cholesterol, which were found both in the extracellular matrix and associated with the cell surface, that we show function in reverse cholesterol transport. Coincubation of AcLDL with 50 μg/ml apoA-I eliminated all extracellular and cell surface-associated cholesterol microdomains, while coincubation with the same concentration of HDL only removed extracellular matrix-associated cholesterol microdomains. Only at an HDL concentration of 200 µg/ml did HDL eliminate the cholesterol microdomains that were cell-surface associated. The deposition of cholesterol microdomains was inhibited by probucol, but it was increased by the liver X receptor (LXR) agonist TO901317, which upregulates ABCA1 and ABCG1. Extracellular cholesterol microdomains did not develop when ABCG1-deficient mouse bone marrow-derived macrophages were enriched with cholesterol. Our findings show that generation of extracellular cholesterol microdomains is mediated by ABCG1 and that reverse cholesterol transport occurs not only at the cell surface but also within the extracellular space. PMID:24212237

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

PubMed Central

Siddle, Hannah V.; Kreiss, Alexandre; Tovar, Cesar; Yuen, Chun Kit; Cheng, Yuanyuan; Belov, Katherine; Swift, Kate; Pearse, Anne-Maree; Hamede, Rodrigo; Jones, Menna E.; Skjødt, Karsten; Woods, Gregory M.; Kaufman, Jim

2013-01-01

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host–immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD. PMID:23479617

Excimer laser annealing: A gold process for CZ silicon junction formation

NASA Technical Reports Server (NTRS)

Wong, David C.; Bottenberg, William R.; Byron, Stanley; Alexander, Paul

1987-01-01

A cold process using an excimer laser for junction formation in silicon has been evaluated as a way to avoid problems associated with thermal diffusion. Conventional thermal diffusion can cause bulk precipitation of SiOx and SiC or fail to completely activate the dopant, leaving a degenerate layer at the surface. Experiments were conducted to determine the feasibility of fabricating high quality p-n junctions using a pulsed excimer laser for junction formation at remelt temperature with ion-implanted surfaces. Solar-cell efficiency exceeding 16 percent was obtained using Czochralski single-crystal silicon without benefit of back surface field or surface passivation. Characterization shows that the formation of uniform, shallow junctions (approximately 0.25 micron) by excimer laser scanning preserves the minority carrier lifetime that leads to high current collection. However, the process is sensitive to initial surface conditions and handling parameters that drive the cost up.

Friction Stir Processing of Stainless Steel for Ascertaining Its Superlative Performance in Bioimplant Applications.

PubMed

Perumal, G; Ayyagari, A; Chakrabarti, A; Kannan, D; Pati, S; Grewal, H S; Mukherjee, S; Singh, S; Arora, H S

2017-10-25

Substrate-cell interactions for a bioimplant are driven by substrate's surface characteristics. In addition, the performance of an implant and resistance to degradation are primarily governed by its surface properties. A bioimplant typically degrades by wear and corrosion in the physiological environment, resulting in metallosis. Surface engineering strategies for limiting degradation of implants and enhancing their performance may reduce or eliminate the need for implant removal surgeries and the associated cost. In the current study, we tailored the surface properties of stainless steel using submerged friction stir processing (FSP), a severe plastic deformation technique. FSP resulted in significant microstructural refinement from 22 μm grain size for the as-received alloy to 0.8 μm grain size for the processed sample with increase in hardness by nearly 1.5 times. The wear and corrosion behavior of the processed alloy was evaluated in simulated body fluid. The processed sample demonstrated remarkable improvement in both wear and corrosion resistance, which is explained by surface strengthening and formation of a highly stable passive layer. The methylthiazol tetrazolium assay demonstrated that the processed sample is better in supporting cell attachment, proliferation with minimal toxicity, and hemolysis. The athrombogenic characteristic of the as-received and processed samples was evaluated by fibrinogen adsorption and platelet adhesion via the enzyme-linked immunosorbent assay and lactate dehydrogenase assay, respectively. The processed sample showed less platelet and fibrinogen adhesion compared with the as-received alloy, signifying its high thromboresistance. The current study suggests friction stir processing to be a versatile toolbox for enhancing the performance and reliability of currently used bioimplant materials.

Multilamellar Structures and Filament Bundles Are Found on the Cell Surface during Bunyavirus Egress

PubMed Central

Sanz-Sánchez, Laura; Risco, Cristina

2013-01-01

Inside cells, viruses build specialized compartments for replication and morphogenesis. We observed that virus release associates with specific structures found on the surface of mammalian cells. Cultured adherent cells were infected with a bunyavirus and processed for oriented sectioning and transmission electron microscopy. Imaging of cell basal regions showed sophisticated multilamellar structures (MLS) and extracellular filament bundles with attached viruses. Correlative light and electron microscopy confirmed that both MLS and filaments proliferated during the maximum egress of new viruses. MLS dimensions and structure were reminiscent of those reported for the nanostructures on gecko fingertips, which are responsible for the extraordinary attachment capacity of these lizards. As infected cells with MLS were more resistant to detachment than control cells, we propose an adhesive function for these structures, which would compensate for the loss of adherence during release of new virus progeny. PMID:23799021

Self-assembling triblock proteins for biofunctional surface modification

NASA Astrophysics Data System (ADS)

Fischer, Stephen E.

Despite the tremendous promise of cell/tissue engineering, significant challenges remain in engineering functional scaffolds to precisely regulate the complex processes of tissue growth and development. As the point of contact between the cells and the scaffold, the scaffold surface plays a major role in mediating cellular behaviors. In this dissertation, the development and utility of self-assembling, artificial protein hydrogels as biofunctional surface modifiers is described. The design of these recombinant proteins is based on a telechelic triblock motif, in which a disordered polyelectrolyte central domain containing embedded bioactive ligands is flanked by two leucine zipper domains. Under moderate conditions of temperature and pH, the leucine zipper end domains form amphiphilic alpha-helices that reversibly associate into homo-trimeric aggregates, driving hydrogel formation. Moreover, the amphiphilic nature of these helical domains enables surface adsorption to a variety of scaffold materials to form biofunctional protein coatings. The nature and stability of these coatings in various solution conditions, and their interaction with mammalian cells is the primary focus of this dissertation. In particular, triblock protein coatings functionalized with cell recognition sequences are shown to produce well-defined surfaces with precise control over ligand density. The impact of this is demonstrated in multiple cell types through ligand density-dependent cell-substrate interactions. To improve the stability of these physically self-assembled coatings, two covalent crosslinking strategies are described---one in which a zero-length chemical crosslinker (EDC) is utilized and a second in which disulfide bonds are engineered into the recombinant proteins. These targeted crosslinking approaches are shown to increase the stability of surface adsorbed protein layers with minimal effect on the presentation of many bioactive ligands. Finally, to demonstrate the versatility of the triblock protein hydrogels, and the ease of introducing multiple functionalities to a substrate surface, a surface coating is tailored for neural stem cell culture in order to improve proliferation on the scaffold, while maintaining the stem cell phenotype. These studies demonstrate the unique advantages of genetic engineering over traditional techniques for surface modification. In addition to their unmatched sequence fidelity, recombinant proteins can easily be modified with bioactive ligands and their organization into coherent, supramolecular structures mimics natural self-assembly processes.

Cell Surface THY-1 Contributes to Human Cytomegalovirus Entry via a Macropinocytosis-Like Process

PubMed Central

Li, Qingxue; Fischer, Elizabeth

2016-01-01

ABSTRACT Previously we showed that THY-1 has a critical role in the initial stage of infection of certain cell types with human cytomegalovirus (HCMV) and that THY-1 is important for HCMV-mediated activation of phosphatidylinositol 3-kinase (PI3K)/Akt during virus entry. THY-1 is known to interact with integrins and is a major cargo protein of clathrin-independent endocytic vesicles. Since macropinocytosis involves integrin signaling, is PI3K/Akt dependent, and is a clathrin-independent endocytic process, we determined whether THY-1 has a role in HCMV entry by macropinocytosis. Using electron microscopy in two cell lines that support HCMV infection in a THY-1-dependent manner, we found that HCMV enters these cells by a macropinocytosis-like process. THY-1 associated with HCMV virions on the cell surface and colocalized with virus inside macropinosomes. 5-(N-Ethyl-N-isopropyl)amiloride (EIPA) and soluble THY-1 blocked HCMV infection in the cell lines by ≥80% and 60%, respectively. HCMV entry into the cells triggered increased influx of extracellular fluid, a marker of macropinocytosis, and this increased fluid uptake was inhibited by EIPA and by soluble THY-1. Blocking actin depolymerization, Na+/H+ exchange, PI3K, and Pak1 kinase, which are critical for macropinocytosis, impaired HCMV infection. Neither internalized HCMV virions nor THY-1 in virus-infected cells colocalized with transferrin as determined by confocal microscopy, indicating that clathrin-mediated endocytosis was not involved in THY-1-associated virus entry. These results suggest that HCMV has adapted to utilize THY-1, a cargo protein of clathrin-independent endocytotic vesicles, to facilitate efficient entry into certain cell types by a macropinocytosis-like process. IMPORTANCE Human cytomegalovirus (HCMV) infects over half of the population and is the most common infectious cause of birth defects. The virus is the most important infection occurring in transplant recipients. The mechanism of how HCMV enters cells is controversial. In this study, we show that THY-1, a cell surface protein that is critical for the early stage of entry of HCMV into certain cell types, contributes to virus entry by macropinocytosis. Our findings suggest that HCMV has adapted to utilize THY-1 to facilitate entry of HCMV into macropinosomes in certain cell types. Further knowledge about the mechanism of HCMV entry into cells may facilitate the development of novel inhibitors of virus infection. PMID:27558416

Ultrastructural analysis of bone nodules formed in vitro by isolated fetal rat calvaria cells

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

Bhargava, U.; Bar-Lev, M.; Bellows, C.G.

When cells enzymatically digested from 21 d fetal rat calvaria are grown in ascorbic acid and Na beta-glycerophosphate, they form discrete three-dimensional nodular structures with the histological and immunohistochemical appearance of woven bone. The present investigation was undertaken to verify that bone-like features were identifiable at the ultrastructural level. The nodules formed on top of a fibroblast-like multilayer of cells. The upper surface of the nodules was lined by a continuous layer of cuboidal osteoblastic cells often seen to be joined by adherens junctions. Numerous microvilli, membrane protrusions, and coated pits could be seen on the upper surface of thesemore » cells, their cytoplasm contained prominent RER and Golgi membranes, and processes extended from their lower surfaces into a dense, highly organized collagenous matrix. Some osteocyte-like cells were completely embedded within this matrix; they also displayed RER and prominent processes which extended through the matrix and often made both adherens and gap junctional contacts with the processes of other cells. The fibroblastic cells not participating in nodule formation were surrounded by a less dense collagenous matrix and, in contrast to the matrix of the nodules, it did not mineralize. An unmineralized osteoid-like layer was seen directly below the cuboidal top layer of cells. A mineralization front was detectable below this in which small, discrete structures resembling matrix vesicles and feathery mineral crystals were evident and frequently associated with the collagen fibrils. More heavily mineralized areas were seen further into the nodule. Electron microprobe and electron and X-ray diffraction analysis confirmed the mineral to be hydroxyapatite.« less

Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding.

PubMed

Fayadat, L; Niccoli-Sire, P; Lanet, J; Franc, J L

1999-04-09

Thyroperoxidase (TPO) is a glycosylated hemoprotein that plays a key role in thyroid hormone synthesis. We previously showed that in CHO cells expressing human TPO (hTPO) only 2% of synthesized hTPO reaches the cell surface. Herein, we investigated the role of heme moiety insertion in the exit of hTPO from the endoplasmic reticulum. Peroxidase activity at the cell surface and cell surface expression of hTPO were decreased by approximately 30 and approximately 80%, respectively, with succinyl acetone, an inhibitor of heme biosynthesis, and were increased by 20% with holotransferrin and aminolevulinic acid, precursors of heme biosynthesis. Results were similar with holotransferrin plus aminolevulinic acid or hemin, but hemin increased cell surface activity more efficiently (+120%) relative to the control. It had been suggested (DePillis, G., Ozaki, S., Kuo, J. M., Maltby, D. A., and Ortiz de Montellano, P. R. (1997) J. Biol. Chem. 272, 8857-8960) that covalent attachment of heme to mammalian peroxidases could be an H2O2-dependent autocatalytic processing. In our study, heme associated intracellularly with hTPO, and we hypothesized that there was insufficient exposure to H2O2 in Chinese hamster ovary cells before hTPO reached the cell surface. After a 10-min incubation, 10 microM H2O2 led to a 65% increase in cell surface activity. In contrast, in thyroid cells, H2O2 was synthesized at the apical cell surface and allowed covalent attachment of heme. Two-day incubation of primocultures of thyroid cells with catalase led to a 30% decrease in TPO activity at the cell surface. In conclusion, we provide compelling evidence for an essential role of 1) heme incorporation in the intracellular trafficking of hTPO and of 2) H2O2 generated at the apical pole of thyroid cells in the autocatalytic covalent heme binding to the TPO molecule.

CD147-targeting siRNA inhibits cell-matrix adhesion of human malignant melanoma cells by phosphorylating focal adhesion kinase.

PubMed

Nishibaba, Rie; Higashi, Yuko; Su, Juan; Furukawa, Tatsuhiko; Kawai, Kazuhiro; Kanekura, Takuro

2012-01-01

CD147/basigin, highly expressed on the surface of malignant tumor cells including malignant melanoma (MM) cells, plays a critical role in the invasiveness and metastasis of MM. Metastasis is an orchestrated process comprised of multiple steps including adhesion and invasion. Integrin, a major adhesion molecule, co-localizes with CD147/basigin on the cell surface. Using the human MM cell line A375 that highly expresses CD147/basigin, we investigated whether CD147/basigin is involved in adhesion in association with integrin. CD147/basigin was knocked-down using siRNA targeting CD147 to elucidate the role of CD147/basigin. Cell adhesion was evaluated by adhesion assay on matrix-coated plates. The localization of integrin was inspected under a confocal microscope and the expression and phosphorylation of focal adhesion kinase (FAK), a downstream kinase of integrin, were examined by western blot analysis. Silencing of CD147/basigin in A375 cells by siRNA induced the phosphorylation of FAK at Y397. Integrin identified on the surface of parental cells was distributed in a speckled fashion in the cytoplasm of CD147 knockdown cells, resulting in morphological changes from a round to a polygonal shape with pseudopodial protrusions. Silencing of CD147/basigin in A375 cells clearly weakened their adhesiveness to collagen I and IV. Our results suggest that CD147/basigin regulates the adhesion of MM cells to extracellular matrices and of integrin β1 signaling via the phosphorylation of FAK. © 2011 Japanese Dermatological Association.

Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

PubMed Central

Li, Cunxi; Hao, Mingming; Cao, Zheng; Ding, Wei; Graves-Deal, Ramona; Hu, Jianyong; Piston, David W.

2007-01-01

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells. PMID:17553928

Correlation of cell surface proteins of distinct Beauveria bassiana cell types and adaption to varied environment and interaction with the host insect.

PubMed

Yang, Zhi; Jiang, Hongyan; Zhao, Xin; Lu, Zhuoyue; Luo, Zhibing; Li, Xuebing; Zhao, Jing; Zhang, Yongjun

2017-02-01

The insect fungal pathogen Beauveria bassiana produces a number of distinct cell types that include aerial conidia, blastospores and haemolymph-derived cells, termed hyphal bodies, to adapt varied environment niches and within the host insect. These cells display distinct biochemical properties and surface structures, and a highly ordered outermost brush-like structure uniquely present on hyphal bodies, but not on any in vitro cells. Here, we found that the outermost structure on the hyphal bodies mainly consisted of proteins associated to structural wall components in that most of it could be removed by dithiothreitol (DTT) or proteinase K. DTT-treatment also caused delayed germination, decreased tolerance to ultraviolet irradiation and virulence of conidia or blastospores, with decreased adherence and alternated carbohydrate epitopes, suggesting involvement in fungal development, stress responses and virulence. To characterize these cell surface molecules, proteins were released from the living cells using DTT, and identified and quantitated using label-free quantitative mass spectrometry. Thereafter, a series of bioinformatics programs were used to predict cell surface-associated proteins (CSAPs), and 96, 166 and 54 CSAPs were predicted from the identified protein pools of conidia, blastospores and hyphal bodies, respectively, which were involved in utilization of carbohydrate, nitrogen, and lipid, detoxification, pathogen-host interaction, and likely other cellular processes. Thirteen, sixty-nine and six CSAPs were exclusive in conidia, blastospores and hyphal bodies, respectively, which were verified by eGFP-tagged proteins at their N-terminus. Our data provide a crucial cue to understand mechanism of B. bassiana to adapt to varied environment and interaction with insect host. Copyright © 2016 Elsevier Inc. All rights reserved.

Fuel cell plates with skewed process channels for uniform distribution of stack compression load

DOEpatents

Granata, Jr., Samuel J.; Woodle, Boyd M.

1989-01-01

An electrochemical fuel cell includes an anode electrode, a cathode electrode, an electrolyte matrix sandwiched between electrodes, and a pair of plates above and below the electrodes. The plate above the electrodes has a lower surface with a first group of process gas flow channels formed thereon and the plate below the electrodes has an upper surface with a second group of process gas flow channels formed thereon. The channels of each group extend generally parallel to one another. The improvement comprises the process gas flow channels on the lower surface of the plate above the anode electrode and the process gas flow channels on the upper surface of the plate below the cathode electrode being skewed in opposite directions such that contact areas of the surfaces of the plates through the electrodes are formed in crisscross arrangements. Also, the plates have at least one groove in areas of the surfaces thereof where the channels are absent for holding process gas and increasing electrochemical activity of the fuel cell. The groove in each plate surface intersects with the process channels therein. Also, the opposite surfaces of a bipolar plate for a fuel cell contain first and second arrangements of process gas flow channels in the respective surfaces which are skewed the same amount in opposite directions relative to the longitudinal centerline of the plate.

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.

Association to HeLa cells and surface behavior of exogenous gangliosides studied with a fluorescent derivative of GM1

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

Masserini, M.; Giuliani, A.; Palestini, P.

1990-01-23

Cultured HeLa cells were incubated with pyrene-GM1/{sup 3}H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of {sup 3}H-GM1. The pyrene-GM1 and {sup 3}H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and {sup 3}H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GDla)more » derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca{sup 2+} salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca{sup 2+} concentration dependent (maximum at 10 mM), and subsequent addition of EDTA has no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties.« less

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

CD45RO enriches for activated, highly mutated human germinal center B cells

PubMed Central

Jackson, Stephen M.; Harp, Natessa; Patel, Darshna; Zhang, Jeffrey; Willson, Savannah; Kim, Yoon J.; Clanton, Christian

2007-01-01

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker. PMID:17644737

Rotavirus RRV associates with lipid membrane microdomains during cell entry.

PubMed

Isa, Pavel; Realpe, Mauricio; Romero, Pedro; López, Susana; Arias, Carlos F

2004-05-01

Rotavirus cell entry is a multistep process, not completely understood, which requires at least four interactions between the virus and cell surface molecules. In this work, we investigated the role of the sphingolipid- and cholesterol-enriched lipid microdomains (rafts) in the entry of rotavirus strain RRV to MA104 cells. We found that ganglioside GM1, integrin subunits alpha2 and beta3, and the heat shock cognate protein 70 (hsc70), all of which have been implicated as rotavirus receptors, are associated with TX-100 and Lubrol WX detergent-resistant membranes (DRMs). Integrin subunits alpha2 and beta3 were found to be particularly enriched in DRMs resistant to lysis by Lubrol WX. When purified RRV particles were incubated with cells at 4 degrees C, about 10% of the total infectious virus was found associated with DRMs, and the DRM-associated virus increased to 37% in Lubrol-resistant membrane domains after 60-min incubation at 37 degrees C. The virus was excluded from DRMs if the cells were treated with methyl-beta-cyclodextrin (MbetaCD). Immunoblot analysis of the viral proteins showed that the virus surface proteins became enriched in DRMs upon incubation at 37 degrees C, being almost exclusively localized in Lubrol-resistant DRMs after 60 min. These data suggest that detergent-resistant membrane domains play an important role in the cell entry of rotaviruses, which could provide a platform to facilitate the efficient interaction of the rotavirus receptors with the virus particle.

Ultrastructural study on the differentiation and the fate of M cells in follicle-associated epithelium of rat Peyer's patch.

PubMed

Onishi, Sachiko; Yokoyama, Toshifumi; Chin, Keigi; Yuji, Midori; Inamoto, Tetsurou; Qi, Wang-Mei; Warita, Katsuhiko; Hoshi, Nobuhiko; Kitagawa, Hiroshi

2007-05-01

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin

PubMed Central

Singh, Bhuminder; Bogatcheva, Galina; Starchenko, Alina; Sinnaeve, Justine; Lapierre, Lynne A.; Williams, Janice A.; Goldenring, James R.; Coffey, Robert J.

2015-01-01

ABSTRACT Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically 156EEMETL161). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization. PMID:26272915

Human cytomegalovirus gH stability and trafficking are regulated by ER-associated degradation and transmembrane architecture.

PubMed

Gardner, Thomas J; Hernandez, Rosmel E; Noriega, Vanessa M; Tortorella, Domenico

2016-03-30

The prototypic betaherpesvirus human cytomegalovirus (CMV) establishes life-long persistence within its human host. While benign in healthy individuals, CMV poses a significant threat to the immune compromised, including transplant recipients and neonates. The CMV glycoprotein complex gH/gL/gO mediates infection of fibroblasts, and together with the gH/gL/UL128/130/131 a pentameric complex permits infection of epithelial, endothethial, and myeloid cells. Given the central role of the gH/gL complex during infection, we were interested in studying cellular trafficking of the gH/gL complex through generation of human cells that stably express gH and gL. When expressed alone, CMV gH and gL were degraded through the ER-associated degradation (ERAD) pathway. However, co-expression of these proteins stabilized the polypeptides and enhanced their cell-surface expression. To further define regulatory factors involved in gH/gL trafficking, a CMV gH chimera in which the gH transmembrane and cytoplasmic tail were replaced with that of human CD4 protein permitted cell surface gH expression in absence of gL. We thus demonstrate the ability of distinct cellular processes to regulate the trafficking of viral glycoproteins. Collectively, the data provide insight into the processing and trafficking requirements of CMV envelope protein complexes and provide an example of the co-opting of cellular processes by CMV.

Involvement of vesicle coat material in casein secretion and surface regeneration

PubMed Central

1976-01-01

The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces. PMID:1254641

Calreticulin is expressed on the cell surface of activated human peripheral blood T lymphocytes in association with major histocompatibility complex class I molecules.

PubMed

Arosa, F A; de Jesus, O; Porto, G; Carmo, A M; de Sousa, M

1999-06-11

Calreticulin is an endoplasmic reticulum resident molecule known to be involved in the folding and assembly of major histocompatibility complex (MHC) class I molecules. In the present study, expression of calreticulin was analyzed in human peripheral blood T lymphocytes. Pulse-chase experiments in [35S]methionine-labeled T cell blasts showed that calreticulin was associated with several proteins in the endoplasmic reticulum and suggested that it was expressed at the cell surface. Indeed, the 60-kDa calreticulin was labeled by cell surface biotinylation and precipitated from the surface of activated T cells together with a protein with an apparent molecular mass of 46 kDa. Cell surface expression of calreticulin by activated T lymphocytes was further confirmed by immunofluorescence and flow cytometry, studies that showed that both CD8+ and CD4+ T cells expressed calreticulin in the plasma membrane. Low amounts of cell surface calreticulin were detected in resting T lymphocytes. By sequential immunoprecipitation using the conformation independent monoclonal antibody HC-10, we provided evidence that the cell surface 46-kDa protein co-precipitated with calreticulin is unfolded MHC I. These results show for the first time that after T cell activation, significant amounts of calreticulin are expressed on the T cell surface, where they are found in physical association with a pool of beta2-free MHC class I molecules.

Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

PubMed

González, Silvia A; Falcón, Juan I; Affranchino, José L

2014-03-01

Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

PubMed

Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

2016-11-25

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

A novel image processing method to determine the nutritional condition of lobsters.

PubMed

Berillis, P; Simon, C; Mente, E; Sofos, F; Karapanagiotidis, I T

2013-02-01

The digestive gland of crustacean is involved in various metabolic activities, including the synthesis and secretion of digestive enzymes that begin the process of food digestion, intracellular digestion and absorption of nutrients, storage of reserves, and disposal of waste products. It consists of two glandular lobes which extensively subdivide to form a complex of blind-ending tubules, whose size, surface area, and digestive cells are associated with intracellular digestion and the nutritional status of the organism. The aim of this paper was to study the morphology of the digestive gland in various lobster species and calculate the surface area of tubules, lumen and digestive cells (R-, F-, and B-cells) and their ratios to total tubule surface area. The similarity in ratios obtained in this study between individual lobsters suggests that the method developed in this study can be successfully applied to a range of species. This study describes a novel image processing algorithm for the automatic measurement of the hepatopancreas structure using stained cross sections of digestive gland tubules. The proposed new methodology could be used for studying the physiology and nutrient metabolism of lobsters and other crustaceans. The computer-aided analysis described in this paper is accurate for the quantitative assessment of the lobster's digestive gland structure. Copyright © 2012 Elsevier Ltd. All rights reserved.

Ion Transport by Pulmonary Epithelia

PubMed Central

Hollenhorst, Monika I.; Richter, Katrin; Fronius, Martin

2011-01-01

The lung surface of air-breathing vertebrates is formed by a continuous epithelium that is covered by a fluid layer. In the airways, this epithelium is largely pseudostratified consisting of diverse cell types such as ciliated cells, goblet cells, and undifferentiated basal cells, whereas the alveolar epithelium consists of alveolar type I and alveolar type II cells. Regulation and maintenance of the volume and viscosity of the fluid layer covering the epithelium is one of the most important functions of the epithelial barrier that forms the outer surface area of the lungs. Therefore, the epithelial cells are equipped with a wide variety of ion transport proteins, among which Na+, Cl−, and K+ channels have been identified to play a role in the regulation of the fluid layer. Malfunctions of pulmonary epithelial ion transport processes and, thus, impairment of the liquid balance in our lungs is associated with severe diseases, such as cystic fibrosis and pulmonary oedema. Due to the important role of pulmonary epithelial ion transport processes for proper lung function, the present paper summarizes the recent findings about composition, function, and ion transport properties of the airway epithelium as well as of the alveolar epithelium. PMID:22131798

Selective binding and transcytosis of Ulex europaeus 1 lectin by mouse Peyer's patch M-cells in vivo.

PubMed

Clark, M A; Jepson, M A; Simmons, N L; Hirst, B H

1995-12-01

The in vivo interaction of the lectin Ulex europaeus agglutinin 1 with mouse Peyer's patch follicle-associated epithelial cells was studied in the mouse Peyer's patch gut loop model by immunofluorescence and electron microscopy. The lectin targets to mouse Peyer's patch M-cells and is rapidly endocytosed and transcytosed. These processes are accompanied by morphological changes in the M-cell microvilli and by redistribution of polymerised actin. The demonstration of selective binding and uptake of a lectin by intestinal M-cells in vivo suggests that M-cell-specific surface glycoconjugates might act as receptors for the selective adhesion/uptake of microorganisms.

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

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.

Examining Merkel Cell Polyomavirus Minor Capsid Proteins | Center for Cancer Research

Cancer.gov

Merkel cell polyomavirus (MCV or MCPyV) is a recently discovered member of the viral family Polyomaviridae. It is a skin-dwelling polyomavirus species that appears to cause a rare but highly lethal form of skin cancer called Merkel cell carcinoma (MCC). Despite MCC being uncommon, chronic MCV infection of human skin is widespread, and most infected people have no known symptoms. The surface of polyomavirus virions is made up of pentameric knobs of the major capsid protein VP1. VP1 enables attachment of the virus to the cell surface, permitting infectious entry and delivery of the viral genome to host cells. The VP1 protein of previously studied polyomaviruses, such as simian virus 40 and murine polyomavirus, associates with two minor capsid proteins, VP2 and VP3, which are considered to play important roles during the infectious entry process.

Application of biofilm bioreactors in white biotechnology.

PubMed

Muffler, K; Lakatos, M; Schlegel, C; Strieth, D; Kuhne, S; Ulber, R

2014-01-01

The production of valuable compounds in industrial biotechnology is commonly done by cultivation of suspended cells or use of (immobilized) enzymes rather than using microorganisms in an immobilized state. Within the field of wastewater as well as odor treatment the application of immobilized cells is a proven technique. The cells are entrapped in a matrix of extracellular polymeric compounds produced by themselves. The surface-associated agglomerate of encapsulated cells is termed biofilm. In comparison to common immobilization techniques, toxic effects of compounds used for cell entrapment may be neglected. Although the economic impact of biofilm processes used for the production of valuable compounds is negligible, many prospective approaches were examined in the laboratory and on a pilot scale. This review gives an overview of biofilm reactors applied to the production of valuable compounds. Moreover, the characteristics of the utilized materials are discussed with respect to support of surface-attached microbial growth.

Screen printing technology applied to silicon solar cell fabrication

NASA Technical Reports Server (NTRS)

Thornhill, J. W.; Sipperly, W. E.

1980-01-01

The process for producing space qualified solar cells in both the conventional and wraparound configuration using screen printing techniques was investigated. Process modifications were chosen that could be easily automated or mechanized. Work was accomplished to optimize the tradeoffs associated with gridline spacing, gridline definition and junction depth. An extensive search for possible front contact metallization was completed. The back surface field structures along with the screen printed back contacts were optimized to produce open circuit voltages of at least an average of 600 millivolts. After all intended modifications on the process sequence were accomplished, the cells were exhaustively tested. Electrical tests at AMO and 28 C were made before and after boiling water immersion, thermal shock, and storage under conditions of high temperature and high humidity.

Investigating dynamic structural and mechanical changes of neuroblastoma cells associated with glutamate-mediated neurodegeneration

NASA Astrophysics Data System (ADS)

Fang, Yuqiang; Iu, Catherine Y. Y.; Lui, Cathy N. P.; Zou, Yukai; Fung, Carmen K. M.; Li, Hung Wing; Xi, Ning; Yung, Ken K. L.; Lai, King W. C.

2014-11-01

Glutamate-mediated neurodegeneration resulting from excessive activation of glutamate receptors is recognized as one of the major causes of various neurological disorders such as Alzheimer's and Huntington's diseases. However, the underlying mechanisms in the neurodegenerative process remain unidentified. Here, we investigate the real-time dynamic structural and mechanical changes associated with the neurodegeneration induced by the activation of N-methyl-D-aspartate (NMDA) receptors (a subtype of glutamate receptors) at the nanoscale. Atomic force microscopy (AFM) is employed to measure the three-dimensional (3-D) topography and mechanical properties of live SH-SY5Y cells under stimulus of NMDA receptors. A significant increase in surface roughness and stiffness of the cell is observed after NMDA treatment, which indicates the time-dependent neuronal cell behavior under NMDA-mediated neurodegeneration. The present AFM based study further advance our understanding of the neurodegenerative process to elucidate the pathways and mechanisms that govern NMDA induced neurodegeneration, so as to facilitate the development of novel therapeutic strategies for neurodegenerative diseases.

Tumor and Endothelial Cell-Derived Microvesicles Carry Distinct CEACAMs and Influence T-Cell Behavior

PubMed Central

Muturi, Harrison T.; Dreesen, Janine D.; Nilewski, Elena; Jastrow, Holger; Giebel, Bernd; Ergun, Suleyman; Singer, Bernhard B.

2013-01-01

Normal and malignant cells release a variety of different vesicles into their extracellular environment. The most prominent vesicles are the microvesicles (MVs, 100-1 000 nm in diameter), which are shed of the plasma membrane, and the exosomes (70-120 nm in diameter), derivates of the endosomal system. MVs have been associated with intercellular communication processes and transport numerous proteins, lipids and RNAs. As essential component of immune-escape mechanisms tumor-derived MVs suppress immune responses. Additionally, tumor-derived MVs have been found to promote metastasis, tumor-stroma interactions and angiogenesis. Since members of the carcinoembryonic antigen related cell adhesion molecule (CEACAM)-family have been associated with similar processes, we studied the distribution and function of CEACAMs in MV fractions of different human epithelial tumor cells and of human and murine endothelial cells. Here we demonstrate that in association to their cell surface phenotype, MVs released from different human epithelial tumor cells contain CEACAM1, CEACAM5 and CEACAM6, while human and murine endothelial cells were positive for CEACAM1 only. Furthermore, MVs derived from CEACAM1 transfected CHO cells carried CEACAM1. In terms of their secretion kinetics, we show that MVs are permanently released in low doses, which are extensively increased upon cellular starvation stress. Although CEACAM1 did not transmit signals into MVs it served as ligand for CEACAM expressing cell types. We gained evidence that CEACAM1-positive MVs significantly increase the CD3 and CD3/CD28-induced T-cell proliferation. All together, our data demonstrate that MV-bound forms of CEACAMs play important roles in intercellular communication processes, which can modulate immune response, tumor progression, metastasis and angiogenesis. PMID:24040308

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.

Glycan Engineering for Cell and Developmental Biology.

PubMed

Griffin, Matthew E; Hsieh-Wilson, Linda C

2016-01-21

Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to modulate the structures of glycans on cell surfaces provides a powerful means not only to understand fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered glycans have been employed to control biological outcomes such as the immune response and stem cell fate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Parallel-processing with surface plasmons, a new strategy for converting the broad solar spectrum

NASA Technical Reports Server (NTRS)

Anderson, L. M.

1982-01-01

A new strategy for efficient solar-energy conversion is based on parallel processing with surface plasmons: guided electromagnetic waves supported on thin films of common metals like aluminum or silver. The approach is unique in identifying a broadband carrier with suitable range for energy transport and an inelastic tunneling process which can be used to extract more energy from the more energetic carriers without requiring different materials for each frequency band. The aim is to overcome the fundamental 56-percent loss associated with mismatch between the broad solar spectrum and the monoenergetic conduction electrons used to transport energy in conventional silicon solar cells. This paper presents a qualitative discussion of the unknowns and barrier problems, including ideas for coupling surface plasmons into the tunnels, a step which has been the weak link in the efficiency chain.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation.

PubMed

Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand; Apte, Shree Kumar

2016-08-15

Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Interaction of Uranium with Bacterial Cell Surfaces: Inferences from Phosphatase-Mediated Uranium Precipitation

PubMed Central

Kulkarni, Sayali; Misra, Chitra Seetharam; Gupta, Alka; Ballal, Anand

2016-01-01

ABSTRACT Deinococcus radiodurans and Escherichia coli expressing either PhoN, a periplasmic acid phosphatase, or PhoK, an extracellular alkaline phosphatase, were evaluated for uranium (U) bioprecipitation under two specific geochemical conditions (GCs): (i) a carbonate-deficient condition at near-neutral pH (GC1), and (ii) a carbonate-abundant condition at alkaline pH (GC2). Transmission electron microscopy revealed that recombinant cells expressing PhoN/PhoK formed cell-associated uranyl phosphate precipitate under GC1, whereas the same cells displayed extracellular precipitation under GC2. These results implied that the cell-bound or extracellular location of the precipitate was governed by the uranyl species prevalent at that particular GC, rather than the location of phosphatase. MINTEQ modeling predicted the formation of predominantly positively charged uranium hydroxide ions under GC1 and negatively charged uranyl carbonate-hydroxide complexes under GC2. Both microbes adsorbed 6- to 10-fold more U under GC1 than under GC2, suggesting that higher biosorption of U to the bacterial cell surface under GC1 may lead to cell-associated U precipitation. In contrast, at alkaline pH and in the presence of excess carbonate under GC2, poor biosorption of negatively charged uranyl carbonate complexes on the cell surface might have resulted in extracellular precipitation. The toxicity of U observed under GC1 being higher than that under GC2 could also be attributed to the preferential adsorption of U on cell surfaces under GC1. This work provides a vivid description of the interaction of U complexes with bacterial cells. The findings have implications for the toxicity of various U species and for developing biological aqueous effluent waste treatment strategies. IMPORTANCE The present study provides illustrative insights into the interaction of uranium (U) complexes with recombinant bacterial cells overexpressing phosphatases. This work demonstrates the effects of aqueous speciation of U on the biosorption of U and the localization pattern of uranyl phosphate precipitated as a result of phosphatase action. Transmission electron microscopy revealed that location of uranyl phosphate (cell associated or extracellular) was primarily influenced by aqueous uranyl species present under the given geochemical conditions. The data would be useful for understanding the toxicity of U under different geochemical conditions. Since cell-associated precipitation of metal facilitates easy downstream processing by simple gravity-based settling down of metal-loaded cells, compared to cumbersome separation techniques, the results from this study are of considerable relevance to effluent treatment using such cells. PMID:27287317

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.

3D surface reconstruction and visualization of the Drosophila wing imaginal disc at cellular resolution

NASA Astrophysics Data System (ADS)

Bai, Linge; Widmann, Thomas; Jülicher, Frank; Dahmann, Christian; Breen, David

2013-01-01

Quantifying and visualizing the shape of developing biological tissues provide information about the morphogenetic processes in multicellular organisms. The size and shape of biological tissues depend on the number, size, shape, and arrangement of the constituting cells. To better understand the mechanisms that guide tissues into their final shape, it is important to investigate the cellular arrangement within tissues. Here we present a data processing pipeline to generate 3D volumetric surface models of epithelial tissues, as well as geometric descriptions of the tissues' apical cell cross-sections. The data processing pipeline includes image acquisition, editing, processing and analysis, 2D cell mesh generation, 3D contourbased surface reconstruction, cell mesh projection, followed by geometric calculations and color-based visualization of morphological parameters. In their first utilization we have applied these procedures to construct a 3D volumetric surface model at cellular resolution of the wing imaginal disc of Drosophila melanogaster. The ultimate goal of the reported effort is to produce tools for the creation of detailed 3D geometric models of the individual cells in epithelial tissues. To date, 3D volumetric surface models of the whole wing imaginal disc have been created, and the apicolateral cell boundaries have been identified, allowing for the calculation and visualization of cell parameters, e.g. apical cross-sectional area of cells. The calculation and visualization of morphological parameters show position-dependent patterns of cell shape in the wing imaginal disc. Our procedures should offer a general data processing pipeline for the construction of 3D volumetric surface models of a wide variety of epithelial tissues.

A cell-based MHC stabilization assay for the detection of peptide binding to the canine classical class I molecule, DLA-88.

PubMed

Ross, Peter; Holmes, Jennifer C; Gojanovich, Gregory S; Hess, Paul R

2012-12-15

Identifying immunodominant CTL epitopes is essential for studying CD8+ T-cell responses in populations, but remains difficult, as peptides within antigens typically are too numerous for all to be synthesized and screened. Instead, to facilitate discovery, in silico scanning of proteins for sequences that match the motif, or binding preferences, of the restricting MHC class I allele - the largest determinant of immunodominance - can be used to predict likely candidates. The high false positive rate with this analysis ideally requires binding confirmation, which is obtained routinely by an assay using cell lines such as RMA-S that have defective transporter associated with antigen processing (TAP) machinery, and consequently, few surface class I molecules. The stabilization and resultant increased life-span of peptide-MHC complexes on the cell surface by the addition of true binders validates their identity. To determine whether a similar assay could be developed for dogs, we transfected a prevalent class I allele, DLA-88*50801, into RMA-S. In the BARC3 clone, the recombinant heavy chain was associated with murine β2-microglobulin, and importantly, could differentiate motif-matched and -mismatched peptides by surface MHC stabilization. This work demonstrates the potential to use RMA-S cells transfected with canine alleles as a tool for CTL epitope discovery in this species. Copyright © 2012 Elsevier B.V. All rights reserved.

Use of a portable hyperspectral imaging system for monitoring the efficacy of sanitation procedures in produce processing plants

USDA-ARS?s Scientific Manuscript database

Cleaning and sanitation of production surfaces and equipment plays a critical role in lowering the risk of food borne illness associated with consumption of fresh-cut produce. Visual observation and sampling methods including ATP tests and cell culturing are commonly used to monitor the effectivenes...

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.

A high volume cost efficient production macrostructuring process. [for silicon solar cell surface treatment

NASA Technical Reports Server (NTRS)

Chitre, S. R.

1978-01-01

The paper presents an experimentally developed surface macro-structuring process suitable for high volume production of silicon solar cells. The process lends itself easily to automation for high throughput to meet low-cost solar array goals. The tetrahedron structure observed is 0.5 - 12 micron high. The surface has minimal pitting with virtually no or very few undeveloped areas across the surface. This process has been developed for (100) oriented as cut silicon. Chemi-etched, hydrophobic and lapped surfaces were successfully texturized. A cost analysis as per Samics is presented.

Development of pulsed processes for the manufacture of solar cells. Quarterly progress report No. 3, April--July 1978

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

Not Available

1978-07-01

Third quarter results under a program to develop ion implantation and specialized, associated processes necessary to achieve automated production of silicon solar cells are described. An ion implantation facility development for solar cell production is described, and a design for an automated production implanter is presented. Also, solar cell development efforts using combined ion implantation and pulsed energy techniques are discussed. Cell performance comparisons have also been made in which junctions and back surface fields were prepared by diffusion and ion implantation. A model is presented to explain the mechanism of ion implantation damage annealing using pulsed energy sources. Functionalmore » requirements have been determined for a pulsed electron beam processor for annealing ion implantation damage at a rate compatible with a 100 milliampere ion implanter. These rates result in a throughput of 100 megawatts of solar cell product per year.« less

An ubiquitin-binding molecule can work as an inhibitor of ubiquitin processing enzymes and ubiquitin receptors.

PubMed

Nguyen, Thanh; Ho, Minh; Ghosh, Ambarnil; Kim, Truc; Yun, Sun Il; Lee, Seung Seo; Kim, Kyeong Kyu

2016-10-07

The ubiquitin pathway plays a critical role in regulating diverse biological processes, and its dysregulation is associated with various diseases. Therefore, it is important to have a tool that can control the ubiquitin pathway in order to improve understanding of this pathway and to develop therapeutics against relevant diseases. We found that Chicago Sky Blue 6B binds directly to the β-groove, a major interacting surface of ubiquitin. Hence, it could successfully inhibit the enzymatic activity of ubiquitin processing enzymes and the binding of ubiquitin to the CXCR4, a cell surface ubiquitin receptor. Furthermore, we demonstrated that this ubiquitin binding chemical could effectively suppress the ubiquitin induced cancer cell migration by blocking ubiquitin-CXCR4 interaction. Current results suggest that ubiquitin binding molecules can be developed as inhibitors of ubiquitin-protein interactions, which will have the value not only in unveiling the biological role of ubiquitin but also in treating related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Real-time and label-free analysis of binding thermodynamics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a QCM biosensor

PubMed Central

Li, Xueming; Song, Siyu; Shuai, Qi; Pei, Yihan; Aastrup, Teodor; Pei, Yuxin; Pei, Zhichao

2015-01-01

A novel approach to the study of binding thermodynamics and kinetics of carbohydrate-protein interactions on unfixed cancer cell surfaces using a quartz crystal microbalance (QCM) biosensor was developed, in which binding events take place at the cell surface, more closely mimicking a biologically relevant environment. In this study, colon adenocarcinoma cells (KM-12) and ovary adenocarcinoma cells (SKOV-3) grew on the optimized polystyrene-coated biosensor chip without fixation. The association and dissociation between the cell surface carbohydrates and a range of lectins, including WGA, Con A, UEA-I, GS-II, PNA and SBA, were monitored in real time and without label for evaluation of cell surface glycosylation. Furthermore, the thermodynamic and kinetic parameters of the interaction between lectins and cell surface glycan were studied, providing detailed information about the interactions, such as the association rate constant, dissociation rate constant, affinity constant, as well as the changes of entropy, enthalpy and Gibbs free energy. This application provides an insight into the cell surface glycosylation and the complex molecular recognition on the intact cell surface, which may have impacts on disease diagnosis and drug discovery. PMID:26369583

Recrystallization as a controlling process in the wear of some f.c.c. metals

NASA Technical Reports Server (NTRS)

Bill, R. C.; Wisander, D.

1977-01-01

Detailed examination of copper specimens after sliding against 440 C steel in liquid methane at speeds up to 25 m/s and loads of up to 2 kg showed the metal comprising the wear surface to possess a fine cell recrystallized structure. Wear proceeded by the plastic shearing of metal in this near surface region without the occurrence of visible metal transfer. A dynamic balance between the intense shear process at the surface and the nucleation of recrystallized grains was proposed to account for the behavior of the metal at the wear surface. Sliding wear experiments were also conducted on Ag, Cu-10% Al, Cu-10% Sn, Ni and Al. It was found that low wear and the absence of heavy metal transfer were associated with those metals observed to undergo recrystallization nucleation without prior recovery.

Automated Array Assembly, Phase 2

NASA Technical Reports Server (NTRS)

Carbajal, B. G.

1979-01-01

The solar cell module process development activities in the areas of surface preparation are presented. The process step development was carried out on texture etching including the evolution of a conceptual process model for the texturing process; plasma etching; and diffusion studies that focused on doped polymer diffusion sources. Cell processing was carried out to test process steps and a simplified diode solar cell process was developed. Cell processing was also run to fabricate square cells to populate sample minimodules. Module fabrication featured the demonstration of a porcelainized steel glass structure that should exceed the 20 year life goal of the low cost silicon array program. High efficiency cell development was carried out in the development of the tandem junction cell and a modification of the TJC called the front surface field cell. Cell efficiencies in excess of 16 percent at AM1 have been attained with only modest fill factors. The transistor-like model was proposed that fits the cell performance and provides a guideline for future improvements in cell performance.

Hydrophobically-associating cationic polymers as micro-bubble surface modifiers in dissolved air flotation for cyanobacteria cell separation.

PubMed

Yap, R K L; Whittaker, M; Diao, M; Stuetz, R M; Jefferson, B; Bulmus, V; Peirson, W L; Nguyen, A V; Henderson, R K

2014-09-15

Dissolved air flotation (DAF), an effective treatment method for clarifying algae/cyanobacteria-laden water, is highly dependent on coagulation-flocculation. Treatment of algae can be problematic due to unpredictable coagulant demand during blooms. To eliminate the need for coagulation-flocculation, the use of commercial polymers or surfactants to alter bubble charge in DAF has shown potential, termed the PosiDAF process. When using surfactants, poor removal was obtained but good bubble adherence was observed. Conversely, when using polymers, effective cell removal was obtained, attributed to polymer bridging, but polymers did not adhere well to the bubble surface, resulting in a cationic clarified effluent that was indicative of high polymer concentrations. In order to combine the attributes of both polymers (bridging ability) and surfactants (hydrophobicity), in this study, a commercially-available cationic polymer, poly(dimethylaminoethyl methacrylate) (polyDMAEMA), was functionalised with hydrophobic pendant groups of various carbon chain lengths to improve adherence of polymer to a bubble surface. Its performance in PosiDAF was contrasted against commercially-available poly(diallyl dimethyl ammonium chloride) (polyDADMAC). All synthesised polymers used for bubble surface modification were found to produce positively charged bubbles. When applying these cationic micro-bubbles in PosiDAF, in the absence of coagulation-flocculation, cell removals in excess of 90% were obtained, reaching a maximum of 99% cell removal and thus demonstrating process viability. Of the synthesised polymers, the polymer containing the largest hydrophobic functionality resulted in highly anionic treated effluent, suggesting stronger adherence of polymers to bubble surfaces and reduced residual polymer concentrations. Copyright © 2014 Elsevier Ltd. All rights reserved.

Studies of hemidesmosomes in human amnion: the use of a detergent extraction protocol for compositional and ultrastructural analysis and preparation of a hemidesmosome-enriched fraction from tissue.

PubMed

Behzad, F; Jones, C J; Ball, S; Alvares, T; Aplin, J D

1995-01-01

A method is described for the sequential detergent and high ionic strength extraction of human amnion with the progressive enrichment of the intermediate filament (IF) cytoskeleton and its associated structures including hemidesmosomes (HD). TEM of the extracted epithelium in situ reveals IF bundles beneath the apical cell surface, around the nucleus and at the lateral edges of the cells where association with desmosomes occurs. IF bundles are also very prominent within basal cell processes where they loop through the cytoplasm adjacent to the HDs. A novel connecting filament network is observed running between the IFs and the hemidesmosomal dense plaque. The adjacent IF network contains both cytokeratin and vimentin, the latter revealed much more fully as a result of the extraction protocol. The hemidesmosomal plasma membrane contains integrin subunits alpha 6 and beta 4 and these are quantitatively retained as the basal cell surface during extraction, while nonjunctional plasma membrane is solubilised. Integrin beta 1 is found at the basolateral cell surface but, like actin, is extracted quantitatively and is not present in HDs. The extracted epithelial cells may be recovered by scraping and the IF network depolymerised to produce a particulate fraction containing short residual IFs, associated thin filaments and plaque material. This fraction contains immunoreactive cytokeratin and vimentin. Integrin alpha 6 beta 4 has been used as a biochemical criterion of the presence of HD material in the fraction. Both subunits are highly enriched. The fraction also contains the hemidesmosomal components HD1, BP230 and BP180. This method is likely to be useful in further characterisation of the HD.

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

Robust Non-Wetting PTFE Surfaces by Femtosecond Laser Machining

PubMed Central

Liang, Fang; Lehr, Jorge; Danielczak, Lisa; Leask, Richard; Kietzig, Anne-Marie

2014-01-01

Nature shows many examples of surfaces with extraordinary wettability, which can often be associated with particular air-trapping surface patterns. Here, robust non-wetting surfaces have been created by femtosecond laser ablation of polytetrafluoroethylene (PTFE). The laser-created surface structure resembles a forest of entangled fibers, which support structural superhydrophobicity even when the surface chemistry is changed by gold coating. SEM analysis showed that the degree of entanglement of hairs and the depth of the forest pattern correlates positively with accumulated laser fluence and can thus be influenced by altering various laser process parameters. The resulting fibrous surfaces exhibit a tremendous decrease in wettability compared to smooth PTFE surfaces; droplets impacting the virgin or gold coated PTFE forest do not wet the surface but bounce off. Exploratory bioadhesion experiments showed that the surfaces are truly air-trapping and do not support cell adhesion. Therewith, the created surfaces successfully mimic biological surfaces such as insect wings with robust anti-wetting behavior and potential for antiadhesive applications. In addition, the fabrication can be carried out in one process step, and our results clearly show the insensitivity of the resulting non-wetting behavior to variations in the process parameters, both of which make it a strong candidate for industrial applications. PMID:25110862

Characterization of the Cell Surface Properties of Drinking Water Pathogens by Microbial Adhesion to Hydrocarbon and Electrophoretic Mobility Measurements

EPA Science Inventory

The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregati...

Co-culture of osteocytes and neurons on a unique patterned surface.

PubMed

Boggs, Mary E; Thompson, William R; Farach-Carson, Mary C; Duncan, Randall L; Beebe, Thomas P

2011-12-01

Neural and skeletal communication is essential for the maintenance of bone mass and transmission of pain, yet the mechanism(s) of signal transduction between these tissues is unknown. The authors established a novel system to co-culture murine long bone osteocyte-like cells (MLO-Y4) and primary murine dorsal root ganglia (DRG) neurons. Assessment of morphology and maturation marker expression on perlecan domain IV peptide (PlnDIV) and collagen type-1 (Col1) demonstrated that PlnDIV was an optimal matrix for MLO-Y4 culture. A novel matrix-specificity competition assay was developed to expose these cells to several extracellular matrix proteins such as PlnDIV, Col1, and laminin (Ln). The competition assay showed that approximately 70% of MLO-Y4 cells preferred either PlnDIV or Col1 to Ln. To co-culture MLO-Y4 and DRG, we developed patterned surfaces using micro-contact printing to create 40 μm × 1 cm alternating stripes of PlnDIV and Ln or PlnDIV and Col1. Co-culture on PlnDIV/Ln surfaces demonstrated that these matrix molecules provided unique cues for each cell type, with MLO-Y4 preferentially attaching to the PlnDIV lanes and DRG neurons to the Ln lanes. Approximately 80% of DRG were localized to Ln. Cellular processes from MLO-Y4 were closely associated with axonal extensions of DRG neurons. Approximately 57% of neuronal processes were in close proximity to nearby MLO-Y4 cells at the PlnDIV-Ln interface. The surfaces in this new assay provided a unique model system with which to study the communication between osteocyte-like cells and neurons in an in vitro environment.

Identification of a New Class of Lipid Droplet-Associated Proteins in Plants1[C][W][OPEN

PubMed Central

Horn, Patrick J.; James, Christopher N.; Gidda, Satinder K.; Kilaru, Aruna; Dyer, John M.; Mullen, Robert T.; Ohlrogge, John B.; Chapman, Kent D.

2013-01-01

Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues. PMID:23821652

Ephrinb1 and Ephrinb2 Are Associated with Interleukin-7 Receptor α and Retard Its Internalization from the Cell Surface*

PubMed Central

Luo, Hongyu; Wu, Zenghui; Qi, Shijie; Jin, Wei; Han, Bing; Wu, Jiangping

2011-01-01

IL-7 plays vital roles in thymocyte development, T cell homeostasis, and the survival of these cells. IL-7 receptor α (IL-7Rα) on thymocytes and T cells is rapidly internalized upon IL-7 ligation. Ephrins (Efns) are cell surface molecules and ligands of the largest receptor kinase family, Eph kinases. We discovered that T cell-specific double gene knock-out (dKO) of Efnb1 and Efnb2 in mice led to reduced IL-7Rα expression in thymocytes and T cells, and that IL-7Rα down-regulation was accelerated in dKO CD4 cells upon IL-7 treatment. On the other hand, Efnb1 and Efnb2 overexpression on T cell lymphoma EL4 cells retarded IL-7Rα down-regulation. dKO T cells manifested compromised STAT5 activation and homeostatic proliferation, an IL-7-dependent process. Fluorescence resonance energy transfer and immunoprecipitation demonstrated that Efnb1 and Efnb2 interacted physically with IL-7Rα. Such interaction likely retarded IL-7Rα internalization, as Efnb1 and Efnb2 were not internalized. Therefore, we revealed a novel function of Efnb1 and Efnb2 in stabilizing IL-7Rα expression at the post-translational level, and a previously unknown modus operandi of Efnbs in the regulation of expression of other vital cell surface receptors. PMID:22069310

Tyrosine phosphorylation–dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

PubMed Central

Kanda, Shoichiro; Harita, Yutaka; Shibagaki, Yoshio; Sekine, Takashi; Igarashi, Takashi; Inoue, Takafumi; Hattori, Seisuke

2011-01-01

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6–PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type–specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity. PMID:21471003

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells.

PubMed

Peters, B P; Hartle, R J; Krzesicki, R F; Kroll, T G; Perini, F; Balun, J E; Goldstein, I J; Ruddon, R W

1985-11-25

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

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

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.

Clearance of Dying Cells by Phagocytes: Mechanisms and Implications for Disease Pathogenesis.

PubMed

Fond, Aaron M; Ravichandran, Kodi S

The efficient clearance of apoptotic cells is an evolutionarily conserved process crucial for homeostasis in multicellular organisms. The clearance involves a series of steps that ultimately facilitates the recognition of the apoptotic cell by the phagocytes and the subsequent uptake and processing of the corpse. These steps include the phagocyte sensing of "find-me" signals released by the apoptotic cell, recognizing "eat-me" signals displayed on the apoptotic cell surface, and then intracellular signaling within the phagocyte to mediate phagocytic cup formation around the corpse and corpse internalization, and the processing of the ingested contents. The engulfment of apoptotic cells by phagocytes not only eliminates debris from tissues but also produces an anti-inflammatory response that suppresses local tissue inflammation. Conversely, impaired corpse clearance can result in loss of immune tolerance and the development of various inflammation-associated disorders such as autoimmunity, atherosclerosis, and airway inflammation but can also affect cancer progression. Recent studies suggest that the clearance process can also influence antitumor immune responses. In this review, we will discuss how apoptotic cells interact with their engulfing phagocytes to generate important immune responses, and how modulation of such responses can influence pathology.

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

Adhesion of Photoactive Microalgae to Surfaces is Switchable by Light

NASA Astrophysics Data System (ADS)

Bäumchen, Oliver; Kreis, Christian; Le Blay, Marine; Linne, Christine; Makowski, Marcin

The natural habitats of many microorganisms are confined geometries, such as the interstitial space of rocks and soil, where interactions with interfaces and surfaces are of paramount importance. We performed in vivo force spectroscopy experiments on the unicellular biflagellated microalga Chlamydomonas, a prime model organism in cell- and microbiology, and discovered that the flagella-mediated adhesion to surfaces can be switched on and off by light. Time-resolved micropipette experiments show that the light-switchable adhesiveness of the flagella is a completely reversible process that is based on a redistribution of adhesion-promoting flagella-membrane proteins within seconds. Light-switchable adhesion enables the cell to regulate the transition between planktonic and surface-associated state, which possibly represents a significant biological advantage for photoactive microorganisms. In terms of the colonization of surfaces and the formation of biofilms, the findings might have immediate economic and environmental relevance in biotechnological settings, such as photo-bioreactors for the sustainable production of biofuels.

Possible ecological role of pseudopterosins G and P-U and seco-pseudopterosins J and K from the gorgonian Pseudopterogorgia elisabethae from Providencia Island (SW Caribbean) in regulating microbial surface communities.

PubMed

Correa, Hebelin; Zorro, Pamela; Arevalo-Ferro, Catalina; Puyana, Monica; Duque, Carmenza

2012-09-01

The gorgonian Pseudopterogorgia elisabethae collected at Providencia Island (Colombia) has an unfouled surface, free of obvious algal and invertebrate growth. This gorgonian produces significant amounts of the glycosilated diterpenes pseudopterosins and seco-pseudopterosins (Ps and seco-Ps). Our previous experiments have shown activity of these compounds against eukaryotic (human cancer cell lines and Candida albicans) and prokaryotic cells (Staphylococcus aureus and Enterococcus faecalis). However, the potential role of pseudopterosins on the regulation of the fouling process is still under study. We evaluated the activity of these compounds against bacteria isolated from heavily fouled marine surfaces as an indicator of antifouling activity. Additionally, we assessed their activity against bacteria isolated from P. elisabethae to determine whether potentially they play a role in preventing surface bacterial colonization, thus impairing presumptively the establishment of further successional stages of fouling communities. Results showed that Ps and seco-Ps seem to modulate bacterial growth (controlling Gram-positive bacterial growth and inducing Gram-negative bacterial associations). We thus hypothesized that Ps and seco-Ps may play a role in controlling microbial fouling communities on the surface of this gorgonian. By using bTEFAP and FISH we showed that the most abundant bacteria present in the microbial communities associated with P. elisabethae are Gram-negative bacteria, with Proteobacteria and Gammaproteobacteria the most representative. To evaluate whether Ps and seco-Ps have a direct effect on the structure of the bacterial community associated with P. elisabethae, we tested these compounds against culturable bacteria associated with the surface of P. elisabethae, finding remarkable selectivity against Gram-positive bacteria. The evidence presented here suggests that Ps and seco-Ps might have a role in the selection of organisms associated with the gorgonian surface and in the regulation of the associated bacterial community composition.

IL-4Rα-Associated Antigen Processing by B Cells Promotes Immunity in Nippostrongylus brasiliensis Infection

PubMed Central

Hoving, Jennifer C.; Nieuwenhuizen, Natalie; McSorley, Henry J.; Ndlovu, Hlumani; Bobat, Saeeda; Kimberg, Matti; Kirstein, Frank; Cutler, Anthony J.; DeWals, Benjamin; Cunningham, Adam F.; Brombacher, Frank

2013-01-01

In this study, B cell function in protective TH2 immunity against N. brasiliensis infection was investigated. Protection against secondary infection depended on IL-4Rα and IL-13; but not IL-4. Protection did not associate with parasite specific antibody responses. Re-infection of B cell-specific IL-4Rα−/− mice resulted in increased worm burdens compared to control mice, despite their equivalent capacity to control primary infection. Impaired protection correlated with reduced lymphocyte IL-13 production and B cell MHC class II and CD86 surface expression. Adoptive transfer of in vivo N. brasiliensis primed IL-4Rα expressing B cells into naïve BALB/c mice, but not IL-4Rα or IL-13 deficient B cells, conferred protection against primary N. brasiliensis infection. This protection required MHC class II compatibility on B cells suggesting cognate interactions by B cells with CD4+ T cells were important to co-ordinate immunity. Furthermore, the rapid nature of these protective effects by B cells suggested non-BCR mediated mechanisms, such as via Toll Like Receptors, was involved, and this was supported by transfer experiments using antigen pulsed Myd88−/− B cells. These data suggest TLR dependent antigen processing by IL-4Rα-responsive B cells producing IL-13 contribute significantly to CD4+ T cell-mediated protective immunity against N. brasiliensis infection. PMID:24204255

Biocompatibility enhancement of rare earth magnesium alloy by laser surface processing

NASA Astrophysics Data System (ADS)

Nie, Shilin; Wang, Yuqing; Liu, Haifeng; Guan, Yingchun

2018-01-01

Although magnesium and magnesium alloys are considered biocompatible and biodegradable, insufficient biocompatibility in body fluid environment is still the major drawback of magnesium alloys for their successful applications as biodegradable orthopaedic implants. In this work, magnesium alloy surface with both enhanced corrosion resistance and better cell adhesion property was directly fabricated by laser surface processing. Laser surface melting was used to improve corrosion resistance of Mg-6Gd-0.6Ca alloy. After laser surface melting, laser surface texturing was utilized on melted surface for better cell adhesion property. The corrosion resistance of laser-treated and as-received samples were evaluated using electrochemical technique. The effect of laser surface treatment on phase and microstructure evolution was evaluated using scanning electron microscopy, optical microscopy and X-ray diffraction. This work investigated the effect of laser treatment on cell distribution across the surface of magnesium alloy substrates. Osteoblast was cultured on the laser-treated surface and as-received surface. Cell morphology was observed with a scanning electron microscopy, and cell viability was evaluated by optical density measurement.

Pulsed Light Treatment of Different Food Types with a Special Focus on Meat: A Critical Review.

PubMed

Heinrich, V; Zunabovic, M; Varzakas, T; Bergmair, J; Kneifel, W

2016-01-01

Today, the increasing demand for minimally processed foods that are at the same moment nutritious, organoleptically satisfactory, and free from microbial hazards challenges the research and development to establish alternative methods to reduce the level of bacterial contamination. As one of the recent emerging nonthermal methods, pulsed light (PL) constitutes a technology for the fast, mild, and residue-free surface decontamination of food and food contact materials in the processing environment. Via high frequency, high intensity pulses of broad-spectrum light rich in the UV fraction, viable cells as well as spores are inactivated in a nonselective multi-target process that rapidly overwhelms cell functions and subsequently leads to cell death. This review provides specific information on the technology of pulsed light and its suitability for unpackaged and packaged meat and meat products as well as food contact materials like production surfaces, cutting tools, and packaging materials. The advantages, limitations, risks, and essential process criteria to work efficiently are illustrated and discussed with relation to implementation on industrial level and future aspects. Other issues addressed by this paper are the need to take care of the associated parameters such as alteration of the product and utilized packaging material to satisfy consumers and other stakeholders.

Cellular membrane trafficking of mesoporous silica nanoparticles

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

Fang, I-Ju

This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulfmore » some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to determine the specific organelle that mesoporous silica nanoparticles could approach via the identification of harvested proteins from exocytosis process. Based on the study of endo- and exocytosis behavior of mesoporous silica nanoparticle materials, we can design smarter drug delivery vehicles for cancer therapy that can be effectively controlled. The destination, uptake efficiency and the cellular distribution of mesoporous silica nanoparticle materials can be programmable. As a result, release mechanism and release rate of drug delivery systems can be a well-controlled process. The deep investigation of an endo- and exocytosis study of mesoporous silica nanoparticle materials promotes the development of drug delivery applications.« less

Comparative and functional characterization of intragenic tandem repeats in 10 Aspergillus genomes.

PubMed

Gibbons, John G; Rokas, Antonis

2009-03-01

Intragenic tandem repeats (ITRs) are consecutive repeats of three or more nucleotides found in coding regions. ITRs are the underlying cause of several human genetic diseases and have been associated with phenotypic variation, including pathogenesis, in several clades of the tree of life. We have examined the evolution and functional role of ITRs in 10 genomes spanning the fungal genus Aspergillus, a clade of relevance to medicine, agriculture, and industry. We identified several hundred ITRs in each of the species examined. ITR content varied extensively between species, with an average 79% of ITRs unique to a given species. For the fraction of conserved ITR regions, sequence comparisons within species and between close relatives revealed that they were highly variable. ITR-containing proteins were evolutionarily less conserved, compositionally distinct, and overrepresented for domains associated with cell-surface localization and function relative to the rest of the proteome. Furthermore, ITRs were preferentially found in proteins involved in transcription, cellular communication, and cell-type differentiation but were underrepresented in proteins involved in metabolism and energy. Importantly, although ITRs were evolutionarily labile, their functional associations appeared. To be remarkably conserved across eukaryotes. Fungal ITRs likely participate in a variety of developmental processes and cell-surface-associated functions, suggesting that their contribution to fungal lifestyle and evolution may be more general than previously assumed.

A new nano-enhanced technology proposed to quantify intracellular detection of radiation-induced metabolic processes.

PubMed

Malak, Henryk; Richmond, Robert; Dicello, J F

2011-02-01

A new approach to intracellular detection and imaging of metabolic processes and pathways is presented that uses surface plasmon resonance to enhance interactions between photon-absorbing metabolites and metal nanoparticles in contact with cells in vitro or in vivo. Photon absorption in the nanoparticles creates plasmon fields, enhancing intrinsic metabolite fluorescence, thereby increasing absorption and emission rates, creating new spectral emission bands, shortening fluorescence lifetimes, becoming more photo-stable and increasing fluorescent resonance energy transfer efficiency. Because the cells remain viable, it is proposed that the method may be used to interrogate cells prior to and after irradiation, with the potential for automated analyses of intracellular interactive pathways associated with radiation exposures at lower doses than existing technologies. The design and concepts of the instrument are presented along with data for unexposed cells.

Radiation-induced immunogenic modulation of tumor enhances antigen processing and calreticulin exposure, resulting in enhanced T-cell killing

PubMed Central

Gameiro, Sofia R.; Jammed, Momodou L.; Wattenberg, Max M.; Tsang, Kwong Y.; Ferrone, Soldano; Hodge, James W.

2014-01-01

Radiation therapy (RT) is used for local tumor control through direct killing of tumor cells. Radiation-induced cell death can trigger tumor antigen-specific immune responses, but these are often noncurative. Radiation has been demonstrated to induce immunogenic modulation (IM) in various tumor types by altering the biology of surviving cells to render them more susceptible to T cell-mediated killing. Little is known about the mechanism(s) underlying IM elicited by sub-lethal radiation dosing. We have examined the molecular and immunogenic consequences of radiation exposure in breast, lung, and prostate human carcinoma cells. Radiation induced secretion of ATP and HMGB1 in both dying and surviving tumor cells. In vitro and in vivo tumor irradiation induced significant upregulation of multiple components of the antigen-processing machinery and calreticulin cell-surface expression. Augmented CTL lysis specific for several tumor-associated antigens was largely dictated by the presence of calreticulin on the surface of tumor cells and constituted an adaptive response to endoplasmic reticulum stress, mediated by activation of the unfolded protein response. This study provides evidence that radiation induces a continuum of immunogenic alterations in tumor biology, from immunogenic modulation to immunogenic cell death. We also expand the concept of immunogenic modulation, where surviving tumor cells recovering from radiation-induced endoplasmic reticulum stress become more sensitive to CTL killing. These observations offer a rationale for the combined use of radiation with immunotherapy, including for patients failing RT alone. PMID:24480782

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus.

PubMed

Senesi, Sonia; Ghelardi, Emilia; Celandroni, Francesco; Salvetti, Sara; Parisio, Eva; Galizzi, Alessandro

2004-02-01

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.

Steamed cake-derived 3D carbon foam with surface anchored carbon nanoparticles as freestanding anodes for high-performance microbial fuel cells.

PubMed

Yuan, Haoran; Dong, Ge; Li, Denian; Deng, Lifang; Cheng, Peng; Chen, Yong

2018-09-15

Anode design is highly significant for microbial fuel cells, since it simultaneously serves as the scaffold for electroactive microorganisms and as a medium for electron migration. In this study, a stiff 3D carbon foam with surface anchored nitrogen-containing carbon nanoparticles was facilely constructed via in-situ polyaniline coating of carbonized steamed cake prior to the carbonization process. The resultant product was determined to be an excellent freestanding anode that enabled the microbial fuel cell to deliver a maximum power density of up to 1307 mW/m 2 , which significantly outperformed its non-coated counterpart, the widely used commercial carbon felt. Further investigations revealed that the overall performance enhancement was associated with the open porosity, enlarged electroactive surface, increased biocompatibility, and decreased electric resistance of the anode scaffold. This promising anode material would offer a green and economical option for fabricating high-performance microbial fuel cell-based devices towards various ends. Copyright © 2018 Elsevier B.V. All rights reserved.

Effect of tissue scaffold topography on protein structure monitored by fluorescence spectroscopy.

PubMed

Portugal, Carla A M; Truckenmüller, Roman; Stamatialis, Dimitrios; Crespo, João G

2014-11-10

The impact of surface topography on the structure of proteins upon adhesion was assessed through non-invasive fluorescence monitoring. This study aimed at obtaining a better understanding about the role of protein structural status on cell-scaffold interactions. The changes induced upon adsorption of two model proteins with different geometries, trypsin (globular conformation) and fibrinogen (rod-shaped conformation) on poly-l-lactic acid (PLLA) scaffolds with different surface topographies, flat, fibrous and surfaces with aligned nanogrooves, were assessed by fluorescence spectroscopy monitoring, using tryptophan as structural probe. Hence, the maximum emission blue shift and the increase of fluorescence anisotropy observed after adsorption of globular and rod-like shaped proteins on surfaces with parallel nanogrooves were ascribed to more intense protein-surface interactions. Furthermore, the decrease of fluorescence anisotropy observed upon adsorption of proteins to scaffolds with fibrous morphology was more significant for rod-shaped proteins. This effect was associated to the ability of these proteins to adjust to curved surfaces. The additional unfolding of proteins induced upon adsorption on scaffolds with a fibrous morphology may be the reason for better cell attachment there, promoting an easier access of cell receptors to initially hidden protein regions (e.g. RGDS sequence), which are known to have a determinant role in cell attaching processes. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification of Proteins Associating with Glycosylphosphatidylinositol- Anchored T-Cadherin on the Surface of Vascular Endothelial Cells: Role for Grp78/BiP in T-Cadherin-Dependent Cell Survival▿ †

PubMed Central

Philippova, Maria; Ivanov, Danila; Joshi, Manjunath B.; Kyriakakis, Emmanouil; Rupp, Katharina; Afonyushkin, Taras; Bochkov, Valery; Erne, Paul; Resink, Therese J.

2008-01-01

There is scant knowledge regarding how cell surface lipid-anchored T-cadherin (T-cad) transmits signals through the plasma membrane to its intracellular targets. This study aimed to identify membrane proteins colocalizing with atypical glycosylphosphatidylinositol (GPI)-anchored T-cad on the surface of endothelial cells and to evaluate their role as signaling adaptors for T-cad. Application of coimmunoprecipitation from endothelial cells expressing c-myc-tagged T-cad and high-performance liquid chromatography revealed putative association of T-cad with the following proteins: glucose-related protein GRP78, GABA-A receptor α1 subunit, integrin β3, and two hypothetical proteins, LOC124245 and FLJ32070. Association of Grp78 and integrin β3 with T-cad on the cell surface was confirmed by surface biotinylation and reciprocal immunoprecipitation and by confocal microscopy. Use of anti-Grp78 blocking antibodies, Grp78 small interfering RNA, and coexpression of constitutively active Akt demonstrated an essential role for surface Grp78 in T-cad-dependent survival signal transduction via Akt in endothelial cells. The findings herein are relevant in the context of both the identification of transmembrane signaling partners for GPI-anchored T-cad as well as the demonstration of a novel mechanism whereby Grp78 can influence endothelial cell survival as a cell surface signaling receptor rather than an intracellular chaperone. PMID:18411300

Swimming motility plays a key role in the stochastic dynamics of cell clumping

NASA Astrophysics Data System (ADS)

Qi, Xianghong; Nellas, Ricky B.; Byrn, Matthew W.; Russell, Matthew H.; Bible, Amber N.; Alexandre, Gladys; Shen, Tongye

2013-04-01

Dynamic cell-to-cell interactions are a prerequisite to many biological processes, including development and biofilm formation. Flagellum induced motility has been shown to modulate the initial cell-cell or cell-surface interaction and to contribute to the emergence of macroscopic patterns. While the role of swimming motility in surface colonization has been analyzed in some detail, a quantitative physical analysis of transient interactions between motile cells is lacking. We examined the Brownian dynamics of swimming cells in a crowded environment using a model of motorized adhesive tandem particles. Focusing on the motility and geometry of an exemplary motile bacterium Azospirillum brasilense, which is capable of transient cell-cell association (clumping), we constructed a physical model with proper parameters for the computer simulation of the clumping dynamics. By modulating mechanical interaction (‘stickiness’) between cells and swimming speed, we investigated how equilibrium and active features affect the clumping dynamics. We found that the modulation of active motion is required for the initial aggregation of cells to occur at a realistic time scale. Slowing down the rotation of flagellar motors (and thus swimming speeds) is correlated to the degree of clumping, which is consistent with the experimental results obtained for A. brasilense.

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

PubMed Central

Lewis, Leanne E.; Bain, Judith M.; Okai, Blessing; Gow, Neil A.R.; Erwig, Lars Peter

2013-01-01

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10. In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms. PMID:23329139

Endoplasmic reticulum aminopeptidase 1 function and its pathogenic role in regulating innate and adaptive immunity in cancer and major histocompatibility complex class I-associated autoimmune diseases.

PubMed

Fruci, D; Romania, P; D'Alicandro, V; Locatelli, F

2014-08-01

Major histocompatibility complex (MHC) class I molecules present antigenic peptides on the cell surface to alert natural killer (NK) cells and CD8(+) T cells for the presence of abnormal intracellular events, such as virus infection or malignant transformation. The generation of antigenic peptides is a multistep process that ends with the trimming of N-terminal extensions in the endoplasmic reticulum (ER) by aminopeptidases ERAP1 and ERAP2. Recent studies have highlighted the potential role of ERAP1 in reprogramming the immunogenicity of tumor cells in order to elicit innate and adaptive antitumor immune responses, and in conferring susceptibility to autoimmune diseases in predisposed individuals. In this review, we will provide an overview of the current knowledge about the role of ERAP1 in MHC class I antigen processing and how its manipulation may constitute a promising tool for cancer immunotherapy and treatment of MHC class I-associated autoimmune diseases. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mesenchymal Stromal Cells Express GARP/LRRC32 on Their Surface: Effects on Their Biology and Immunomodulatory Capacity

PubMed Central

Carrillo-Galvez, Ana Belén; Cobo, Marién; Cuevas-Ocaña, Sara; Gutiérrez-Guerrero, Alejandra; Sánchez-Gilabert, Almudena; Bongarzone, Pierpaolo; García-Pérez, Angélica; Muñoz, Pilar; Benabdellah, Karim; Toscano, Miguel G; Martín, Francisco; Anderson, Per

2015-01-01

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3+ regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. Stem Cells 2015;33:183–195 PMID:25182959

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.

High-resolution imaging of cellular processes across textured surfaces using an indexed-matched elastomer.

PubMed

Ravasio, Andrea; Vaishnavi, Sree; Ladoux, Benoit; Viasnoff, Virgile

2015-03-01

Understanding and controlling how cells interact with the microenvironment has emerged as a prominent field in bioengineering, stem cell research and in the development of the next generation of in vitro assays as well as organs on a chip. Changing the local rheology or the nanotextured surface of substrates has proved an efficient approach to improve cell lineage differentiation, to control cell migration properties and to understand environmental sensing processes. However, introducing substrate surface textures often alters the ability to image cells with high precision, compromising our understanding of molecular mechanisms at stake in environmental sensing. In this paper, we demonstrate how nano/microstructured surfaces can be molded from an elastomeric material with a refractive index matched to the cell culture medium. Once made biocompatible, contrast imaging (differential interference contrast, phase contrast) and high-resolution fluorescence imaging of subcellular structures can be implemented through the textured surface using an inverted microscope. Simultaneous traction force measurements by micropost deflection were also performed, demonstrating the potential of our approach to study cell-environment interactions, sensing processes and cellular force generation with unprecedented resolution. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Multigenerational memory and adaptive adhesion in early bacterial biofilm communities.

PubMed

Lee, Calvin K; de Anda, Jaime; Baker, Amy E; Bennett, Rachel R; Luo, Yun; Lee, Ernest Y; Keefe, Joshua A; Helali, Joshua S; Ma, Jie; Zhao, Kun; Golestanian, Ramin; O'Toole, George A; Wong, Gerard C L

2018-04-24

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.

Investigation of Low Cost Substrate Approaches for III-V Solar Cells

NASA Astrophysics Data System (ADS)

Lichty, Marlene Lydia

With the need for cleaner energy sources, which can displace fossil fuel, the solar cell industry is of particular interest due to the abundancy of the Sun. Silicon currently dominates terrestrial applications, but efficiency improvements have saturated. III-V based solar cells have reported the highest efficiencies, however, high costs due to substrates and fabrication processes have limited these devices to specialty applications, such as space. In order to reduce the cost associated with fabricating III-V semiconductor substrate material, two different approaches were taken in this work with a particular focus on making III-Vs more applicable outside of specialty applications, including InP, InAsnd Ge. Typical material characterization techniques were used to analyze the samples and processes studied in this thesis. The first process examined was the direct epitaxial growth of III-V materials by MOCVD on cheaper substrates. More specifically, the direct growth of InP and InAs on metal foils. A growth time study and surface coverage analysis was performed for the growth of InP. A characterization study was then conducted on the second process, the aluminum- induced crystallization of germanium to determine the effects this process had on the surface. Crystalline InP, InAs and Ge were successfully characterized in this work, and show promise for use in cheaper III-V alternatives to terrestrial energy solutions.

Calcium and initial surface binding phase of pinocytosis in Amoeba proteus.

PubMed

Prusch, R D

1986-08-01

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca2+ in the external medium. Increasing external Ca2+ to approximately 10(-4) M increases pinocytotic intensity, while increases in Ca2+ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca2+ at the surface of Amoeba proteus was therefore examined. Alcian blue and Na+, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sites on the amoeba surface. Low levels of external Ca2+ in the range of 3 X 10(-5) to 1.5 X 10(-4) M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca2+ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity.

Deciliation Is Associated with Dramatic Remodeling of Epithelial Cell Junctions and Surface Domains

PubMed Central

Overgaard, Christian E.; Sanzone, Kaitlin M.; Spiczka, Krystle S.; Sheff, David R.; Sandra, Alexander

2009-01-01

Stress-induced shedding of motile cilia (autotomy) has been documented in diverse organisms and likely represents a conserved cellular reaction. However, little is known about whether primary cilia are shed from mammalian epithelial cells and what impact deciliation has on polarized cellular organization. We show that several chemically distinct agents trigger autotomy in epithelial cells. Surprisingly, deciliation is associated with a significant, but reversible increase in transepithelial resistance. This reflects substantial reductions in tight junction proteins associated with “leaky” nephron segments (e.g., claudin-2). At the same time, apical trafficking of gp80/clusterin and gp114/CEACAM becomes randomized, basal-lateral delivery of Na,K-ATPase is reduced, and expression of the nonciliary apical protein gp135/podocalyxin is greatly decreased. However, ciliogenesis-impaired MDCK cells do not undergo continual junction remodeling, and mature cilia are not required for autotomy-associated remodeling events. Deciliation and epithelial remodeling may be mechanistically linked processes, because RNAi-mediated reduction of Exocyst subunit Sec6 inhibits ciliary shedding and specifically blocks deciliation-associated down-regulation of claudin-2 and gp135. We propose that ciliary autotomy represents a signaling pathway that impacts the organization and function of polarized epithelial cells. PMID:19005211

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

PubMed Central

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-01-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface. PMID:27353000

Cargo binding promotes KDEL receptor clustering at the mammalian cell surface

NASA Astrophysics Data System (ADS)

Becker, Björn; Shaebani, M. Reza; Rammo, Domenik; Bubel, Tobias; Santen, Ludger; Schmitt, Manfred J.

2016-06-01

Transmembrane receptor clustering is a ubiquitous phenomenon in pro- and eukaryotic cells to physically sense receptor/ligand interactions and subsequently translate an exogenous signal into a cellular response. Despite that receptor cluster formation has been described for a wide variety of receptors, ranging from chemotactic receptors in bacteria to growth factor and neurotransmitter receptors in mammalian cells, a mechanistic understanding of the underlying molecular processes is still puzzling. In an attempt to fill this gap we followed a combined experimental and theoretical approach by dissecting and modulating cargo binding, internalization and cellular response mediated by KDEL receptors (KDELRs) at the mammalian cell surface after interaction with a model cargo/ligand. Using a fluorescent variant of ricin toxin A chain as KDELR-ligand (eGFP-RTAH/KDEL), we demonstrate that cargo binding induces dose-dependent receptor cluster formation at and subsequent internalization from the membrane which is associated and counteracted by anterograde and microtubule-assisted receptor transport to preferred docking sites at the plasma membrane. By means of analytical arguments and extensive numerical simulations we show that cargo-synchronized receptor transport from and to the membrane is causative for KDELR/cargo cluster formation at the mammalian cell surface.

Method for processing silicon solar cells

DOEpatents

Tsuo, Y.S.; Landry, M.D.; Pitts, J.R.

1997-05-06

The instant invention teaches a novel method for fabricating silicon solar cells utilizing concentrated solar radiation. The solar radiation is concentrated by use of a solar furnace which is used to form a front surface junction and back-surface field in one processing step. The present invention also provides a method of making multicrystalline silicon from amorphous silicon. The invention also teaches a method of texturing the surface of a wafer by forming a porous silicon layer on the surface of a silicon substrate and a method of gettering impurities. Also contemplated by the invention are methods of surface passivation, forming novel solar cell structures, and hydrogen passivation. 2 figs.

Method for processing silicon solar cells

DOEpatents

Tsuo, Y. Simon; Landry, Marc D.; Pitts, John R.

1997-01-01

The instant invention teaches a novel method for fabricating silicon solar cells utilizing concentrated solar radiation. The solar radiation is concentrated by use of a solar furnace which is used to form a front surface junction and back-surface field in one processing step. The present invention also provides a method of making multicrystallline silicon from amorphous silicon. The invention also teaches a method of texturing the surface of a wafer by forming a porous silicon layer on the surface of a silicon substrate and a method of gettering impurities. Also contemplated by the invention are methods of surface passivation, forming novel solar cell structures, and hydrogen passivation.

In situ proteolysis of the Vibrio cholerae matrix protein RbmA promotes biofilm recruitment.

PubMed

Smith, Daniel R; Maestre-Reyna, Manuel; Lee, Gloria; Gerard, Harry; Wang, Andrew H-J; Watnick, Paula I

2015-08-18

The estuarine gram-negative rod and human diarrheal pathogen Vibrio cholerae synthesizes a VPS exopolysaccharide-dependent biofilm matrix that allows it to form a 3D structure on surfaces. Proteins associated with the matrix include, RbmA, RbmC, and Bap1. RbmA, a protein whose crystallographic structure suggests two binding surfaces, associates with cells by means of a VPS-dependent mechanism and promotes biofilm cohesiveness and recruitment of cells to the biofilm. Here, we show that RbmA undergoes limited proteolysis within the biofilm. This proteolysis, which is carried out by the hemagglutinin/protease and accessory proteases, yields the 22-kDa C-terminal polypeptide RbmA*. RbmA* remains biofilm-associated. Unlike full-length RbmA, the association of RbmA* with cells is no longer VPS-dependent, likely due to an electropositive surface revealed by proteolysis. We provide evidence that this proteolysis event plays a role in recruitment of VPS(-) cells to the biofilm surface. Based on our findings, we propose that association of RbmA with the matrix reinforces the biofilm structure and leads to limited proteolysis of RbmA to RbmA*. RbmA*, in turn, promotes recruitment of cells that have not yet initiated VPS synthesis to the biofilm surface. The assignment of two functions to RbmA, separated by a proteolytic event that depends on matrix association, dictates an iterative cycle in which reinforcement of recently added biofilm layers precedes the recruitment of new VPS(-) cells to the biofilm.

Living together in biofilms: the microbial cell factory and its biotechnological implications.

PubMed

Berlanga, Mercedes; Guerrero, Ricardo

2016-10-01

In nature, bacteria alternate between two modes of growth: a unicellular life phase, in which the cells are free-swimming (planktonic), and a multicellular life phase, in which the cells are sessile and live in a biofilm, that can be defined as surface-associated microbial heterogeneous structures comprising different populations of microorganisms surrounded by a self-produced matrix that allows their attachment to inert or organic surfaces. While a unicellular life phase allows for bacterial dispersion and the colonization of new environments, biofilms allow sessile cells to live in a coordinated, more permanent manner that favors their proliferation. In this alternating cycle, bacteria accomplish two physiological transitions via differential gene expression: (i) from planktonic cells to sessile cells within a biofilm, and (ii) from sessile to detached, newly planktonic cells. Many of the innate characteristics of biofilm bacteria are of biotechnological interest, such as the synthesis of valuable compounds (e.g., surfactants, ethanol) and the enhancement/processing of certain foods (e.g., table olives). Understanding the ecology of biofilm formation will allow the design of systems that will facilitate making products of interest and improve their yields.

Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

PubMed

Stemmer, Nina; Strekalova, Elena; Djogo, Nevena; Plöger, Frank; Loers, Gabriele; Lutz, David; Buck, Friedrich; Michalak, Marek; Schachner, Melitta; Kleene, Ralf

2013-01-01

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.

Enabling aqueous processing for crack-free thick electrodes

DOE PAGES

Du, Zhijia; Rollag, K. M.; Li, J.; ...

2017-04-14

Aqueous processing of thick electrodes for Li-ion cells promises to increase energy density due to increased volume fraction of active materials, and to reduce cost due to the elimination of the toxic solvents. Here in this paper this work reports the processing and characterization of aqueous processed electrodes with high areal loading and associated full pouch cell performance. Cracking of the electrode coatings becomes a critical issue for aqueous processing of the positive electrode as areal loading increases above 20–25 mg/cm 2 (~4 mAh/cm 2). Crack initiation and propagation, which was observed during drying via optical microscopy, is related tomore » the build-up of capillary pressure during the drying process. The surface tension of water was reduced by the addition of isopropyl alcohol (IPA), which led to improved wettability and decreased capillary pressure during drying. The critical thickness (areal loading) without cracking increased gradually with increasing IPA content. The electrochemical performance was evaluated in pouch cells. Electrodes processed with water/IPA (80/20 wt%) mixture exhibited good structural integrity with good rate performance and cycling performance.« less

Regulation of Mesenchymal Stem Cell Differentiation by Nanopatterning of Bulk Metallic Glass.

PubMed

Loye, Ayomiposi M; Kinser, Emily R; Bensouda, Sabrine; Shayan, Mahdis; Davis, Rose; Wang, Rui; Chen, Zheng; Schwarz, Udo D; Schroers, Jan; Kyriakides, Themis R

2018-06-08

Mesenchymal stem cell (MSC) differentiation is regulated by surface modification including texturing, which is applied to materials to enhance tissue integration. Here, we used Pt 57.5 Cu 14.7 Ni 5.3 P 22.5 bulk metallic glass (Pt-BMG) with nanopatterned surfaces achieved by thermoplastic forming to influence differentiation of human MSCs. Pt-BMGs are a unique class of amorphous metals with high strength, elasticity, corrosion resistance, and an unusual plastic-like processability. It was found that flat and nanopattened Pt-BMGs induced osteogenic and adipogenic differentiation, respectively. In addition, osteogenic differentiation on flat BMG exceeded that observed on medical grade titanium and was associated with increased formation of focal adhesions and YAP nuclear localization. In contrast, cells on nanopatterned BMGs exhibited rounded morphology, formed less focal adhesions and had mostly cytoplasmic YAP. These changes were preserved on nanopatterns made of nanorods with increased stiffness due to shorter aspect ratios, suggesting that MSC differentiation was primarily influenced by topography. These observations indicate that both elemental composition and nanotopography can modulate biochemical cues and influence MSCs. Moreover, the processability and highly tunable nature of Pt-BMGs enables the creation of a wide range of surface topographies that can be reproducibly and systematically studied, leading to the development of implants capable of engineering MSC functions.

The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans

PubMed Central

Moffat, Laura L.; Robinson, Ryan E.; Bakoulis, Anastasia; Clark, Scott G.

2014-01-01

Wnts control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. We identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror and LIN-18/Ryk. PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. We propose that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development. PMID:24401370

Osseointegrated dental implants produced via microwave processing

NASA Astrophysics Data System (ADS)

Kutty, Muralithran G.

This research is a comprehensive effort to develop osseointegrated dental implants via microwave processing. A net-shape microwave sintering procedure was employed to fabricate dental implants. Commercial pure titanium powders (-100, -200 and -325 mesh sizes) were used in this work. This process eliminates the need for machining of implants and prevents contamination. The idea was to take advantage of the peculiar way microwave couple with metallic powders, i.e. generating heat in the interior of the sample and dissipating it away through the surface. The desired features for an implant, a dense core with surface pores, is not possible via conventional sintering. Coating with hydroxyapatite via electrodeposition and chemical combustion vapor deposition was also attempted to further enhance the bioactivity of this layer. Surface roughness and area were measured using a non-contact surface profilometer to further describe the unique surface. In-vitro studies, conducted using osteoblast cells extracted from neonatal rat calvarial, showed improved cell growth on all the uncoated porous samples. However, the highest cell growth was observed on the -200 mesh size samples. The higher surface area of the -200 mesh samples is attributed to this observation. This work was able to identify the processing parameters for titanium in microwave and establishes the importance of surface area as a key parameter for cell growth on porous surfaces as compared to surface roughness.

Clustering T cell GM1 Lipid Rafts Increases Cellular Resistance to Shear on Fibronectin through Changes in Integrin Affinity and Cytoskeletal Dynamics

PubMed Central

Mitchell, Jason S.; Brown, Wells S.; Woodside, Darren G.; Vanderslice, Peter; McIntyre, Bradley W.

2008-01-01

Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T cell functions. The aggregation of specific GM1 lipid rafts can control many T cell activation events, including their novel association with T cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin “inside-out” signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1 suggesting the induction of high affinity integrin conformations. The activation of these adhesion strengthening characteristics appear to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion strengthening processes. PMID:19139760

The p14 fusion-associated small transmembrane (FAST) protein effects membrane fusion from a subset of membrane microdomains.

PubMed

Corcoran, Jennifer A; Salsman, Jayme; de Antueno, Roberto; Touhami, Ahmed; Jericho, Manfred H; Clancy, Eileen K; Duncan, Roy

2006-10-20

The reovirus fusion-associated small transmembrane (FAST) proteins are a unique family of viral membrane fusion proteins. These nonstructural viral proteins induce efficient cell-cell rather than virus-cell membrane fusion. We analyzed the lipid environment in which the reptilian reovirus p14 FAST protein resides to determine the influence of the cell membrane on the fusion activity of the FAST proteins. Topographical mapping of the surface of fusogenic p14-containing liposomes by atomic force microscopy under aqueous conditions revealed that p14 resides almost exclusively in thickened membrane microdomains. In transfected cells, p14 was found in both Lubrol WX- and Triton X-100-resistant membrane complexes. Cholesterol depletion of donor cell membranes led to preferential disruption of p14 association with Lubrol WX (but not Triton X-100)-resistant membranes and decreased cell-cell fusion activity, both of which were reversed upon subsequent cholesterol repletion. Furthermore, co-patching analysis by fluorescence microscopy indicated that p14 did not co-localize with classical lipid-anchored raft markers. These data suggest that the p14 FAST protein associates with heterogeneous membrane microdomains, a distinct subset of which is defined by cholesterol-dependent Lubrol WX resistance and which may be more relevant to the membrane fusion process.

A Glycosylphosphatidylinositol Anchor Is Required for Membrane Localization but Dispensable for Cell Wall Association of Chitin Deacetylase 2 in Cryptococcus neoformans

PubMed Central

Gilbert, Nicole M.; Baker, Lorina G.; Specht, Charles A.; Lodge, Jennifer K.

2012-01-01

ABSTRACT Cell wall proteins (CWPs) mediate important cellular processes in fungi, including adhesion, invasion, biofilm formation, and flocculation. The current model of fungal cell wall organization includes a major class of CWPs covalently bound to β-1,6-glucan via a remnant of a glycosylphosphatidylinositol (GPI) anchor. This model was established by studies of ascomycetes more than a decade ago, and relatively little work has been done with other fungi, although the presumption has been that proteins identified in the cell wall which contain a predicted GPI anchor are covalently linked to cell wall glucans. The pathogenic basidiomycete Cryptococcus neoformans encodes >50 putatively GPI-anchored proteins, some of which have been identified in the cell wall. One of these proteins is chitin deacetylase 2 (Cda2), an enzyme responsible for converting chitin to chitosan, a cell wall polymer recently established as a virulence factor for C. neoformans infection of mammalian hosts. Using a combination of biochemistry, molecular biology, and genetics, we show that Cda2 is GPI anchored to membranes but noncovalently associated with the cell wall by means independent of both its GPI anchor and β-1,6-glucan. We also show that Cda2 produces chitosan when localized to the plasma membrane, but association with the cell wall is not essential for this process, thereby providing insight into the mechanism of chitosan biosynthesis. These results increase our understanding of the surface of C. neoformans and provide models of cell walls likely applicable to other undercharacterized basidiomycete pathogenic fungi. PMID:22354955

Enhancement of endothelialisation of coronary stents by laser surface engineering.

PubMed

Li, Lin; Mirhosseini, Nazanin; Michael, Alun; Liu, Zhu; Wang, Tao

2013-11-01

Coronary stents have been widely used in the treatment of coronary heart disease. However, complications have hampered the long-term success of the device. Bare-metal stents (BMS) have a high rate of restenosis and poor endothelialisation. The drug-eluting stents (DES), although dramatically reduce restenosis, significantly prevent endothelialisation leading to late thrombosis and behave the same way as BMS after drug releasing. Rapid adhesion and growth of endothelial cells on the stent surface is a key process for early vascular healing after coronary stenting which contributes to the reduction of major complications. Surface properties manipulate cell growth and directly determine the success and life-span of the implants. However, the ideal surface properties of coronary stents are not yet fully understood. The objective of this research is to understand how surface micro/nano textures and associated material chemistry changes generated by a laser beam affect the behavior of endothelial cells on bare metal 316L stents. A high power laser beam was applied to modifying the surface properties of 316L coronary stent material and the commercial coronary stents, followed by examination of the adhesion and proliferation of human coronary endothelial cells that were growing on the surfaces. Surface properties were examined by scanning electron microscopy, contact angle measurement, and X-ray photoelectron spectroscopy. A novel surface with combined micro/nano features was created on stent material 316L and coronary stent with a specific surface chemistry. This surface gives rise to a threefold increase in the adhesion and eightfold increase in the proliferation of endothelial cells. Interestingly, such effects were only observed when the surface texture was produced in the nitrogen atmosphere suggesting the importance of the surface chemistry, including the dramatic increase of chromium nitride, for the interaction of endothelial cells with the material surface. This novel surface is also super-hydrophilic with close to zero water/cell culture fluid contact angles and low cytotoxicity. A novel surface created by laser surface-engineering with a combination of defined surface texture and surface chemistry was found beneficial for the improvement of coronary stent endothelialisation. The technology presented here could work with both DES and BMS with added benefit for the improvement of the biocompatibility of current coronary stents. © 2013 Wiley Periodicals, Inc.

Germinal Center T Follicular Helper Cell IL-4 Production Is Dependent on Signaling Lymphocytic Activation Molecule Receptor (CD150)

PubMed Central

Yusuf, Isharat; Kageyama, Robin; Monticelli, Laurel; Johnston, Robert J.; DiToro, Daniel; Hansen, Kyle; Barnett, Burton; Crotty, Shane

2010-01-01

CD4 T cell help is critical for the generation and maintenance of germinal centers (GCs), and T follicular helper (TFH) cells are the CD4 T cell subset required for this process. Signaling lymphocytic activation molecule (SLAM)-associated protein (SAP [SH2D1A]) expression in CD4 T cells is essential for GC development. However, SAP-deficient mice have only a moderate defect in TFH differentiation, as defined by common TFH surface markers. CXCR5+ TFH cells are found within the GC, as well as along the boundary regions of T/B cell zones. In this study, we show that GC-associated T follicular helper (GC TFH) cells can be identified by their coexpression of CXCR5 and the GL7 epitope, allowing for phenotypic and functional analysis of TFH and GC TFH populations. GC TFH cells are a functionally discrete subset of further polarized TFH cells, with enhanced B cell help capacity and a specialized ability to produce IL-4 in a TH2-independent manner. Strikingly, SAP-deficient mice have an absence of the GC TFH cell subset and SAP− TFH cells are defective in IL-4 and IL-21 production. We further demonstrate that SLAM (Slamf1, CD150), a surface receptor that uses SAP signaling, is specifically required for IL-4 production by GC TFH cells. GC TFH cells require IL-4 and -21 production for optimal help to B cells. These data illustrate complexities of SAP-dependent SLAM family receptor signaling, revealing a prominent role for SLAM receptor ligation in IL-4 production by GC CD4 T cells but not in TFH cell and GC TFH cell differentiation. PMID:20525889

Silicon solar cell process. Development, fabrication and analysis

NASA Technical Reports Server (NTRS)

Yoo, H. I.; Iles, P. A.; Tanner, D. P.

1978-01-01

Solar cells were fabricated from unconventional silicon sheets, and the performances were characterized with an emphasis on statistical evaluation. A number of solar cell fabrication processes were used and conversion efficiency was measured under AMO condition at 25 C. Silso solar cells using standard processing showed an average efficiency of about 9.6%. Solar cells with back surface field process showed about the same efficiency as the cells from standard process. Solar cells from grain boundary passivation process did not show any improvements in solar cell performance.

The RSV F and G glycoproteins interact to form a complex on the surface of infected cells

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

Low, Kit-Wei; Tan, Timothy; Ng, Ken

2008-02-08

In this study, the interaction between the respiratory syncytial virus (RSV) fusion (F) protein, attachment (G) protein, and small hydrophobic (SH) proteins was examined. Immunoprecipitation analysis suggested that the F and G proteins exist as a protein complex on the surface of RSV-infected cells, and this conclusion was supported by ultracentrifugation analysis that demonstrated co-migration of surface-expressed F and G proteins. Although our analysis provided evidence for an interaction between the G and SH proteins, no evidence was obtained for a single protein complex involving all three of the virus proteins. These data suggest the existence of multiple virus glycoproteinmore » complexes within the RSV envelope. Although the stimulus that drives RSV-mediated membrane fusion is unknown, the association between the G and F proteins suggest an indirect role for the G protein in this process.« less

Biosynthesis and processing of a human T lymphocyte antigen.

PubMed

Bergman, Y; Levy, R

1982-03-01

The biosynthesis and processing of Leu-1, a human T lymphocyte antigen, has been studied with the use of a monoclonal antibody. This molecule exists on the cell surface as a 67,000 m.w. glycoprotein. Through a series of pulse-labeling studies, in conjunction with the use of the antibiotic tunicamycin and the enzyme Endo-H, the details of glycosylation, processing, and deposition at the cell membrane were examined. The protein backbone of the molecule is 58,000 m.w. High-mannose sugars are added to asparagine residues during synthesis. Within 20 min, these high mannose sugars are converted to complex type carbohydrates, including fucose. The fully processed glycoprotein appears at the cell surface within 30 min after synthesis. This sequence of events is similar to that for other cell surface glycoproteins, including HLA and vesicular stomatitus virus glycoprotein.

Small organelle, big responsibility: the role of centrosomes in development and disease

PubMed Central

Chavali, Pavithra L.; Pütz, Monika; Gergely, Fanni

2014-01-01

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. PMID:25047622

Corrosion of Mg alloy AZ91D in the presence of living cells.

PubMed

Seuss, F; Seuss, S; Turhan, M C; Fabry, B; Virtanen, S

2011-11-01

Mg and Mg alloys are of interest for biodegradable implants as they readily corrode in biological fluids, and dissolved Mg ions are nontoxic. Even though it is well known that Mg dissolution leads to pH increase in the surroundings, the effect of the corrosion-induced alkalization on the biological environment has not been studied in detail. We therefore explored the interactions between corrosion-induced pH increase and cell growth on Mg alloy AZ91D surface. Cell adhesion and spreading on the alloy surface is unimpeded initially. However, with time a large fraction of cells de-adhere. We attribute this to the observed increase of the pH in the cell culture medium in the process of alloy dissolution. Cytotoxicity tests with HeLa cells grown on glass surfaces confirm that cell death increases with increasing alkalinity of the cell culture medium. We also show that a the cells that adhere on the Mg alloy surface act as a corrosion-blocking surface layer. In consequence, a slower pH increase in the medium takes place when the alloy surface is covered with cells. Electrochemical impedance spectroscopy measurements (EIS) verify that a cell layer slows down the corrosion process. 2011 Wiley Periodicals, Inc.

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells.

PubMed

Maxson, Julia E; Enns, Caroline A; Zhang, An-Sheng

2009-02-19

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release.

Processing of hemojuvelin requires retrograde trafficking to the Golgi in HepG2 cells

PubMed Central

Maxson, Julia E.; Enns, Caroline A.

2009-01-01

Hemojuvelin (HJV) was recently identified as a critical regulator of iron homeostasis. It is either associated with cell membranes through a glycosylphosphatidylinositol anchor or released as a soluble form. Membrane-anchored HJV acts as a coreceptor for bone morphogenetic proteins and activates the transcription of hepcidin, a hormone that regulates iron efflux from cells. Soluble HJV antagonizes bone morphogenetic protein signaling and suppresses hepcidin expression. In this study, we examined the trafficking and processing of HJV. Cellular HJV reached the plasma membrane without obtaining complex oligosaccharides, indicating that HJV avoided Golgi processing. Secreted HJV, in contrast, has complex oligosaccharides and can be derived from HJV with high-mannose oligosaccharides at the plasma membrane. Our results support a model in which retrograde trafficking of HJV before cleavage is the predominant processing pathway. Release of HJV requires it to bind to the transmembrane receptor neogenin. Neogenin does not, however, play a role in HJV trafficking to the cell surface, suggesting that it could be involved either in retrograde trafficking of HJV or in cleavage leading to HJV release. PMID:19029439

Fabrication and hydrophobic characteristics of micro / nanostructures on polydimethylsiloxane surface prepared by picosecond laser

NASA Astrophysics Data System (ADS)

Bin, Wang; Dong, Shiyun; Yan, Shixing; Gang, Xiao; Xie, Zhiwei

2018-03-01

Picosecond laser has ultrashort pulse width and ultrastrong peak power, which makes it widely used in the field of micro-nanoscale fabrication. polydimethylsiloxane (PDMS) is a typical silicone elastomer with good hydrophobicity. In order to further improve the hydrophobicity of PDMS, the picosecond laser was used to fabricate a grid-like microstructure on the surface of PDMS, and the relationship between hydrophobicity of PDMS with surface microstructure and laser processing parameters, such as processing times and cell spacing was studied. The results show that: compared with the unprocessed PDMS, the presence of surface microstructure significantly improved the hydrophobicity of PDMS. When the number of processing is constant, the hydrophobicity of PDMS decreases with the increase of cell spacing. However, when the cell spacing is fixed, the hydrophobicity of PDMS first increases and then decreases with the increase of processing times. In particular, when the times of laser processing is 6 and the cell spacing is 50μm, the contact angle of PDMS increased from 113° to 154°, which reached the level of superhydrophobic.

Mesenchymal stromal cells express GARP/LRRC32 on their surface: effects on their biology and immunomodulatory capacity.

PubMed

Carrillo-Galvez, Ana Belén; Cobo, Marién; Cuevas-Ocaña, Sara; Gutiérrez-Guerrero, Alejandra; Sánchez-Gilabert, Almudena; Bongarzone, Pierpaolo; García-Pérez, Angélica; Muñoz, Pilar; Benabdellah, Karim; Toscano, Miguel G; Martín, Francisco; Anderson, Per

2015-01-01

Mesenchymal stromal cells (MSCs) represent a promising tool for therapy in regenerative medicine, transplantation, and autoimmune disease due to their trophic and immunomodulatory activities. However, we are still far from understanding the mechanisms of action of MSCs in these processes. Transforming growth factor (TGF)-β1 is a pleiotropic cytokine involved in MSC migration, differentiation, and immunomodulation. Recently, glycoprotein A repetitions predominant (GARP) was shown to bind latency-associated peptide (LAP)/TGF-β1 to the cell surface of activated Foxp3(+) regulatory T cells (Tregs) and megakaryocytes/platelets. In this manuscript, we show that human and mouse MSCs express GARP which presents LAP/TGF-β1 on their cell surface. Silencing GARP expression in MSCs increased their secretion and activation of TGF-β1 and reduced their proliferative capacity in a TGF-β1-independent manner. Importantly, we showed that GARP expression on MSCs contributed to their ability to inhibit T-cell responses in vitro. In summary, we have found that GARP is an essential molecule for MSC biology, regulating their immunomodulatory and proliferative activities. We envision GARP as a new target for improving the therapeutic efficacy of MSCs and also as a novel MSC marker. © 2014 AlphaMed Press.

Solar cell modules with improved backskin and methods for forming same

DOEpatents

Hanoka, Jack I.

1998-04-21

A laminated solar cell module with a backskin layer that reduces the materials and labor required during the manufacturing process. The solar cell module includes a rigid front support layer formed of light transmitting material having first and second surfaces. A transparent encapsulant layer has a first surface disposed adjacent the second surface of the front support layer. A plurality of interconnected solar cells have a first surface disposed adjacent a second surface of the transparent encapsulant layer. The backskin layer is formed of a thermoplastic olefin, which includes first ionomer, a second ionomer, glass fiber, and carbon black. A first surface of the backskin layer is disposed adjacent a second surface of the interconnected solar cells. The transparent encapsulant layer and the backskin layer, in combination, encapsulate the interconnected solar cells. An end portion of the backskin layer can be wrapped around the edge of the module for contacting the first surface of the front support layer to form an edge seal. A laminated solar cell module with a backskin layer that reduces the materials and labor required during the manufacturing process. The solar cell module includes a rigid front support layer formed of light transmitting material having first and second surfaces. A transparent encapsulant layer has a first surface disposed adjacent the second surface of the front support layer. A plurality of interconnected solar cells have a first surface disposed adjacent a second surface of the transparent encapsulant layer. The backskin layer is formed of a thermoplastic olefin, which includes first ionomer, a second ionomer, glass fiber, and carbon black. A first surface of the backskin layer is disposed adjacent a second surface of the interconnected solar cells. The transparent encapsulant layer and the backskin layer, in combination, encapsulate the interconnected solar cells. An end portion of the backskin layer can be wrapped around the edge of the module for contacting the first surface of the front support layer to form an edge seal.

Mucin characteristics of human corneal-limbal epithelial cells that exclude the rose bengal anionic dye.

PubMed

Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K

2006-01-01

Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye

PubMed Central

Argüeso, Pablo; Tisdale, Ann; Spurr-Michaud, Sandra; Sumiyoshi, Mika; Gipson, Ilene K.

2005-01-01

Purpose Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. Methods HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. Results The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. Conclusions These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia. PMID:16384952

Thin silicon solar cell performance characteristics

NASA Technical Reports Server (NTRS)

Gay, C. F.

1978-01-01

Refined techniques for surface texturizing, back surface field and back surface reflector formation were evaluated for use with shallow junction, single-crystal silicon solar cells. Each process was characterized individually and collectively as a function of device thickness and bulk resistivity. Among the variables measured and reported are open circuit voltage, short circuit current and spectral response. Substantial improvements were obtained by the utilization of a low cost aluminum paste process to simultaneously remove the unwanted n(+) diffused region, form the back surface field and produce an ohmic contact metallization. The highly effective BSF which results from applying this process has allowed fabrication of cells 0.05 mm thick with initial outputs as high as 79.5 mW/4 sq cm (28 C, AM0) and superior electron radiation tolerance. Cells of 0.02 mm to 0.04 mm thickness have been fabricated with power to mass ratios well in excess of 2 watts per gram.

Beyond the hydrophobic effect: Critical function of water at biological phase boundaries--A hypothesis.

PubMed

Damodaran, Srinivasan

2015-07-01

Many life-sustaining processes in living cells occur at the membrane-water interface. The pertinent questions that need to be asked are what is the evolutionary reason for biology to choose the membrane-water interface as the site for performing and/or controlling crucial biological reactions and what is the key physical principle that is singular to the membrane-water interface that biology exploits for regulating metabolic processes in cells? In this review, a hypothesis is developed, which espouses that cells control activities of membrane-bound enzymes and receptor activated processes via manipulating the thermodynamic activity of water at the membrane-water interfacial region. In support of this hypothesis, first we establish that the surface pressure of a lipid monolayer is a direct measure of a reduction in the thermodynamic activity of interfacial water. Second, we show that the surface pressure-dependent activation/inactivation of interfacial enzymes is fundamentally related to their dependence on interfacial water activity. We extend this argument to infer that cells might manipulate activities of membrane-associated biological processes via manipulating the activity of interfacial water via localized compression or expansion of the interface. In this paper, we critically analyze literature data on mechano-activation of large pore ion channels in Escherichia coli spheroplasts and G-proteins in reconstituted lipid vesicles, and show that these pressure-induced activation processes are fundamentally and quantitatively related to changes in the thermodynamic state of interfacial water, caused by mechanical stretching of the bilayer. Copyright © 2015 Elsevier B.V. All rights reserved.

Gas-solid carbonation as a current alternative origin for carbonates in Martian regolith

NASA Astrophysics Data System (ADS)

Garenne, A.; Montes-Hernandez, G.; Beck, P.; Schmitt, B.; Brissaud, O.

2011-12-01

Carbonates are abundant sedimentary minerals at the surface and sub-surface of Earth and they have been proposed as tracers of liquid water in extraterrestrial environments (e.g. at Mars surface). Its formation mechanism is since generally associated with aqueous alteration processes. Recently, carbonates minerals have been discovered on Mars surface by different orbital or rovers missions. In particular, the phoenix mission has measured from 1 to 5% of calcium carbonate (calcite type). These occurrences have been reported in area were the relative humidity is significantly high (Boynton et al., 2009). The small concentration of carbonates suggests an alternative process than carbonation in aqueous conditions. Such an observation might rather point toward a possible formation mechanism by dust-gas reaction under current Martian conditions. For this reason, in the present study, we designed an experimental setup consisting of an infrared microscope coupled to a cryogenic reaction cell (IR-CryoCell setup) in order to investigate the gas-solid carbonation of three different mineral precursors for carbonates (Ca and Mg hydroxides, and a hydrated Ca silicate formed from Ca2SiO4) at low temperature (from -10 to 25°C) and at reduced CO2 pressure (from 100 to 1000 mbar). These mineral materials are crucial precursors to form respective Ca and Mg carbonates in humid environments (0 < relative humidity < 100%) at dust-CO2 or dust-water ice-CO2 interfaces. The results have revealed a significant and fast carbonation process for Ca hydroxide and hydrated Ca silicate. Conversely, slight carbonation process was observed for Mg hydroxide. These results suggest that gas-solid carbonation process or carbonate formation at the dust-water ice-CO2 interfaces could be a currently active Mars surface process. We note that the carbonation process at low temperature (<0°C) described in the present study could also have important implications on the dust-water ice-CO2 interactions in cold terrestrial environments (e.g. Antarctic).

Anionic lipids and the maintenance of membrane electrostatics in eukaryotes.

PubMed

Platre, Matthieu Pierre; Jaillais, Yvon

2017-02-01

A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells.

ARP2/3 localization in Arabidopsis leaf pavement cells: a diversity of intracellular pools and cytoskeletal interactions.

PubMed

Zhang, Chunhua; Mallery, Eileen L; Szymanski, Daniel B

2013-01-01

In plant cells the actin cytoskeleton adopts many configurations, but is best understood as an unstable, interconnected track that rearranges to define the patterns of long distance transport of organelles during growth. Actin filaments do not form spontaneously; instead filament nucleators, such as the evolutionarily conserved actin-related protein (ARP) 2/3 complex, can efficiently generate new actin filament networks when in a fully activated state. A growing number of genetic experiments have shown that ARP2/3 is necessary for morphogenesis in processes that range from tip growth during root nodule formation to the diffuse polarized growth of leaf trichomes and pavement cells. Although progress has been rapid in the identification of proteins that function in series to positively regulate ARP2/3, less has been learned about the actual function of ARP2/3 in cells. In this paper, we analyze the localization of ARP2/3 in Arabidopsis leaf pavement cells. We detect a pool of ARP2/3 in the nucleus, and also find that ARP2/3 is efficiently and specifically clustered on multiple organelle surfaces and associates with both the actin filament and microtubule cytoskeletons. Our mutant analyses and ARP2/3 and actin double labeling experiments indicate that the clustering of ARP2/3 on organelle surfaces and an association with actin bundles does not necessarily reflect an active pool of ARP2/3, and instead most of the complex appears to exist as a latent organelle-associated pool.

Nanoscale Structure at Mineral-Fluid Interfaces

NASA Astrophysics Data System (ADS)

Sturchio, N. C.; Sturchio, N. C.; Fenter, P.; Cheng, L.; Park, C.; Zhang, Z.; Zhang, Z.; Nagy, K. L.; Schlegel, M. L.

2001-12-01

The nature of nanoparticles and their role in the natural environment is currently a subject of renewed interest. The high surface area (and surface area-to-volume ratio) of nanoparticles exerts a widespread influence on geochemical reactions and transport processes. A thorough understanding of the nanoscale world remains largely hypothetical, however, because of the challenges associated with characterizing nanoscale structures and processes. Recent insights gained from high-resolution synchrotron x-ray reflectivity measurements at the solid-fluid interfaces of macroscopic (i.e., mm-scale) mineral particles may provide relevant guidelines for expected nanoparticle surface structures. For example, at calcite-water and barite-water interfaces, undercoordinated surface cations bond with water species of variable protonation, and modest relaxations (to several hundredths of a nanometer) affect the outermost unit cells [1,2]. Undercoordinated tetrahedral ions at aluminosilicate surfaces also bond with water species, whereas interstitial or interlayer alkali or alkaline earth ions at the surface may readily exchange with hydronium or other ions; modest relaxations also affect the outermost unit cells [3,4]. Modulation of liquid water structure out to about one nanometer has been observed at the (001) cleavage surface of muscovite in deionized water, and may be present at other mineral-fluid interfaces [4]. Dissolution mechanisms at the orthoclase-water interface have been clarified by combining x-ray reflectivity and scanning force microscopy measurements [5]. Further progress in understanding nanoscale structures and processes at macroscopic mineral-water interfaces is likely to benefit nanoparticle studies. [1] Fenter et al. (2000) Geochim. Cosmochim. Acta 64, 1221-1228. [2] Fenter et al. (2001) J. Phys. Chem. B 105(34), 8112-8119. [3] Fenter et al. (2000) Geochim. Cosmochim. Acta 64, 3663-3673. [4] Cheng et al. (2001) Phys. Rev. Lett., (in press). [5] Teng et al. (2001) Geochim. Cosmochim. Acta 65, (in press).

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.

Xylella fastidiosa Afimbrial Adhesins Mediate Cell Transmission to Plants by Leafhopper Vectors▿

PubMed Central

Killiny, Nabil; Almeida, Rodrigo P. P.

2009-01-01

The interactions between the economically important plant-pathogenic bacterium Xylella fastidiosa and its leafhopper vectors are poorly characterized. We used different approaches to determine how X. fastidiosa cells interact with the cuticular surface of the foreguts of vectors. We demonstrate that X. fastidiosa binds to different polysaccharides with various affinities and that these interactions are mediated by cell surface carbohydrate-binding proteins. In addition, competition assays showed that N-acetylglucosamine inhibits bacterial adhesion to vector foregut extracts and intact wings, demonstrating that attachment to leafhopper surfaces is affected in the presence of specific polysaccharides. In vitro experiments with several X. fastidiosa knockout mutants indicated that hemagglutinin-like proteins are associated with cell adhesion to polysaccharides. These results were confirmed with biological experiments in which hemagglutinin-like protein mutants were transmitted to plants by vectors at lower rates than that of the wild type. Furthermore, although these mutants were defective in adhesion to the cuticle of vectors, their growth rate once attached to leafhoppers was similar to that of the wild type, suggesting that these proteins are important for initial adhesion of X. fastidiosa to leafhoppers. We propose that X. fastidiosa colonization of leafhopper vectors is a complex, stepwise process similar to the formation of biofilms on surfaces. PMID:19011051

Cytochemical localization of phosphatases in the germ- and Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae).

PubMed

Fernandes, A P; Báo, S N

1998-08-01

Ultrastructural cytochemical techniques were used for the localization of phosphatases in spermatid and spermatozoon, as well as in Sertoli cells of Odontophrynus cultripes (Amphibia, Anura, Leptodactylidae). Acid phosphatase was found in the acrosome. Thiamine pyrophosphatase was observed in the Golgi cisternae and in the tail spermatozoon surface. Glucose-6-phosphatase was located in the membrane complex of the acrosomal region. Already, in the Sertoli cells acid phosphatase was located in the lysosomes and glucose-6-phosphatase was observed in association with the endoplasmic reticulum and Golgi complex. These observations support the idea that various phosphatases may play some role in spermatid differentiation and in the interactions germ cells--Sertoli cells during spermiogenesis process.

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763

Cellular and Molecular Level Responses After Radiofrequency Radiation Exposure, Alone or in Combination with X-rays or Chemicals

DTIC Science & Technology

1992-07-27

cell surface marker CD22 , which plays a role in early B-cell activation, is present within the cytoplasm of all B- cells, but expressed only on the...surface of a subpopulation of those cells. CD22 is an activation receptor associated with cell proliferation of small resting B-cells, and acts as an...adhesion molecule mediating the binding of B-cells to other hematopoietic cells (Stamenkovic & Seed, 1990). The CD22 surface glycoprotein is the putative

Photovoltaic cell and production thereof

DOEpatents

Narayanan, Srinivasamohan [Gaithersburg, MD; Kumar, Bikash [Bangalore, IN

2008-07-22

An efficient photovoltaic cell, and its process of manufacture, is disclosed wherein the back surface p-n junction is removed from a doped substrate having an oppositely doped emitter layer. A front surface and edges and optionally the back surface periphery are masked and a back surface etch is performed. The mask is not removed and acts as an anti-reflective coating, a passivating agent, or both. The photovoltaic cell retains an untextured back surface whether or not the front is textured and the dopant layer on the back surface is removed to enhance the cell efficiency. Optionally, a back surface field is formed.

Growth Inhibition of Tumour Implants by Associated Surface Active Agents

PubMed Central

Altman, R. F. A.; Spoladore, L. G.; Esch, E. L.

1970-01-01

Whereas dilute solutions of surface active agents modify the properties of cell membranes, particularly in relation to their electrical behaviour, moderate and strong solutions provoke more serious structural damage of the membrane, leading to an increase of its permeability and, finally, to cytolysis. These phenomena have inspired some authors to apply detergents as possible cancer chemotherapeuticals so far, however, with only poor results. The disintegrating effect of tumour emboli into single cells by certain detergents, and the ingenious discovery that the mutual adhesiveness between cancer cells is much less than between normal cells, have led the present authors to investigate the action of some biological surface active agents, alone as well as in some of their associations on the “take” of Yoshida sarcoma implants. Certain associations showed, in contradistinction to the separately applied components, surprisingly favourable activity. It could be established that a correlation actually exists between inhibitory effect and surface activity. PMID:4394469

Linear glandular trichomes of Helianthus (Asteraceae): morphology, localization, metabolite activity and occurrence

PubMed Central

Aschenbrenner, Anna-Katharina; Horakh, Silke; Spring, Otmar

2013-01-01

Capitate glandular trichomes of sunflower are well investigated, but detailed studies are lacking for the linear glandular trichomes (LGT), a second type of physiologically active plant hair present on the surface of sunflowers. Light, fluorescence and scanning electron microscopy as well as histochemical staining were used to investigate the structure and metabolite deposition of LGT. Consisting of 6–11 linearly arranged cells, LGT were found on the surface of most plant organs of Helianthus annuus. They were associated with the leaf vascular system, and also occurred along petioles, stems and the abaxial surface of chaffy bracts, ray and disc florets. The highest density was found on the abaxial surface of phyllaries. Phenotypically similar LGT were common in all species of the genus, but also occurred in most other genera of the Helianthinae so far screened. Brownish and fluorescent metabolites of an as yet unknown chemical structure, together with terpenoids, were produced and stored in apical cells of LGT. The deposition of compounds gradually progressed from the tip cell to the basal cells of older trichomes. This process was accompanied by nucleus degradation in metabolite-accumulating cells. The localization of these trichomes on prominent plant parts of the apical bud and the capitulum combined with the accumulation of terpenoids and other as yet unknown compounds suggests a chemo-ecological function of the LGT in plant–insect or plant–herbivore interaction.

Force Generation upon T Cell Receptor Engagement

PubMed Central

Husson, Julien; Chemin, Karine; Bohineust, Armelle; Hivroz, Claire; Henry, Nelly

2011-01-01

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca2+ concentration ([Ca2+]i) was monitored simultaneously to verify the cell commitment in the activation process. [Ca2+]i increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process. PMID:21572959

Force generation upon T cell receptor engagement.

PubMed

Husson, Julien; Chemin, Karine; Bohineust, Armelle; Hivroz, Claire; Henry, Nelly

2011-05-10

T cells are major players of adaptive immune response in mammals. Recognition of an antigenic peptide in association with the major histocompatibility complex at the surface of an antigen presenting cell (APC) is a specific and sensitive process whose mechanism is not fully understood. The potential contribution of mechanical forces in the T cell activation process is increasingly debated, although these forces are scarcely defined and hold only limited experimental evidence. In this work, we have implemented a biomembrane force probe (BFP) setup and a model APC to explore the nature and the characteristics of the mechanical forces potentially generated upon engagement of the T cell receptor (TCR) and/or lymphocyte function-associated antigen-1 (LFA-1). We show that upon contact with a model APC coated with antibodies towards TCR-CD3, after a short latency, the T cell developed a timed sequence of pushing and pulling forces against its target. These processes were defined by their initial constant growth velocity and loading rate (force increase per unit of time). LFA-1 engagement together with TCR-CD3 reduced the growing speed during the pushing phase without triggering the same mechanical behavior when engaged alone. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was monitored simultaneously to verify the cell commitment in the activation process. [Ca(2+)](i) increased a few tens of seconds after the beginning of the pushing phase although no strong correlation appeared between the two events. The pushing phase was driven by actin polymerization. Tuning the BFP mechanical properties, we could show that the loading rate during the pulling phase increased with the target stiffness. This indicated that a mechanosensing mechanism is implemented in the early steps of the activation process. We provide here the first quantified description of force generation sequence upon local bidimensional engagement of TCR-CD3 and discuss its potential role in a T cell mechanically-regulated activation process.

Overview of processing activities aimed at higher efficiencies and economical production

NASA Technical Reports Server (NTRS)

Bickler, D. B.

1985-01-01

An overview of processing activities aimed at higher efficiencies and economical production were presented. Present focus is on low-cost process technology for higher-efficiency cells of up to 18% or higher. Process development concerns center on the use of less than optimum silicon sheet, the control of production yields, and making uniformly efficient large-area cells. High-efficiency cell factors that require process development are bulk material perfection, very shallow junction formation, front-surface passivation, and finely detailed metallization. Better bulk properties of the silicon sheet and the keeping of those qualities throughout large areas during cell processing are required so that minority carrier lifetimes are maintained and cell performance is not degraded by high doping levels. When very shallow junctions are formed, the process must be sensitive to metallizatin punch-through, series resisitance in the cell, and control of dopant leaching during surface passivation. There is a need to determine the sensitivity to processing by mathematical modeling and experimental activities.

Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity.

PubMed

Ogawa, Mikako; Tomita, Yusuke; Nakamura, Yuko; Lee, Min-Jung; Lee, Sunmin; Tomita, Saori; Nagaya, Tadanobu; Sato, Kazuhide; Yamauchi, Toyohiko; Iwai, Hidenao; Kumar, Abhishek; Haystead, Timothy; Shroff, Hari; Choyke, Peter L; Trepel, Jane B; Kobayashi, Hisataka

2017-02-07

Immunogenic cell death (ICD) is a form of cell death that activates an adaptive immune response against dead-cell-associated antigens. Cancer cells killed via ICD can elicit antitumor immunity. ICD is efficiently induced by near-infrared photo-immunotherapy (NIR-PIT) that selectively kills target-cells on which antibody-photoabsorber conjugates bind and are activated by NIR light exposure. Advanced live cell microscopies showed that NIR-PIT caused rapid and irreversible damage to the cell membrane function leading to swelling and bursting, releasing intracellular components due to the influx of water into the cell. The process also induces relocation of ICD bio markers including calreticulin, Hsp70 and Hsp90 to the cell surface and the rapid release of immunogenic signals including ATP and HMGB1 followed by maturation of immature dendritic cells. Thus, NIR-PIT is a therapy that kills tumor cells by ICD, eliciting a host immune response against tumor.

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.

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.

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.

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

Effects of Coating Materials and Processing Conditions on Flow Enhancement of Cohesive Acetaminophen Powders by High-Shear Processing With Pharmaceutical Lubricants.

PubMed

Wei, Guoguang; Mangal, Sharad; Denman, John; Gengenbach, Thomas; Lee Bonar, Kevin; Khan, Rubayat I; Qu, Li; Li, Tonglei; Zhou, Qi Tony

2017-10-01

This study has investigated the surface coating efficiency and powder flow improvement of a model cohesive acetaminophen powder by high-shear processing with pharmaceutical lubricants through 2 common equipment, conical comil and high-shear mixer. Effects of coating materials and processing parameters on powder flow and surface coating coverage were evaluated. Both Carr's index and shear cell data indicated that processing with the lubricants using comil or high-shear mixer substantially improved the flow of the cohesive acetaminophen powder. Flow improvement was most pronounced for those processed with 1% wt/wt magnesium stearate, from "cohesive" for the V-blended sample to "easy flowing" for the optimally coated sample. Qualitative and quantitative characterizations demonstrated a greater degree of surface coverage for high-shear mixing compared with comilling; nevertheless, flow properties of the samples at the corresponding optimized conditions were comparable between 2 techniques. Scanning electron microscopy images demonstrated different coating mechanisms with magnesium stearate or l-leucine (magnesium stearate forms a coating layer and leucine coating increases surface roughness). Furthermore, surface coating with hydrophobic magnesium stearate did not retard the dissolution kinetics of acetaminophen. Future studies are warranted to evaluate tableting behavior of such dry-coated pharmaceutical powders. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Biochemical and ultrastructural characterization of a novel cell structure associated with immunoglobulin secretion in B-lymphocytes.

PubMed

Mazzaferro, P K; Repasky, E A; Black, J; Kubo, R T; Bankert, R B

1987-01-01

In the companion paper, it was established that a secretory form of immunoglobulin, sIg, is present at or near the cell surface. This unexpected occurrence of sIg was postulated to be due to the labelling of sIg which remains temporarily associated with the cell packaged in a vesicle which appears to bud from the plasma membrane at a single pole of the cell. The question that is addressed in this report is whether or not this polar accumulation of sIg represents a common pathway for the exit of this protein which is used by antibody-producing cells. This question is important since, in spite of the fact that the intracellular events associated with immunoglobulin synthesis (processing and movement between subcellular compartments) have been defined, very little data exists on how or where immunoglobulin finally leaves the plasma cell. This question was approached here by first demonstrating that the polar immunoglobulin secretory vesicles (ISV) are associated with several sIg-producing cells including other hybridomas, B-cell lines, and mitogen-activated spleen cells. The second approach was to characterize the ISV on the cell ultrastructurally and to establish that these vesicles are released from the cell carrying with them sIg. Isolated vesicles released from biosynthetically labeled Ig-producing cells were analyzed by SDS-PAGE in order to confirm the presence of sIg and to determine the number of other proteins associated with the ISV, their molecular weights, and the degree of disulfide crosslinking of the molecules comprising this structure. Finally, the kinetics of sIg release was established by a pulse chase protocol for biosynthetically labeled cells, and by monitoring the release of radioactive Ig from surface iodinated cells. As was predicted from our biochemical studies of the ISV, we observed a very slow phase of sIg release as well as a rapid release phase. Our studies have established that at least one of the pathways for the release of Ig from hybridomas, B-cell lines, and normal splenic B-cells is via a polar multivesiculated structure that we have termed ISV, and that the sIg can be released either as a free form of the protein or packaged within a satellite vesicle which may release the sIg later and perhaps at considerable distance from the cell that produced it.

Effects of Bacillus subtilis endospore surface reactivity on the rate of forsterite dissolution

NASA Astrophysics Data System (ADS)

Harrold, Z.; Gorman-Lewis, D.

2013-12-01

Primary mineral dissolution products, such as silica (Si), calcium (Ca) and magnesium (Mg), play an important role in numerous biologic and geochemical cycles including microbial metabolism, plant growth and secondary mineral precipitation. The flux of these and other dissolution products into the environment is largely controlled by the rate of primary silicate mineral dissolution. Bacteria, a ubiquitous component in water-rock systems, are known to facilitate mineral dissolution and may play a substantial role in determining the overall flux of dissolution products into the environment. Bacterial cell walls are complex and highly reactive organic surfaces that can affect mineral dissolution rates directly through microbe-mineral adsorption or indirectly by complexing dissolution products. The effect of bacterial surface adsorption on chemical weathering rates may even outweigh the influence of active processes in environments where a high proportion of cells are metabolically dormant or cell metabolism is slow. Complications associated with eliminating or accounting for ongoing metabolic processes in long-term dissolution studies have made it challenging to isolate the influence of cell wall interactions on mineral dissolution rates. We utilized Bacillus subtilis endospores, a robust and metabolically dormant cell type, to isolate and quantify the effects of bacterial surface reactivity on forsterite (Mg2SiO4) dissolution rates. We measured the influence of both direct and indirect microbe-mineral interactions on forsterite dissolution. Indirect pathways were isolated using dialysis tubing to prevent mineral-microbe contact while allowing free exchange of dissolved mineral products and endospore-ion adsorption. Homogenous experimental assays allowed both direct microbe-mineral and indirect microbe-ion interactions to affect forsterite dissolution rates. Dissolution rates were calculated based on silica concentrations and zero-order dissolution kinetics. Additional analyses including Mg concentrations, microprobe and BET analyses support mineral dissolution rate calculations and stoichiometry considerations. All experimental assays containing endospores show increased forsterite dissolution rates relative to abiotic controls. Forsterite dissolution rates increased by approximately one order of magnitude in dialysis bound, biotic experiments relative to abiotic assays. Homogenous biotic assays exhibited a more complex dissolution rate profile that changes over time. All microbially mediated forsterite dissolution rates returned to abiotic control rates after 10 to 15 days of incubation. This shift in dissolution rate likely corresponds to maximum endospore surface adsorption capacity. The Bacillus subtilis endospore surface serves as a first-order proxy for studying the effect of metabolizing microbe surfaces on silicate dissolution rates. Comparisons with published abiotic, microbial, and organic acid mediated forsterite dissolution rates will provide insight on the importance of bacterial surfaces in primary mineral dissolution processes.

A Versatile Class of Cell Surface Directional Motors Gives Rise to Gliding Motility and Sporulation in Myxococcus xanthus

PubMed Central

Wartel, Morgane; Czerwinski, Fabian; Le Gall, Anne-Valérie; Mauriello, Emilia M. F.; Bergam, Ptissam; Brun, Yves V.; Shaevitz, Joshua; Mignot, Tâm

2013-01-01

Eukaryotic cells utilize an arsenal of processive transport systems to deliver macromolecules to specific subcellular sites. In prokaryotes, such transport mechanisms have only been shown to mediate gliding motility, a form of microbial surface translocation. Here, we show that the motility function of the Myxococcus xanthus Agl-Glt machinery results from the recent specialization of a versatile class of bacterial transporters. Specifically, we demonstrate that the Agl motility motor is modular and dissociates from the rest of the gliding machinery (the Glt complex) to bind the newly expressed Nfs complex, a close Glt paralogue, during sporulation. Following this association, the Agl system transports Nfs proteins directionally around the spore surface. Since the main spore coat polymer is secreted at discrete sites around the spore surface, its transport by Agl-Nfs ensures its distribution around the spore. Thus, the Agl-Glt/Nfs machineries may constitute a novel class of directional bacterial surface transporters that can be diversified to specific tasks depending on the cognate cargo and machinery-specific accessories. PMID:24339744

Thiol-facilitated cell export and desorption of methylmercury by anaerobic bacteria

DOE PAGES

Lin, Hui; Lu, Xia; Liang, Liyuan; ...

2015-09-04

Neurotoxic methylmercury (MeHg), formed by anaerobic bacteria, is shown to be rapidly excreted from the cell, but the mechanism of this process is unclear. Using both Geobacter sulfurreducens PCA and Desulfovibrio desulfuricans ND132 strains, we investigated the factors affecting export and distribution of MeHg in mercury methylation and MeHg sorption-desorption assays. Thiols, such as cysteine, were found to greatly facilitate desorption and export of MeHg, particularly by PCA cells. However, in cysteine-free assays (4 h) >90% of the synthesized MeHg was associated with PCA, among which ~73% was sorbed on the cell surface and 19% remained inside the cells. Inmore » comparison, a majority of the MeHg (70%) was exported by ND132, leaving ~20% of the MeHg sorbed on the surface and 10% inside the cells. When MeHg was added directly to the cell suspensions, ND132 adsorbed much lower MeHg but took up more MeHg inside cells than PCA did. These results demonstrate that MeHg export is bacteria strain-specific, time dependent, and is influenced by thiols, implicating important roles of ligand complexation in facilitating MeHg production and mobilization in the environment.« less

Monoclonal TCR-redirected tumor cell killing.

PubMed

Liddy, Nathaniel; Bossi, Giovanna; Adams, Katherine J; Lissina, Anna; Mahon, Tara M; Hassan, Namir J; Gavarret, Jessie; Bianchi, Frayne C; Pumphrey, Nicholas J; Ladell, Kristin; Gostick, Emma; Sewell, Andrew K; Lissin, Nikolai M; Harwood, Naomi E; Molloy, Peter E; Li, Yi; Cameron, Brian J; Sami, Malkit; Baston, Emma E; Todorov, Penio T; Paston, Samantha J; Dennis, Rebecca E; Harper, Jane V; Dunn, Steve M; Ashfield, Rebecca; Johnson, Andy; McGrath, Yvonne; Plesa, Gabriela; June, Carl H; Kalos, Michael; Price, David A; Vuidepot, Annelise; Williams, Daniel D; Sutton, Deborah H; Jakobsen, Bent K

2012-06-01

T cell immunity can potentially eradicate malignant cells and lead to clinical remission in a minority of patients with cancer. In the majority of these individuals, however, there is a failure of the specific T cell receptor (TCR)–mediated immune recognition and activation process. Here we describe the engineering and characterization of new reagents termed immune-mobilizing monoclonal TCRs against cancer (ImmTACs). Four such ImmTACs, each comprising a distinct tumor-associated epitope-specific monoclonal TCR with picomolar affinity fused to a humanized cluster of differentiation 3 (CD3)-specific single-chain antibody fragment (scFv), effectively redirected T cells to kill cancer cells expressing extremely low surface epitope densities. Furthermore, these reagents potently suppressed tumor growth in vivo. Thus, ImmTACs overcome immune tolerance to cancer and represent a new approach to tumor immunotherapy.

EARP, a multisubunit tethering complex involved in endocytic recycling

PubMed Central

Schindler, Christina; Chen, Yu; Pu, Jing; Guo, Xiaoli; Bonifacino, Juan S.

2015-01-01

Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named Endosome-Associated Recycling Protein (EARP) that is structurally related to the previously described Golgi-Associated Retrograde Protein (GARP) complex. Both complexes share the Ang2, Vps52 and Vps53 subunits, but EARP comprises an uncharacterized protein, Syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target-SNARE Syntaxin 6 and various cognate SNAREs. Depletion of Syndetin or Syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling. PMID:25799061

Control of Listeria innocua Biofilms on Food Contact Surfaces with Slightly Acidic Electrolyzed Water and the Risk of Biofilm Cells Transfer to Duck Meat.

PubMed

Jeon, Hye Ri; Kwon, Mi Jin; Yoon, Ki Sun

2018-04-01

Biofilm formation on food contact surfaces is a potential hazard leading to cross-contamination during food processing. We investigated Listeria innocua biofilm formation on various food contact surfaces and compared the washing effect of slightly acidic electrolyzed water (SAEW) at 30, 50, 70, and 120 ppm with that of 200 ppm of sodium hypochlorite (NaClO) on biofilm cells. The risk of L. innocua biofilm transfer and growth on food at retail markets was also investigated. The viability of biofilms that formed on food contact surfaces and then transferred cells to duck meat was confirmed by fluorescence microscopy. L. innocua biofilm formation was greatest on rubber, followed by polypropylene, glass, and stainless steel. Regardless of sanitizer type, washing removed biofilms from polypropylene and stainless steel better than from rubber and glass. Among the various SAEW concentrations, washing with 70 ppm of SAEW for 5 min significantly reduced L. innocua biofilms on food contact surfaces during food processing. Efficiency of transfer of L. innocua biofilm cells was the highest on polypropylene and lowest on stainless steel. The transferred biofilm cells grew to the maximum population density, and the lag time of transferred biofilm cells was longer than that of planktonic cells. The biofilm cells that transferred to duck meat coexisted with live, injured, and dead cells, which indicates that effective washing is essential to remove biofilm on food contact surfaces during food processing to reduce the risk of foodborne disease outbreaks.

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

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.

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

Thermal stability of gallium arsenide solar cells

NASA Astrophysics Data System (ADS)

Papež, Nikola; Škvarenina, Ľubomír.; Tofel, Pavel; Sobola, Dinara

2017-12-01

This article summarizes a measurement of gallium arsenide (GaAs) solar cells during their thermal processing. These solar cells compared to standard silicon cells have better efficiency and high thermal stability. However, their use is partly limited due to high acquisition costs. For these reasons, GaAs cells are deployed only in the most demanding applications where their features are needed, such as space applications. In this work, GaAs solar cells were studied in a high temperature range within 30-650 °C where their functionality and changes in surface topology were monitored. These changes were recorded using an electron microscope which determined the position of the defects; using an atomic force microscope we determined the roughness of the surface and an infrared camera that showed us the thermal radiated places of the defected parts of the cell. The electrical characteristics of the cells during processing were determined by its current-voltage characteristics. Despite the occurrence of subtle changes on the solar cell with newly created surface features after 300 °C thermal processing, its current-voltage characteristic remained without a significant change.

Extracellular enzymes facilitate electron uptake in biocorrosion and bioelectrosynthesis.

PubMed

Deutzmann, Jörg S; Sahin, Merve; Spormann, Alfred M

2015-04-21

Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer. The intriguing trait of some microbial organisms to engage in direct electron transfer is thought to be widespread in nature. Consequently, direct uptake of electrons into microbial cells from solid surfaces is assumed to have a significant impact not only on fundamental microbial and biogeochemical processes but also on applied bioelectrochemical systems, such as microbial electrosynthesis and biocorrosion. This study provides a simple mechanistic explanation for frequently observed fast electron uptake kinetics in microbiological systems without a direct transfer: free, cell-derived enzymes can interact with cathodic surfaces and catalyze the formation of intermediates that are rapidly consumed by microbial cells. This electron transfer mechanism likely plays a significant role in various microbial electron transfer reactions in the environment. Copyright © 2015 Deutzmann et al.

Efficacy of atmospheric pressure dielectric barrier discharge for inactivating airborne pathogens

DOE PAGES

Romero-Mangado, Jaione; Dey, Avishek; Diaz-Cartagena, Diana C.; ...

2017-07-05

Atmospheric pressure plasmas have gained attention in recent years for several environmental applications. This technology could potentially be used to deactivate airborne microorganisms, surface-bound microorganisms, and biofilms. Here, the authors explore the efficacy of the atmospheric pressure dielectric barrier discharge (DBD) to inactivate airborne Staphylococcus epidermidis and Aspergillus niger that are opportunistic pathogens associated with nosocomial infections. This technology uses air as the source of gas and does not require any process gas such as helium, argon, nitrogen, or hydrogen. Moreover, the effect of DBD was studied on aerosolized S. epidermidis and aerosolized A. niger spores via scanning electron microscopymore » (SEM). The morphology observed on the SEM micrographs showed deformations in the cellular structure of both microorganisms. Cell structure damage upon interaction with the DBD suggests leakage of vital cellular materials, which is a key mechanism for microbial inactivation. The chemical structure of the cell surface of S. epidermidis was also analyzed by near edge x-ray absorption fine structure spectroscopy before and after DBD exposure. Our results from surface analysis revealed that reactive oxygen species from the DBD discharge contributed to alterations on the chemistry of the cell membrane/cell wall of S. epidermidis.« less

Efficacy of atmospheric pressure dielectric barrier discharge for inactivating airborne pathogens

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

Romero-Mangado, Jaione; Dey, Avishek; Diaz-Cartagena, Diana C.

Atmospheric pressure plasmas have gained attention in recent years for several environmental applications. This technology could potentially be used to deactivate airborne microorganisms, surface-bound microorganisms, and biofilms. Here, the authors explore the efficacy of the atmospheric pressure dielectric barrier discharge (DBD) to inactivate airborne Staphylococcus epidermidis and Aspergillus niger that are opportunistic pathogens associated with nosocomial infections. This technology uses air as the source of gas and does not require any process gas such as helium, argon, nitrogen, or hydrogen. Moreover, the effect of DBD was studied on aerosolized S. epidermidis and aerosolized A. niger spores via scanning electron microscopymore » (SEM). The morphology observed on the SEM micrographs showed deformations in the cellular structure of both microorganisms. Cell structure damage upon interaction with the DBD suggests leakage of vital cellular materials, which is a key mechanism for microbial inactivation. The chemical structure of the cell surface of S. epidermidis was also analyzed by near edge x-ray absorption fine structure spectroscopy before and after DBD exposure. Our results from surface analysis revealed that reactive oxygen species from the DBD discharge contributed to alterations on the chemistry of the cell membrane/cell wall of S. epidermidis.« less

New insights into proton surface mobility processes in PEMFC catalysts using isotopic exchange methods.

PubMed

Ferreira-Aparicio, Paloma

2009-09-01

The surface chemistry and the adsorption/desorption/exchange behavior of a proton-exchange membrane fuel cell catalyst are analyzed as a case study for the development of tailor-made support materials of enhanced performance and stability. By using H2, D2, and CO as probe molecules, the relevance of some surface functional groups of the catalyst support on several diffusion processes taking place during the adsorption is shown. Sulfonic groups associated with the vulcanized carbon black surface have been detected by means of spectroscopic techniques (X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy) and by analysis of the desorbed products during temperature-programmed desorption tests by mass spectrometry. Such hydrophilic species have been observed to favor proton surface mobility and exchange with Pt-adsorbed deuterium even in the presence of adsorbed CO. This behavior is relevant both for the proper characterization of these kinds of catalysts using adsorption probes and for the design of new surface-modified carbon supports, enabling alternative proton-transfer pathways throughout the catalytic layers toward the membrane.

Forward Genetic Dissection of Biofilm Development by Fusobacterium nucleatum: Novel Functions of Cell Division Proteins FtsX and EnvC.

PubMed

Wu, Chenggang; Al Mamun, Abu Amar Mohamed; Luong, Truc Thanh; Hu, Bo; Gu, Jianhua; Lee, Ju Huck; D'Amore, Melissa; Das, Asis; Ton-That, Hung

2018-04-24

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the Δ ftsX and Δ envC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of Δ ftsX and Δ envC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated. IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum , due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the Δ ftsX and Δ envC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen. Copyright © 2018 Wu et al.

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

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

Sakwe, Amos M., E-mail: asakwe@mmc.edu; Koumangoye, Rainelli; Guillory, Bobby

2011-04-01

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagenmore » type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.« less

Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

NASA Astrophysics Data System (ADS)

Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

2010-06-01

Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

Time-Resolved Analysis of Cytosolic and Surface-Associated Proteins of Staphylococcus aureus HG001 under Planktonic and Biofilm Conditions.

PubMed

Moche, Martin; Schlüter, Rabea; Bernhardt, Jörg; Plate, Kristina; Riedel, Katharina; Hecker, Michael; Becher, Dörte

2015-09-04

Staphylococcal biofilms are associated with persistent infections due to their capacity to protect bacteria against the host's immune system and antibiotics. Cell-surface-associated proteins are of great importance during biofilm formation. In the present study, an optimized biotinylation approach for quantitative GeLC-MS-based analysis of the staphylococcal cell-surface proteome was applied and the cytoplasmic protein fraction was analyzed to elucidate proteomic differences between colony biofilms and planktonic cells. The experimental setup enabled a time-resolved monitoring of the proteome under both culture conditions and the comparison of biofilm cells to planktonic cells at several time points. This allowed discrimination of differences attributed to delayed growth phases from responses provoked by biofilm conditions. Biofilm cells expressed CcpA-dependent catabolic proteins earlier than planktonic cells and strongly accumulated proteins that belong to the SigB stress regulon. The amount of the cell-surface protein and virulence gene regulator Rot decreased within biofilms and MgrA-dependent regulations appeared more pronounced. Biofilm cells simultaneously up-regulated activators (e.g., SarZ) as well as repressors (e.g., SarX) of RNAIII. A decreased amount of high-affinity iron uptake systems and an increased amount of the iron-storage protein FtnA possibly indicated a lower demand of iron in biofilms.

Effects of cancer cell permeability control on the efficiency of cell damage through surface plasmon resonance of gold nanoparticle (Conference Presentation)

NASA Astrophysics Data System (ADS)

Hsiao, Jen-Hung; Yu, Jian-He; He, Yulu; Tu, Yi-Chou; Hua, Wei-Hsiang; Low, Meng Chun; Hsieh, Cheng-Che; Kiang, Yean-Woei; Yang, Chih-Chung

2017-02-01

Cancer cell killing efficiencies based on the photothermal effect caused by the surface plasmon resonance of metal nanoparticles (NPs) and the photodynamic effect caused by the singlet oxygen generation of a photosensitizer rely on the cell uptake efficiency of metal NP and photosensitizer. Perforation and heating can increase cell membrane permeability and hence can increase the cell uptake efficiency of NPs and drugs. In this paper, we demonstrate the variations of the cell damage efficiency under the illuminations of different lasers, which can produce mainly photothermal effect, mainly photodynamic effect, and mixed effect, when a pre-perforation and a pre-heating processes are applied. Au nanorings (NRIs) with their localized surface plasmon resonance wavelength around 1064 nm are used. The perforation process is undertaken by illuminating the cell samples by a femtosecond laser at 1064 nm with the power density lower than the cell damage threshold intensity. The heating process is implemented by illuminating cells with a low power continuous laser at 1064 nm. It is found that with the pre-perforation and pre-heating processes, the photodynamic effect is enhanced because the internalized Au NRI number and hence the internalized photosensitizer (AlPcS) molecule number are increased. However, the photothermal effect can be reduced because the adsorbed Au NRIs on cell membrane are effectively internalized during the pre-perforation and pre-heating processes. The photothermal effect is more effective when Au NRIs are adsorbed on cell membrane.

Calcium and initial surface binding phase of pinocytosis in Amoeba proteus

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

Prusch, R.D.

1986-08-01

The uptake of membrane-bound solute and external medium by bulk-phase pinocytosis in Amoeba proteus is influenced by the level of Ca/sup 2 +/ in the external medium. Increasing external Ca/sup 2 +/ to approx.10/sup -4/ M increases pinocytotic intensity, while increases in Ca/sup 2 +/ above this level decrease the intensity of pinocytosis. The initial interaction of pinocytotic inducers and Ca/sup +2/ at the surface of A moeba proteus was therefore examined. Alcain blue and Na/sup +/, both inducers of pinocytosis, differ in the manner with which they associate with the amoeba surface, suggesting the possibility of different pinocytosis-inducing sitesmore » on the amoeba surface. Low levels of external Ca/sup 2 +/ in the range of 3 x 10/sup -5/ to 4.5 x 10/sup -4/ M increase the amount of cationic inducer associated with the cell surface while, at the same time, decreasing anion association with the cell surface. It is suggested that Ca/sup 2 +/ influences ion association with the cell surface by controlling the availability of negative surface sites, which in turn influences pinocytotic intensity. Surface binding of Na/sup +/, Ca/sup 2 +/ and Cl/sup -/ was determined by adding /sup 22/Na, /sup 45/Ca or /sup 36/Cl.« less

Influence of liposome charge on the association of liposomes with Kupffer cells in vitro. Effects of divalent cations and competition with latex particles.

PubMed

Dijkstra, J; van Galen, M; Scherphof, G

1985-03-14

We studied the interaction of large unilamellar liposomes carrying different surface charges with rat Kupffer cells in maintenance culture. In addition to 14C-labeled phosphatidylcholine, all liposome preparations contained either 3H-labeled inulin or 125I-labeled bovine serum albumin as a non-degradable or a degradable aqueous space marker, respectively. With vesicles carrying no net charge, intracellular processing of internalized liposomes caused nearly complete release of protein label into the medium in acid-soluble form, while phospholipid label was predominantly retained by the cells, only about one third being released. The presence of the lysosomotropic agent, ammonia, inhibited the release of both labels from the cells. At 4 degrees C, the association and degradation of the vesicles were strongly reduced. These results are very similar to what we reported on negatively charged liposomes (Dijkstra, J., Van Galen, W.J.M., Hulstaert, C.E., Kalicharan, D., Roerdink, F.H. and Scherphof, G.L. (1984) Exp. Cell Res. 150, 161-176). The interaction of both types of vesicles apparently proceeds by adsorption to the cell surface followed by virtually complete internalization by endocytosis. Similar experiments with positively charged vesicles indicated that only about half of the liposomes were taken up by the endocytic route, the other half remaining adsorbed to the cell-surface. Attachment of all types of liposomes to the cells was strongly dependent on the presence of divalent cations; Ca2+ appeared to be required for optimal binding. Neutral liposomes only slightly competed with the uptake of negatively charged vesicles, both at 4 degrees and 37 degrees C, whereas negatively charged small unilamellar vesicles and negatively charged latex beads were found to compete very effectively with the large negatively charged liposomes. Neutral vesicles competed effectively for uptake with positively charged ones. These results suggest that neutral and positively charged liposomes are largely bound by the same cell-surface binding sites, while negatively charged vesicles attach mainly to other binding sites.

AFM probing in aqueous environment of Staphylococcus epidermidis cells naturally immobilised on glass: physico-chemistry behind the successful immobilisation.

PubMed

Méndez-Vilas, A; Gallardo-Moreno, A M; Calzado-Montero, R; González-Martín, M L

2008-05-01

AFM probing of microbial cells in liquid environments usually requires them to be physically or chemically attached to a solid surface. The fixation mechanisms may influence the nanomechanical characterization done by force curve mapping using an AFM. To study the response of a microbial cell surface to this kind of local measurement this study attempts to overcome the problem associated to the uncertainties introduced by the different fixation treatments by analysing the surface of Staphylococcus epidermidis cells naturally (non-artificially mediated) immobilised on a glass support surface. The particularities of this natural bacterial fixation process for AFM surface analysis are discussed in terms of theoretical predictions of the XDLVO model applied to the systems bacteria/support substratum and bacteria/AFM tip immersed in water. In this sense, in the first part of this study the conditions for adequate natural fixation of three S. epidermidis strains have been analyzed by taking into account the geometries of the bacterium, substrate and tip. In the second part, bacteria are probed without the risk of any possible artefacts due to the mechanical or chemical fixation procedures. Forces measured over the successfully adhered cells have (directly) shown that the untreated bacterial surface suffers from a combination of both reversible and non-reversible deformations during acquisition of force curves all taken under the same operational conditions. This is revealed directly through high-resolution tapping-mode imaging of the bacterial surface immediately following force curve mapping. The results agree with the two different types of force curves that were repeatedly obtained. Interestingly, one type of these force curves suggests that the AFM tip is breaking (rather than pushing) the cell surface during acquisition of the force curve. In this case, adhesive peaks were always observed, suggesting a mechanical origin of the measured pull-off forces. The other type of force curves shows no adhesive peaks and exhibits juxtaposing of approaching and retraction curves, reflecting elastic deformations.

Modification of surface and enzymatic properties of Achromobacter denitrificans and Stenotrophomonas maltophilia in association with diesel oil biodegradation enhanced with alkyl polyglucosides.

PubMed

Sałek, Karina; Zgoła-Grześkowiak, Agnieszka; Kaczorek, Ewa

2013-11-01

The article concerns the influence of selected alkyl polyglucosides on biodegradation, cell surface and enzymatic properties of Stenotrophomonas maltophilia and Achromobacter denitrificans. The biodegradation of diesel oil depends on several factors including type and the amount of surfactant as well as bacterial genera used in the process. Nevertheless, a careful selection of these variables must be made as some bacterial strains prefer to use surfactants as their carbon source. This leads to the lowered biodegradation of diesel oil as can be observed for the tested S. maltophilia strain. Alkyl polyglucosides influenced the cell surface properties of both of the tested strains in slightly different ways. Especially for A. denitrificans, for which the hydrophobicity increased with concentration of both--Lutensol GD 70 and Glucopon 215 in diesel oil-surfactant systems. Moreover, judging by the efficiency of biodegradation, the most effective process was observed in the presence of Lutensol GD 70 (240 and 360 mg L(-1)) with biodegradation rising from 32% (without surfactant) to 68%. No such relation was observed for S. maltophilia. Copyright © 2013 Elsevier B.V. All rights reserved.

Bactericidal activity of biomimetic diamond nanocone surfaces.

PubMed

Fisher, Leanne E; Yang, Yang; Yuen, Muk-Fung; Zhang, Wenjun; Nobbs, Angela H; Su, Bo

2016-03-17

The formation of biofilms on implant surfaces and the subsequent development of medical device-associated infections are difficult to resolve and can cause considerable morbidity to the patient. Over the past decade, there has been growing recognition that physical cues, such as surface topography, can regulate biological responses and possess bactericidal activity. In this study, diamond nanocone-patterned surfaces, representing biomimetic analogs of the naturally bactericidal cicada fly wing, were fabricated using microwave plasma chemical vapor deposition, followed by bias-assisted reactive ion etching. Two structurally distinct nanocone surfaces were produced, characterized, and the bactericidal ability examined. The sharp diamond nanocone features were found to have bactericidal capabilities with the surface possessing the more varying cone dimension, nonuniform array, and decreased density, showing enhanced bactericidal ability over the more uniform, highly dense nanocone surface. Future research will focus on using the fabrication process to tailor surface nanotopographies on clinically relevant materials that promote both effective killing of a broader range of microorganisms and the desired mammalian cell response. This study serves to introduce a technology that may launch a new and innovative direction in the design of biomaterials with capacity to reduce the risk of medical device-associated infections.

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Ku??tzing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L-1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method. ?? Springer 2005.

A direct immunoassay for detecting diatoms in groundwater as an indicator of the direct influence of surface water

USGS Publications Warehouse

Walker, C.E.; Schrock, R.M.; Reilly, T.J.; Baehr, A.L.

2005-01-01

Groundwater under the direct influence of surface water (GWUDISW) is of concern in communities where growing public demand on groundwater resources has resulted in increased withdrawals and hydraulic stress near surface water bodies. Under these conditions, contaminants such as methyl-tert butyl ether (MTBE) and biological materials have been detected in domestic wells. Other contaminants and pathogens associated with surface water are not routinely tested for in groundwater-supplied systems. To address the need for methods to easily identify potentially vulnerable supplies, a direct immunoassay for the quantitative detection of diatoms in raw water samples was developed as a measure of surface water influence on groundwater. Cell wall preparations from Nitzschia palea Kützing, a freshwater diatom found throughout North America, were used to produce a polyclonal antibody that was applied in a direct enzyme-linked immunosorbent assay (ELISA) developed to detect the presence of N. palea cell wall components. The direct immunoassay allows detection at 500 cells L−1, a level similar to diatom concentrations observed in samples of groundwater collected near the test site. This investigation was the first attempt to utilize an ELISA as an indicator of surface water influence on groundwater. Further research is needed to develop more specific diatom-based monoclonal antibodies, determine cross-reactivity, and optimize sample processing and ELISA procedures for development of a standardized method.

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

PubMed Central

Xiao, Z; Devreotes, P N

1997-01-01

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures. Images PMID:9168471

Identification of detergent-resistant plasma membrane microdomains in dictyostelium: enrichment of signal transduction proteins.

PubMed

Xiao, Z; Devreotes, P N

1997-05-01

Unlike most other cellular proteins, the chemoattractant receptor, cAR1, of Dictyostelium is resistant to extraction by the zwitterionic detergent, CHAPS. We exploited this property to isolate a subcellular fraction highly enriched in cAR1 by flotation of CHAPS lysates of cells in sucrose density gradients. Immunogold electron microscopy studies revealed a homogeneous preparation of membrane bilayer sheets. This preparation, designated CHAPS-insoluble floating fraction (CHIEF), also contained a defined set of 20 other proteins and a single uncharged lipid. Cell surface biotinylation and preembedding immunoelectron microscopy both confirmed the plasma membrane origin of this preparation. The cell surface phosphodiesterase (PDE) and a downstream effector of cAR1, adenylate cyclase (ACA), were specifically localized in these structures, whereas the cell adhesion molecule gp80, most of the major cell surface membrane proteins, cytoskeletal components, the actin-binding integral membrane protein ponticulin, and G-protein alpha- and beta-subunits were absent. Overall, CHIFF represents about 3-5% of cell externally exposed membrane proteins. All of these results indicate that CHIFF is derived from specialized microdomains of the plasma membrane. The method of isolation is analogous to that of caveolae. However, we were unable to detect distinct caveolae-like structures on the cell surface associated with cAR1, which showed a diffuse staining profile. The discovery of CHIFF facilitates the purification of cAR1 and related signaling proteins and the biochemical characterization of receptor-mediated processes such as G-protein activation and desensitization. It also has important implications for the "fluid mosaic" model of the plasma membrane structures.

The biochemistry and immunology of non-canonical forms of HLA-B27.

PubMed

Shaw, Jacqueline; Hatano, Hiroko; Kollnberger, Simon

2014-01-01

HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 is expressed at the cell surface of antigen presenting cells (APC) both as canonical β2m-associated and non-canonical β2m-free heavy chain (FHC) forms which include B27 dimers (termed B272). B27 FHC forms arise in an endosomal compartment from recycling β2m-associated B27. Formation of cell surface FHC dimers is critically dependent on an unpaired reactive cysteine 67 in the α1 helix of the class I heavy chain. HLA-B27 also form redox-inducible β2m-associated dimers on exosomes and apoptosing cells. By contrast with cell surface expressed cysteine 67-dependent heavy chain dimers these dimers are dependent on a cytoplasmic cysteine 325 for their formation. HLA-B27 binds to immunoregulatory receptors including members of the Killer cell Immunoglobulin-like (KIR) and Leukocyte Immunoglobulin-like receptor family. B27 FHC bind to different but overlapping sets of these immunoreceptors compared to classical β2m-associated HLA-B27. B27 FHC bind more strongly to KIR3DL2 and LILRB2 immune receptor than other β2m-associated HLA-class I ligands. Genetic studies have implicated genes which control production of the important proinflammatory cytokine IL-17 in the pathogenesis of spondyloarthritis. Cell surface HLA-B27 FHC binding to these immune receptors or acting through other mechanisms could impact on the pathogenesis of spondyloarthritis by promoting immune cell production of IL-17. Here we review the literature on these non-canonical forms of HLA-B27 and the immune receptors they bind to and discuss the possible relevance of these interactions to the pathogenesis of spondyloarthropathy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Effect of manufacturing and experimental conditions on the mechanical and surface properties of silicone elastomer scaffolds used in endothelial mechanobiological studies.

PubMed

Campeau, Marc-Antoine; Lortie, Audrey; Tremblay, Pierrick; Béliveau, Marc-Olivier; Dubé, Dominic; Langelier, Ève; Rouleau, Léonie

2017-07-14

Mechanobiological studies allow the characterization of cell response to mechanical stresses. Cells need to be supported by a material with properties similar to the physiological environment. Silicone elastomers have been used to produce various in vitro scaffolds of different geometries for endothelial cell studies given its relevant mechanical, optical and surface properties. However, obtaining defined and repeatable properties is a challenge as depending on the different manufacturing and processing steps, mechanical and surface properties may vary significantly between research groups. The impact of different manufacturing and processing methods on the mechanical and surface properties was assessed by measuring the Young's modulus and the contact angle. Silicone samples were produced using different curing temperatures and processed with different sterilization techniques and hydrophilization conditions. Different curing temperatures were used to obtain materials of different stiffness with a chosen silicone elastomer, i.e. Sylgard 184 ® . Sterilization by boiling had a tendency to stiffen samples cured at lower temperatures whereas UV and ethanol did not alter the material properties. Hydrophilization using sulphuric acid allowed to decrease surface hydrophobicity, however this effect was lost over time as hydrophobic recovery occurred. Extended contact with water maintained decreased hydrophobicity up to 7 days. Mechanobiological studies require complete cell coverage of the scaffolds used prior to mechanical stresses exposure. Different concentrations of fibronectin and collagen were used to coat the scaffolds and cell seeding density was varied to optimize cell coverage. This study highlights the potential bias introduced by manufacturing and processing conditions needed in the preparation of scaffolds used in mechanobiological studies involving endothelial cells. As manufacturing, processing and cell culture conditions are known to influence cell adhesion and function, they should be more thoroughly assessed by research groups that perform such mechanobiological studies using silicone.

Immunization with SV40-transformed cells yields mainly MHC-restricted monoclonal antibodies

PubMed Central

1986-01-01

Recognition of antigens on cell surfaces only in the context of the MHC- encoded alloantigens of the presenting cell (self + X) has classically been considered the province of T cells. However, evidence from several sources has indicated that B cells and antibodies can exhibit self + X- restricted recognition as well. This report concerns the mAb response to SV40-transformed H-2b fibroblast cell lines. The specificities of the antibodies obtained have been analyzed for binding to a panel of SV40-transformed H-2-syngeneic, H-2-allogeneic, and H-2b mutant fibroblast cell lines, as well as cell lines not bearing cell surface SV40 transformation-associated antigens. A large proportion of primary C57BL/6 (71%) and BALB/c (68%) splenic B cells responding to in vitro stimulation with SV40-transformed H-2b cells recognize cell surface antigens associated with SV40 transformation only when coexpressed with MHC antigens of the immunizing cell, particularly the Kb molecule, on transformed cells. To extensively define the nature of antigen recognition by these antibodies, we have generated and characterized nine hybridoma antibodies specific for SV40-transformed H-2-syngeneic cell lines. Seven of these hybridoma antibodies recognize SV40- associated transformation antigens in the context of H-2b molecules. Six of these are restricted by the Kb molecule and discriminate among a panel of SV40-transformed Kb mutant cell lines, thus confirming the participation of class I MHC-encoded molecules in the recognition by B cells of cell surface antigens. PMID:3014034

Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine phosphorylation.

PubMed

Santos, Susana G; Powis, Simon J; Arosa, Fernando A

2004-12-17

Knowledge of the origin and biochemical status of beta(2)-microglobulin-free or misfolded major histocompatibility complex (MHC)-I molecules is essential for understanding their pleiotropic properties. Here we show that in normal human T cells, misfolding of MHC-I molecules is turned on upon activation and cell division and is proportional to the level of proliferation. Immunoprecipitation showed that a number of proteins are associated with MHC-I heavy chains at the surface of activated T cells, including the CD8alphabeta receptor and the chaperone tandem calreticulin/ERp57, associations that rely upon the existence of a pool of HC-10-reactive molecules. Biochemical analysis showed that misfolded MHC-I molecules present at the cell surface are fully glycosylated mature molecules. Importantly, misfolded MHC-I molecules are tyrosine phosphorylated and are associated with kinase activity. In vitro kinase assays followed by reprecipitation indicated that tyrosine phosphorylation of the class I heavy chain is probably mediated by a Src tyrosine kinase because Lck was found associated with HC-10 immunocomplexes. Finally, we show that inhibition of tyrosine phosphorylation by using the Src-family tyrosine kinase inhibitor PP2 resulted in enhanced release of MHC-I heavy chains from the cell surface of activated T cells and a slight down-regulation of cell surface W6/32-reactive molecules. This study provides new insights into the biology of MHC-I molecules and suggests that tyrosine phosphorylation may be involved in the regulation of MHC-I misfolding and expression.

Scanning electron microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175.

PubMed

Rahim, Zubaidah Haji Abdul; Thurairajah, Nalina

2011-04-01

Previous studies have shown that Piper betle L. leaves extract inhibits the adherence of Streptococcus mutans to glass surface, suggesting its potential role in controlling dental plaque development. In this study, the effect of the Piper betle L. extract towards S. mutans (with/without sucrose) using scanning electron microscopy (SEM) and on partially purified cell-associated glucosyltransferase activity were determined. S. mutans were allowed to adhere to glass beads suspended in 6 different Brain Heart Infusion broths [without sucrose; with sucrose; without sucrose containing the extract (2 mg mL(-1) and 4 mg mL(-1)); with sucrose containing the extract (2 mg mL(-1) and 4 mg mL(-1))]. Positive control was 0.12% chlorhexidine. The glass beads were later processed for SEM viewing. Cell surface area and appearance and, cell population of S. mutans adhering to the glass beads were determined upon viewing using the SEM. The glucosyltransferase activity (with/without extract) was also determined. One- and two-way ANOVA were used accordingly. It was found that sucrose increased adherence and cell surface area of S. mutans (p<0.001). S. mutans adhering to 100 µm² glass surfaces (with/without sucrose) exhibited reduced cell surface area, fluffy extracellular appearance and cell population in the presence of the Piper betle L. leaves extract. It was also found that the extract inhibited glucosyltransferase activity and its inhibition at 2.5 mg mL(-1) corresponded to that of 0.12% chlorhexidine. At 4 mg mL(-1) of the extract, the glucosyltransferase activity was undetectable and despite that, bacterial cells still demonstrated adherence capacity. The SEM analysis confirmed the inhibitory effects of the Piper betle L. leaves extract towards cell adherence, cell growth and extracellular polysaccharide formation of S. mutans visually. In bacterial cell adherence, other factors besides glucosyltransferase are involved.

Scanning Electron Microscopic study of Piper betle L. leaves extract effect against Streptococcus mutans ATCC 25175

PubMed Central

RAHIM, Zubaidah Haji Abdul; THURAIRAJAH, Nalina

2011-01-01

Introduction Previous studies have shown that Piper betle L. leaves extract inhibits the adherence of Streptococcus mutans to glass surface, suggesting its potential role in controlling dental plaque development. Objectives: In this study, the effect of the Piper betle L. extract towards S. mutans (with/without sucrose) using scanning electron microscopy (SEM) and on partially purified cell-associated glucosyltransferase activity were determined. Material and Methods S. mutans were allowed to adhere to glass beads suspended in 6 different Brain Heart Infusion broths [without sucrose; with sucrose; without sucrose containing the extract (2 mg mL-1 and 4 mg mL-1); with sucrose containing the extract (2 mg mL-1 and 4 mg mL-1)]. Positive control was 0.12% chlorhexidine. The glass beads were later processed for SEM viewing. Cell surface area and appearance and, cell population of S. mutans adhering to the glass beads were determined upon viewing using the SEM. The glucosyltransferase activity (with/without extract) was also determined. One- and two-way ANOVA were used accordingly. Results It was found that sucrose increased adherence and cell surface area of S. mutans (p<0.001). S. mutans adhering to 100 µm2 glass surfaces (with/without sucrose) exhibited reduced cell surface area, fluffy extracellular appearance and cell population in the presence of the Piper betle L. leaves extract. It was also found that the extract inhibited glucosyltransferase activity and its inhibition at 2.5 mg mL-1 corresponded to that of 0.12% chlorhexidine. At 4 mg mL-1 of the extract, the glucosyltransferase activity was undetectable and despite that, bacterial cells still demonstrated adherence capacity. Conclusion The SEM analysis confirmed the inhibitory effects of the Piper betle L. leaves extract towards cell adherence, cell growth and extracellular polysaccharide formation of S. mutans visually. In bacterial cell adherence, other factors besides glucosyltransferase are involved. PMID:21552715

Inactivation of Clostridium perfringens spores adhered onto stainless steel surface by agents used in a clean-in-place procedure.

PubMed

Alzubeidi, Yasmeen S; Udompijitkul, Pathima; Talukdar, Prabhat K; Sarker, Mahfuzur R

2018-07-20

Enterotoxigenic Clostridium perfringens, a leading foodborne pathogen can be cross-contaminated from food processing stainless steel (SS) surfaces to the finished food products. This is mostly due to the high resistance of C. perfringens spores adhered onto SS surfaces to various disinfectants commonly used in food industries. In this study, we aimed to investigate the survivability and adherence of C. perfringens spores onto SS surfaces and then validate the effectiveness of a simulated Clean-in-Place (CIP) regime on inactivation of spores adhered onto SS surfaces. Our results demonstrated that, 1) C. perfringens spores adhered firmly onto SS surfaces and survived for at-least 48 h, unlike their vegetative cells who died within 30 min, after aerobic incubation at refrigerated and ambient temperatures; 2) Spores exhibited higher levels of hydrophobicity than vegetative cells, suggesting a correlation between cell surface hydrophobicity and adhesion to solid surfaces; 3) Intact spores were more hydrophobic than the decoated spores, suggesting a positive role of spore coat components on spores' hydrophobicity and thus adhesion onto SS surfaces; and finally 4) The CIP regime (NaOH + HNO 3 ) successfully inactivated C. perfringens spores adhered onto SS surfaces, and most of the effect of CIP regime appeared to be due to the NaOH. Collectively, our current findings may well contribute towards developing a strategy to control cross-contamination of C. perfringens spores into food products, which should help reducing the risk of C. perfringens-associated food poisoning outbreaks. Copyright © 2018 Elsevier B.V. All rights reserved.

Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells.

PubMed

Shin, HyeRim; Kim, Dayoung; Helfman, David M

2017-11-10

Metastasis dissemination is the result of various processes including cell migration and cell aggregation. These processes involve alterations in the expression and organization of cytoskeletal and adhesion proteins in tumor cells. Alterations in actin filaments and their binding partners are known to be key players in metastasis. Downregulation of specific tropomyosin (Tpm) isoforms is a common characteristic of transformed cells. In this study, we examined the role of Tpm2.1 in non-transformed MCF10A breast epithelial cells in cell migration and cell aggregation, because this isoform is downregulated in primary and metastatic breast cancer as well as various breast cancer cell lines. Downregulation of Tpm2.1 using siRNA or shRNA resulted in retardation of collective cell migration but increase in single cell migration and invasion. Loss of Tpm2.1 is associated with enhanced actomyosin contractility and increased expression of E-cadherin and β-catenin. Furthermore, inhibition of Rho-associated kinase (ROCK) recovered collective cell migration in Tpm2.1-silenced cells. We also found that Tpm2.1-silenced cells formed more compacted spheroids and exhibited faster cell motility when spheroids were re-plated on 2D surfaces coated with fibronectin and collagen. When Tpm2.1 was downregulated, we observed a decrease in the level of AXL receptor tyrosine kinase, which may explain the increased levels of E-cadherin and β-catenin. These studies demonstrate that Tpm2.1 functions as an important regulator of cell migration and cell aggregation in breast epithelial cells. These findings suggest that downregulation of Tpm2.1 may play a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function?

PubMed Central

Herrmann, Jens; Gressner, Axel M; Weiskirchen, Ralf

2007-01-01

Abstract At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts. PMID:17760834

Immortal hepatic stellate cell lines: useful tools to study hepatic stellate cell biology and function?

PubMed

Herrmann, Jens; Gressner, Axel M; Weiskirchen, Ralf

2007-01-01

At the cellular level, the activation and transdifferentiation of quiescent hepatic stellate cells (HSC) into myofibroblasts is the key process involved in hepatic fibrogenesis that is associated with an increased and altered deposition of extracellular matrix components in the liver. The temporal sequence of molecular events associated with stellate cell activation turned out to be appropriately mimicked when HSC isolated from normal livers are cultured on uncoated plastic surface. Therefore, cultured primary cells isolated from rodents and human beings are common in vitro models in investigations addressing these issues of hepatic stellate biology and function. However, the limited supply, cost-effective isolation procedure and the ever growing need have resulted in efforts to establish immortalized stellate cell lines having the advantage of virtually unlimited access. They allow rapid screening for disease-associated factors and restrict the necessary number of animal experiments. From the first description of an immortal HSC line in 1986, a huge number of studies were conducted with these established cell lines. However, differences in morphology, growth characteristics and anomalies of chromosome number and structure make the applications of these models questionable. Here, we summarize the history and cellular characteristics of respective cell lines and discuss the differences of continuous HSC lines and their primary counterparts.

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

Microarray analysis of genes associated with cell surface NIS protein levels in breast cancer.

PubMed

Beyer, Sasha J; Zhang, Xiaoli; Jimenez, Rafael E; Lee, Mei-Ling T; Richardson, Andrea L; Huang, Kun; Jhiang, Sissy M

2011-10-11

Na+/I- symporter (NIS)-mediated iodide uptake allows radioiodine therapy for thyroid cancer. NIS is also expressed in breast tumors, raising potential for radionuclide therapy of breast cancer. However, NIS expression in most breast cancers is low and may not be sufficient for radionuclide therapy. We aimed to identify biomarkers associated with NIS expression such that mechanisms underlying NIS modulation in human breast tumors may be elucidated. Published oligonucleotide microarray data within the National Center for Biotechnology Information Gene Expression Omnibus database were analyzed to identify gene expression tightly correlated with NIS mRNA level among human breast tumors. NIS immunostaining was performed in a tissue microarray composed of 28 human breast tumors which had corresponding oligonucleotide microarray data available for each tumor such that gene expression associated with cell surface NIS protein level could be identified. NIS mRNA levels do not vary among breast tumors or when compared to normal breast tissues when detected by Affymetrix oligonucleotide microarray platforms. Cell surface NIS protein levels are much more variable than their corresponding NIS mRNA levels. Despite a limited number of breast tumors examined, our analysis identified cysteinyl-tRNA synthetase as a biomarker that is highly associated with cell surface NIS protein levels in the ER-positive breast cancer subtype. Further investigation on genes associated with cell surface NIS protein levels within each breast cancer molecular subtype may lead to novel targets for selectively increasing NIS expression/function in a subset of breast cancers patients.

Urban runoff (URO) process for MODFLOW 2005: simulation of sub-grid scale urban hydrologic processes in Broward County, FL

USGS Publications Warehouse

Decker, Jeremy D.; Hughes, J.D.

2013-01-01

Climate change and sea-level rise could cause substantial changes in urban runoff and flooding in low-lying coast landscapes. A major challenge for local government officials and decision makers is to translate the potential global effects of climate change into actionable and cost-effective adaptation and mitigation strategies at county and municipal scales. A MODFLOW process is used to represent sub-grid scale hydrology in urban settings to help address these issues. Coupled interception, surface water, depression, and unsaturated zone storage are represented. A two-dimensional diffusive wave approximation is used to represent overland flow. Three different options for representing infiltration and recharge are presented. Additional features include structure, barrier, and culvert flow between adjacent cells, specified stage boundaries, critical flow boundaries, source/sink surface-water terms, and the bi-directional runoff to MODFLOW Surface-Water Routing process. Some abilities of the Urban RunOff (URO) process are demonstrated with a synthetic problem using four land uses and varying cell coverages. Precipitation from a hypothetical storm was applied and cell by cell surface-water depth, groundwater level, infiltration rate, and groundwater recharge rate are shown. Results indicate the URO process has the ability to produce time-varying, water-content dependent infiltration and leakage, and successfully interacts with MODFLOW.

Study of surface damage on cell envelope assessed by AFM and flow cytometry of Lactobacillus plantarum exposed to ethanol and dehydration.

PubMed

Bravo-Ferrada, B M; Gonçalves, S; Semorile, L; Santos, N C; Tymczyszyn, E E; Hollmann, A

2015-06-01

In this work, we evaluated freeze-drying damage at the surface level of oenological strain Lactobacillus plantarum UNQLp155, as well as its ability to grow in a synthetic wine with and without pre-acclimation. Damage on cell surface was studied by flow cytometry, zeta potential and atomic force microscopy, and cell survival was analysed by plate count. Results showed that beside cells acclimated at lower ethanol concentration (6% v/v) became more susceptible to drying than nonacclimated ones, after rehydration they maintain their increased ability to grow in a synthetic wine. Acclimation at a higher ethanol concentration (10% v/v) produces several damages on the cell surface losing its ability to grow in a synthetic wine. In this work, we showed for the first time that sublethal alterations on bacterial surface induced by a pre-acclimation with a low ethanol concentration (6%), upon a freeze-drying process, result in a better bacterial adaptation to the stress conditions of wine-like medium, as well as to the preservation process. Understanding the adaptation to ethanol of oenological strains and their effects on the preservation process has a strong impact on winemaking process and allows to define the most appropriate conditions to obtain malolactic starters cultures. © 2015 The Society for Applied Microbiology.

Surface roughness mediated adhesion forces between borosilicate glass and gram-positive bacteria.

PubMed

Preedy, Emily; Perni, Stefano; Nipiĉ, Damijan; Bohinc, Klemen; Prokopovich, Polina

2014-08-12

It is well-known that a number of surface characteristics affect the extent of adhesion between two adjacent materials. One of such parameters is the surface roughness as surface asperities at the nanoscale level govern the overall adhesive forces. For example, the extent of bacterial adhesion is determined by the surface topography; also, once a bacteria colonizes a surface, proliferation of that species will take place and a biofilm may form, increasing the resistance of bacterial cells to removal. In this study, borosilicate glass was employed with varying surface roughness and coated with bovine serum albumin (BSA) in order to replicate the protein layer that covers orthopedic devices on implantation. As roughness is a scale-dependent process, relevant scan areas were analyzed using atomic force microscope (AFM) to determine Ra; furthermore, appropriate bacterial species were attached to the tip to measure the adhesion forces between cells and substrates. The bacterial species chosen (Staphylococci and Streptococci) are common pathogens associated with a number of implant related infections that are detrimental to the biomedical devices and patients. Correlation between adhesion forces and surface roughness (Ra) was generally better when the surface roughness was measured through scanned areas with size (2 × 2 μm) comparable to bacteria cells. Furthermore, the BSA coating altered the surface roughness without correlation with the initial values of such parameter; therefore, better correlations were found between adhesion forces and BSA-coated surfaces when actual surface roughness was used instead of the initial (nominal) values. It was also found that BSA induced a more hydrophilic and electron donor characteristic to the surfaces; in agreement with increasing adhesion forces of hydrophilic bacteria (as determined through microbial adhesion to solvents test) on BSA-coated substrates.

Process for manufacturing a lithium alloy electrochemical cell

DOEpatents

Bennett, William R.

1992-10-13

A process for manufacturing a lithium alloy, metal sulfide cell tape casts slurried alloy powders in an organic solvent containing a dissolved thermoplastic organic binder onto casting surfaces. The organic solvent is then evaporated to produce a flexible tape removable adhering to the casting surface. The tape is densified to increase its green strength and then peeled from the casting surface. The tape is laminated with a separator containing a lithium salt electrolyte and a metal sulfide electrode to form a green cell. The binder is evaporated from the green cell at a temperature lower than the melting temperature of the lithium salt electrolyte. Lithium alloy, metal sulfide and separator powders may be tape cast.

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.

Isolation of mesenchymal stromal/stem cells from cryopreserved umbilical cord blood cells.

PubMed

Fujii, Sumie; Miura, Yasuo; Iwasa, Masaki; Yoshioka, Satoshi; Fujishiro, Aya; Sugino, Noriko; Kaneko, Hitomi; Nakagawa, Yoko; Hirai, Hideyo; Takaori-Kondo, Akifumi; Ichinohe, Tatsuo; Maekawa, Taira

2017-07-05

Umbilical cord blood (UCB) has advantages over other tissues because it can be obtained without an invasive procedure and complex processing. We explored the availability of cryopreserved UCB cells as a source of mesenchymal stromal/stem cells (MSCs). MSCs were successfully isolated from six of 30 UCB units (median volume, 34.0 mL; median nucleated cell number, 4.4×10 8 ) that were processed and cryopreserved using CP-1/human serum albumin. This isolation rate was lower than that (57%) from non-cryopreserved UCB cells. The number of nucleated cells before and after hydroxyethyl starch separation, UCB unit volume, and cell viability after thawing did not significantly differ between UCB units from which MSCs were successfully isolated and those from which they were not. When CryoSure-DEX40 was used as a cryoprotectant, MSCs were isolated from two of ten UCB units. Logistic regression analysis demonstrated that the cryopreservation method was not significantly associated with the success of MSC isolation. The isolated MSCs had a similar morphology and surface marker expression profile as bone marrow-derived MSCs and were able to differentiate into osteogenic, adipogenic, and chondrogenic cells. In summary, MSCs can be isolated from cryopreserved UCB cells. However, the cryopreservation process reduces the isolation rate; therefore, freshly donated UCB cells are preferable for the isolation of MSCs.

Process boundaries of irreversible scCO2 -assisted phase separation in biphasic whole-cell biocatalysis.

PubMed

Brandenbusch, Christoph; Glonke, Sebastian; Collins, Jonathan; Hoffrogge, Raimund; Grunwald, Klaudia; Bühler, Bruno; Schmid, Andreas; Sadowski, Gabriele

2015-11-01

The formation of stable emulsions in biphasic biotransformations catalyzed by microbial cells turned out to be a major hurdle for industrial implementation. Recently, a cost-effective and efficient downstream processing approach, using supercritical carbon dioxide (scCO2 ) for both irreversible emulsion destabilization (enabling complete phase separation within minutes of emulsion treatment) and product purification via extraction has been proposed by Brandenbusch et al. (2010). One of the key factors for a further development and scale-up of the approach is the understanding of the mechanism underlying scCO2 -assisted phase separation. A systematic approach was applied within this work to investigate the various factors influencing phase separation during scCO2 treatment (that is pressure, exposure of the cells to CO2 , and changes of cell surface properties). It was shown that cell toxification and cell disrupture are not responsible for emulsion destabilization. Proteins from the aqueous phase partially adsorb to cells present at the aqueous-organic interface, causing hydrophobic cell surface characteristics, and thus contribute to emulsion stabilization. By investigating the change in cell-surface hydrophobicity of these cells during CO2 treatment, it was found that a combination of catastrophic phase inversion and desorption of proteins from the cell surface is responsible for irreversible scCO2 mediated phase separation. These findings are essential for the definition of process windows for scCO2 -assisted phase separation in biphasic whole-cell biocatalysis. © 2015 Wiley Periodicals, Inc.

Volume Labeling with Alexa-Fluor Dyes and Surface Functionalization of Highly Sensitive Fluorescent SiO2 Nanoparticles

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

Wang, Wei; Foster, Carmen M; Morrell-Falvey, Jennifer L

2013-01-01

A new synthesis approach is described that allows the direct incorporation of fluorescent labels into the volume or body of SiO2 nanoparticles. In this process, fluorescent Alexa Fluor dyes with different emission wavelengths were covalently incorporated into the SiO2 nanoparticles during their formation by the hydrolysis of tetraethoxysilane. The dye molecules were homogeneously distributed throughout the SiO2 nanoparticles. The quantum yields of the Alexa Fluor volume-labeled SiO2 nanoparticles were much higher than nanoparticles labeled using conventional organic dyes. The size of the resulting nanoparticles was controlled using microemulsion reaction media with sizes in the range of 20-100 nm and amore » polydispersity of <15%. In comparison with conventional surface tagged particles created by post-synthesis modification, this process maintains the physical and surface chemical properties that have the most pronounced effect on colloidal stability and interactions with their surroundings. These volume-labeled nanoparticles have proven to be extremely robust, showing excellent signal strength, negligible photobleaching, and minimal loss of functional organic components. The native or free surface of the volume-labeled particles can be altered to achieve a specific surface functionality without altering fluorescence. Their utility was demonstrated for visualizing the association of surface modified fluorescent particles with cultured macrophages. Differences in particle agglomeration and cell association were clearly associated with differences in observed nanoparticle toxicity. The capacity to maintain particle fluorescence while making significant changes to surface chemistry makes these particles extremely versatile and useful for studies of particle agglomeration, uptake, and transport in environmental and biological systems.« less

Topography and behavior of Sertoli cells in sparse culture during the transitional remodeling phase.

PubMed

Tung, P S; Choi, A H; Fritz, I B

1988-01-01

We report observations on the behavior of Sertoli cells in sparse culture during the period from the time of plating to the time of initial confluence (the transitional remodeling phase). Changes in shape, structure, and polarity of cells, as well as changes in migration patterns and cell-cell association patterns, have been followed during the transitional remodeling phase with the aid of topographical markers. These markers are based upon differences between ultrastructural features of the basolateral and apicolateral surfaces. The basolateral surface is characterized by plasmalemmal blebs, whereas the apicolateral surface is characterized by filopodial extensions. Structural differences observed in situ remain evident in Sertoli cells isolated by sequential enzymatic treatments that are described. Another marker is provided by laminin-binding sites, which are detected exclusively on the blebbed, basolateral surfaces of freshly prepared Sertoli cell aggregates. The orientation described is sustained during the initial radial migration of Sertoli cells explanted on uncoated glass coverslips. Under these conditions, blebs are detected only on the dorsal surfaces, and filopodial extensions are evident only on the ventral surfaces. In contrast, Sertoli cells sparsely plated on a reconstituted basement membrane (air-dried Matrigel) migrate rapidly, display an extraordinary capacity to form elaborate cytoplasmic extensions for cell-cell and cell-substratum contacts, and readily retract blebs and filopodial extensions. These cells do not form mosaic borders, whereas cells plated on uncoated glass do form a monolayer with mosaic-like borders. Cells sparsely seeded on gelated Matrigel migrate preferentially at gaps between adjacent cell explants, and develop a compact cell-cell association pattern. These cells display few, if any, cytoplasmic extensions. We compare the behavior of Sertoli cells sparsely plated on Matrigel with the behavior of Sertoli cells in situ during different stages of development.

A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: to enhance the efficiency of recombinant DNA utilization.

PubMed

Mozley, Olivia L; Thompson, Ben C; Fernandez-Martell, Alejandro; James, David C

2014-01-01

In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl-PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process. © 2014 American Institute of Chemical Engineers.

Surface dynamics of aerolysin on the plasma membrane of living cells.

PubMed

Abrami, L; Fivaz, M; van der Goot, F G

2000-10-01

Aerolysin secreted by the human pathogen Aeromonas hydrophila belongs to a group of bacterial toxins that are hemolytic and form channels in biological membranes. The toxin is secreted as an inactive precursor proaerolysin that must be proteolytically processed at its C-terminus to become active. The toxin then polymerizes into a heptameric ring that is amphipathic and can insert into a lipid bilayer and form a pore. We have examined these various steps at the surface of target cells. The toxin binds to specific receptors. Various receptors have been identified, all of which are anchored to the plasma membrane via a glycosylphosphatidyl inositol (GPI)-anchored moiety. The GPI anchor confers to the protein that is linked to it two usual properties: (i) the protein has a higher lateral mobility in a phospholipid bilayer than its transmembrane counterpart, (ii) the protein has the capacity to transiently associate with cholesterol-glycosphingolipid-rich microdomains. We have shown that both these properties of GPI-anchored proteins are exploited by proaerolysin bound to its receptor. The high lateral mobility within the phosphoglyceride region of the plasma membrane favors the encounter of the protoxin with its converting enzyme furin. The ability to associate with microdomains on the other hand favors the oligomerization process presumably by concentrating the toxin locally.

Modeled Microgravity Inhibits Apoptosis in Peripheral Blood Lymphocytes

NASA Technical Reports Server (NTRS)

Risin, Diana; Pellis, Neal R.

1999-01-01

Impairment of the immunity in astronauts and cosmonauts even in short term flights is a recognized risk. Long term orbital space missions and anticipated interplanetary flights increase the concern for more pronounced effects on the immune system with potential clinical consequences. Impairment of the immunity in space may be due tonumerous physiological changes caused by space-related factors, which in turn affect the immune system, or alternatively, it may be due to direct effects of different factors encountered in space on lymphoid cells and their interactions. Indeed, in modeled microgravity (MMG) experiments on Earth we and others showed that microgravity directly affects multiple lymphocyte functions. It interferes with expression of cell surface molecules, causes inhibition of lymphocyte locomotion, suppresses polyclopal and antigen-specific lymphocyte activation, selectively inhibits protein kinase C (PKC) isoforms. Some of these effects were also confirmed in cell culture experiments in real space conditions during Spacelab, Biokosmos and Shuttle Missions. The results of these studies, taken together, strongly indicated that microgravity interferes with fundamental biological processes associated with functional and structural changes in cell surface membranes, cell surface molecules and in their interaction. Based on the data and on their interpretation, we hypothesized that microgravity in addition to observed functional changes affects programmed cell death (PCD) in lymphocyte populations and that this mechanism could contribute to the impairment of the immunity.

Defining the role of syndecan-4 in mechanotransduction using surface-modification approaches

PubMed Central

Bellin, Robert M.; Kubicek, James D.; Frigault, Matthew J.; Kamien, Andrew J.; Steward, Robert L.; Barnes, Hillary M.; DiGiacomo, Michael B.; Duncan, Luke J.; Edgerly, Christina K.; Morse, Elizabeth M.; Park, Chan Young; Fredberg, Jeffrey J.; Cheng, Chao-Min; LeDuc, Philip R.

2009-01-01

The ability of cells to respond to external mechanical stimulation is a complex and robust process involving a diversity of molecular interactions. Although mechanotransduction has been heavily studied, many questions remain regarding the link between physical stimulation and biochemical response. Of significant interest has been the contribution of the transmembrane proteins involved, and integrins in particular, because of their connectivity to both the extracellular matrix and the cytoskeleton. Here, we demonstrate the existence of a mechanically based initiation molecule, syndecan-4. We first demonstrate the ability of syndecan-4 molecules to support cell attachment and spreading without the direct extracellular binding of integrins. We also examine the distribution of focal adhesion-associated proteins through controlling surface interactions of beads with molecular specificity in binding to living cells. Furthermore, after adhering cells to elastomeric membranes via syndecan-4-specific attachments we mechanically strained the cells via our mechanical stimulation and polymer surface chemical modification approach. We found ERK phosphorylation similar to that shown for mechanotransductive response for integrin-based cell attachments through our elastomeric membrane-based approach and optical magnetic twisting cytometry for syndecan-4. Finally, through the use of cytoskeletal disruption agents, this mechanical signaling was shown to be actin cytoskeleton dependent. We believe that these results will be of interest to a wide range of fields, including mechanotransduction, syndecan biology, and cell–material interactions. PMID:20080785

Gold nano-popcorn-based targeted diagnosis, nanotherapy treatment, and in situ monitoring of photothermal therapy response of prostate cancer cells using surface-enhanced Raman spectroscopy.

PubMed

Lu, Wentong; Singh, Anant Kumar; Khan, Sadia Afrin; Senapati, Dulal; Yu, Hongtao; Ray, Paresh Chandra

2010-12-29

Prostate cancer is the second leading cause of cancer-related death among the American male population, and the cost of treating prostate cancer patients is about $10 billion/year in the United States. Current treatments are mostly ineffective against advanced-stage prostate cancer and are often associated with severe side effects. Driven by these factors, we report a multifunctional, nanotechnology-driven, gold nano-popcorn-based surface-enhanced Raman scattering (SERS) assay for targeted sensing, nanotherapy treatment, and in situ monitoring of photothermal nanotherapy response during the therapy process. Our experimental data show that, in the presence of LNCaP human prostate cancer cells, multifunctional popcorn-shaped gold nanoparticles form several hot spots and provide a significant enhancement of the Raman signal intensity by several orders of magnitude (2.5 × 10(9)). As a result, it can recognize human prostate cancer cells at the 50-cells level. Our results indicate that the localized heating that occurs during near-infrared irradiation can cause irreparable cellular damage to the prostate cancer cells. Our in situ time-dependent results demonstrate for the first time that, by monitoring SERS intensity changes, one can monitor photothermal nanotherapy response during the therapy process. Possible mechanisms and operating principles of our SERS assay are discussed. Ultimately, this nanotechnology-driven assay could have enormous potential applications in rapid, on-site targeted sensing, nanotherapy treatment, and monitoring of the nanotherapy process, which are critical to providing effective treatment of cancer.

Gold Nano-Popcorn Based Targeted Diagonosis, Nanotherapy Treatment and In-Situ Monitoring of Photothermal Therapy Response of Prostate Cancer Cells Using Surface Enhanced Raman Spectroscopy

PubMed Central

Lu, Wentong; Singh, Anant Kumar; Khan, Sadia Afrin; Senapati, Dulal; Yu, Hongtao; Ray, Paresh Chandra

2010-01-01

Prostate cancer is the second leading cause of cancer-related death among the American male population and the cost of treating prostate cancer patients is about $10 billion/year in the US. Current treatments are mostly ineffective against advanced stage prostate cancer disease and are often associated with severe side effects. Driven by the need, in this manuscript, we report multifunctional nanotechnology-driven gold nano-popcorn based surface enhanced Raman scattering (SERS) assay for targeted sensing, nanotherapy treatment and in-situ monitoring of photothermal nanotherapy response during the therapy process. Our experimental data show that in the presence of LNCaP human prostate cancer cell, multifunctional popcorn shape gold nanoparticle forms several hot spots and provides a significant enhancement of the Raman signal intensity by several orders of magnitude (2.5 × 109). As a result, it can recognize human prostate cancer cell in 50 cells level. Our results indicate that the localized heating that occurs during NIR irradiation is able to cause irreparable cellular damage of the prostate cancer cell. Our in-situ time dependent results demonstrates for the first time that by monitoring SERS intensity change, one can monitor photo thermal nanotherapy response during therapy process. Possible mechanisms and operating principle of our SERS assay have been discussed. Ultimately, this nanotechnology driven assay could have enormous potential applications in rapid, on-site targeted sensing, nanotherapy treatment and monitoring of nanotherapy process which is critical to providing effective treatment of cancer disease. PMID:21128627

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.

ARP2/3 localization in Arabidopsis leaf pavement cells: a diversity of intracellular pools and cytoskeletal interactions

PubMed Central

Zhang, Chunhua; Mallery, Eileen L.; Szymanski, Daniel B.

2013-01-01

In plant cells the actin cytoskeleton adopts many configurations, but is best understood as an unstable, interconnected track that rearranges to define the patterns of long distance transport of organelles during growth. Actin filaments do not form spontaneously; instead filament nucleators, such as the evolutionarily conserved actin-related protein (ARP) 2/3 complex, can efficiently generate new actin filament networks when in a fully activated state. A growing number of genetic experiments have shown that ARP2/3 is necessary for morphogenesis in processes that range from tip growth during root nodule formation to the diffuse polarized growth of leaf trichomes and pavement cells. Although progress has been rapid in the identification of proteins that function in series to positively regulate ARP2/3, less has been learned about the actual function of ARP2/3 in cells. In this paper, we analyze the localization of ARP2/3 in Arabidopsis leaf pavement cells. We detect a pool of ARP2/3 in the nucleus, and also find that ARP2/3 is efficiently and specifically clustered on multiple organelle surfaces and associates with both the actin filament and microtubule cytoskeletons. Our mutant analyses and ARP2/3 and actin double labeling experiments indicate that the clustering of ARP2/3 on organelle surfaces and an association with actin bundles does not necessarily reflect an active pool of ARP2/3, and instead most of the complex appears to exist as a latent organelle-associated pool. PMID:23874346

Evaluation of Biofilms and the Effects of Biocides Thereon

NASA Technical Reports Server (NTRS)

Pierson, Duane L. (Inventor); Koenig, David W. (Inventor); Mishra, Saroj K. (Inventor)

2002-01-01

Biofilm formation is monitored by real-time continuous measurement. Images are formed of sessile cells on a surface and planktonic cells adjacent the surface. The attachment of cells to the surface is measured and quantitated, and sessile and planktonic cells are distinguished using image processing techniques. Single cells as well as colonies are monitored on or adjacent a variety of substrates. Flowing streams may be monitored. The effects of biocides on biofilms commonly isolated from recyclable water systems are measured.

LRP1 influences trafficking of N-type calcium channels via interaction with the auxiliary α2δ-1 subunit

PubMed Central

Kadurin, Ivan; Rothwell, Simon W.; Lana, Beatrice; Nieto-Rostro, Manuela; Dolphin, Annette C.

2017-01-01

Voltage-gated Ca2+ (CaV) channels consist of a pore-forming α1 subunit, which determines the main functional and pharmacological attributes of the channel. The CaV1 and CaV2 channels are associated with auxiliary β- and α2δ-subunits. The molecular mechanisms involved in α2δ subunit trafficking, and the effect of α2δ subunits on trafficking calcium channel complexes remain poorly understood. Here we show that α2δ-1 is a ligand for the Low Density Lipoprotein (LDL) Receptor-related Protein-1 (LRP1), a multifunctional receptor which mediates trafficking of cargoes. This interaction with LRP1 is direct, and is modulated by the LRP chaperone, Receptor-Associated Protein (RAP). LRP1 regulates α2δ binding to gabapentin, and influences calcium channel trafficking and function. Whereas LRP1 alone reduces α2δ-1 trafficking to the cell-surface, the LRP1/RAP combination enhances mature glycosylation, proteolytic processing and cell-surface expression of α2δ-1, and also increase plasma-membrane expression and function of CaV2.2 when co-expressed with α2δ-1. Furthermore RAP alone produced a small increase in cell-surface expression of CaV2.2, α2δ-1 and the associated calcium currents. It is likely to be interacting with an endogenous member of the LDL receptor family to have these effects. Our findings now provide a key insight and new tools to investigate the trafficking of calcium channel α2δ subunits. PMID:28256585

Method of making a back contacted solar cell

DOEpatents

Gee, James M.

1995-01-01

A back-contacted solar cell having laser-drilled vias connecting the front-surface carrier-collector junction to an electrode grid on the back surface. The structure may also include a rear surface carrier-collector junction connected to the same grid. The substrate is connected to a second grid which is interdigitated with the first. Both grids are configured for easy series connection with neighboring cells. Several processes are disclosed to produce the cell.

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

Soybean agglutinin binding to corneal endothelial cell surfaces disrupts in situ monolayer integrity and actin organization and interferes with wound repair.

PubMed

Gordon, Sheldon R; Wood, Meredith

2009-03-01

Rat corneal endothelium demonstrates cell-surface soybean agglutinin (SBA) binding during organ-culture or injury. When organ-cultured in medium containing SBA, the endothelial monolayer is disrupted because of cell-cell and cell-matrix alterations. SBA binding disorganizes the circumferential microfilament bundles (CMBs), an effect that is partially prevented by phallacidin preincubation. This disruption is reversible if tissues are returned to standard culture medium. Serum heightens SBA binding, whereas puromycin prevents it. Neither actinomycin D nor alpha-amanitin inhibits SBA binding, suggesting that SBA-binding protein(s) may be post-transcriptionally regulated. During injury-induced cell migration in the presence of SBA, cellular processes are blunted and fail to extend significantly outward. By 72 h post-injury, cells of SBA-treated tissues repopulate the wound but demonstrate little association with neighboring cells. Cells migrating in the presence of N-acetylgalactosamine appear normal but also fail to reassociate with other cells in the jury zone. Immunofluorescent staining for ZO-1 reveals punctuate patterns in cells of control tissues, whereas neither SBA- nor N-acetylgalactosamine-treated tissues exhibit ZO-1 staining. Terminal N-acetylgalactosamine removal fails to affect cell morphology, actin organization, or migration but prevents lectin binding. Our results suggest that SBA binding reflects the synthesis of a stress-induced protein(s) that may play a role in reestablishing cell-cell relationships during monolayer reorganization following injury.

In vitro adhesion of fibroblastic cells to titanium alloy discs treated with sodium hydroxide.

PubMed

Al Mustafa, Maisa; Agis, Hermann; Müller, Heinz-Dieter; Watzek, Georg; Gruber, Reinhard

2015-01-01

Adhesion of osteogenic cells on titanium surfaces is a prerequisite for osseointegration. Alkali treatment can increase the hydrophilicity of titanium implant surfaces, thereby supporting the adhesion of blood components. However, it is unclear if alkali treatment also supports the adhesion of cells with a fibroblastic morphology to titanium. Here, we have used a titanium alloy (Ti-6AL-4V) processed by alkali treatment to demonstrate the impact of hydrophilicity on the adhesion of primary human gingival fibroblast and bone cells. Also included were the osteosarcoma and fibroblastoma cell lines, MG63 and L929, respectively. Cell adhesion was determined by scanning electron microscopy. We also measured viability, proliferation, and protein synthesis of the adherent cells. Alkali treatment increased the adhesion of gingival fibroblasts, bone cells, and the two cell lines when seeded onto the titanium alloy surface for 1 h. At 3 h, no significant changes in cell adhesion were observed. Cells grown for 1 day on the titanium alloy surfaces processed by alkali treatment behave similarly to untreated controls with regard to viability, proliferation, and protein synthesis. Based on these preliminary In vitro findings, we conclude that alkali treatment can support the early adhesion of cells with fibroblastic characteristics to a titanium alloy surface. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Effects of Build Orientation on Surface Morphology and Bone Cell Activity of Additively Manufactured Ti6Al4V Specimens.

PubMed

Weißmann, Volker; Drescher, Philipp; Seitz, Hermann; Hansmann, Harald; Bader, Rainer; Seyfarth, Anika; Klinder, Annett; Jonitz-Heincke, Anika

2018-05-29

Additive manufacturing of lightweight or functional structures by selective laser beam (SLM) or electron beam melting (EBM) is widespread, especially in the field of medical applications. SLM and EBM processes were applied to prepare Ti6Al4V test specimens with different surface orientations (0°, 45° and 90°). Roughness measurements of the surfaces were conducted and cell behavior on these surfaces was analyzed. Hence, human osteoblasts were seeded on test specimens to determine cell viability (metabolic activity, live-dead staining) and gene expression of collagen type 1 (Col1A1), matrix metalloprotease (MMP) 1 and its natural inhibitor, TIMP1, after 3 and 7 days. The surface orientation of specimens during the manufacturing process significantly influenced the roughness. Surface roughness showed significant impact on cellular viability, whereas differences between the time points day 3 and 7 were not found. Collagen type 1 mRNA synthesis rates in human osteoblasts were enhanced with increasing roughness. Both manufacturing techniques further influenced the induction of bone formation process in the cell culture. Moreover, the relationship between osteoblastic collagen type 1 mRNA synthesis rates and specimen orientation during the building process could be characterized by functional formulas. These findings are useful in the designing of biomedical applications and medical devices.

Antibody Therapeutics in Oncology.

PubMed

Wold, Erik D; Smider, Vaughn V; Felding, Brunhilde H

2016-03-01

One of the newer classes of targeted cancer therapeutics is monoclonal antibodies. Monoclonal antibody therapeutics are a successful and rapidly expanding drug class due to their high specificity, activity, favourable pharmacokinetics, and standardized manufacturing processes. Antibodies are capable of recruiting the immune system to attack cancer cells through complement-dependent cytotoxicity or antibody dependent cellular cytotoxicity. In an ideal scenario the initial tumor cell destruction induced by administration of a therapeutic antibody can result in uptake of tumor associated antigens by antigen-presenting cells, establishing a prolonged memory effect. Mechanisms of direct tumor cell killing by antibodies include antibody recognition of cell surface bound enzymes to neutralize enzyme activity and signaling, or induction of receptor agonist or antagonist activity. Both approaches result in cellular apoptosis. In another and very direct approach, antibodies are used to deliver drugs to target cells and cause cell death. Such antibody drug conjugates (ADCs) direct cytotoxic compounds to tumor cells, after selective binding to cell surface antigens, internalization, and intracellular drug release. Efficacy and safety of ADCs for cancer therapy has recently been greatly advanced based on innovative approaches for site-specific drug conjugation to the antibody structure. This technology enabled rational optimization of function and pharmacokinetics of the resulting conjugates, and is now beginning to yield therapeutics with defined, uniform molecular characteristics, and unprecedented promise to advance cancer treatment.

Quantification of chemical transport processes from the soil to surface runoff.

PubMed

Tian, Kun; Huang, Chi-Hua; Wang, Guang-Qian; Fu, Xu-Dong; Parker, Gary

2013-01-01

There is a good conceptual understanding of the processes that govern chemical transport from the soil to surface runoff, but few studies have actually quantified these processes separately. Thus, we designed a laboratory flow cell and experimental procedures to quantify the chemical transport from soil to runoff water in the following individual processes: (i) convection with a vertical hydraulic gradient, (ii) convection via surface flow or the Bernoulli effect, (iii) diffusion, and (iv) soil loss. We applied different vertical hydraulic gradients by setting the flow cell to generate different seepage or drainage conditions. Our data confirmed the general form of the convection-diffusion equation. However, we now have additional quantitative data that describe the contribution of each individual chemical loading process in different surface runoff and soil hydrological conditions. The results of this study will be useful for enhancing our understanding of different geochemical processes in the surface soil mixing zone. Copyright © by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Inc.

Immobilization of concanavalin A receptors during differentiation of neuroblastoma cells.

PubMed

Fishman, M C; Dragsten, P R; Spector, I

1981-04-30

Neuroblastoma cells serve as a useful model of neuronal development because compounds such as dimethyl sulphoxide (DMSO) and dibutyryl cyclic AMP cause them to undergo a process of controlled differentiation in tissue culture, during which they can extend long processes, develop characteristic excitability mechanisms, synthesize neurotransmitters and form synapses. We have used the technique of fluorescence photobleaching recovery to study the lateral mobility of cell-surface constituents during the differentiation of neuroblastoma clone N1E-115 cells. The concanavalin A (Con A) binding sites appear as discrete patches distributed over the entire cell surface and exhibit lateral mobility in undifferentiated cells comparable with that of surface glycoproteins of other cells. After induction of differentiation, however, the vast majority of Con A binding sites become immobilized, and we present data which suggest that the mechanism of this immobilization may involve linkage to the internal actin network.

The two subunits of the human asialoglycoprotein receptor have different fates when expressed alone in fibroblasts

PubMed Central

Shia, Michael A.; Lodish, Harvey F.

1989-01-01

Two related polypeptides, H1 and H2, comprise the human asialoglycoprotein receptor (ASGP-R). Stable lines of murine NIH 3T3 fibroblasts expressing H1 alone or H2 alone do not bind or internalize the ligand asialoorosomucoid (ASOR), which contains triantennary oligosaccharides. In contrast, cells expressing H1 and H2 together bind and degrade ASOR with properties indistinguishable from those of the ASPG-R in human hepatoma HepG2 cells. Whether or not H2 is coexpressed, H1 is synthesized as a 40-kDa precursor bearing high-mannose oligosaccharides, processed to its mature 46-kDa form, and transported to the cell surface. In cells expressing only H1, homodimers and -trimers of H1 are formed. In contrast, when expressed in 3T3 cells without H1, H2 is synthesized as its 43-kDa precursor, bearing high-mannose oligosaccharides, but is rapidly degraded. When H1 and H2 are coexpressed in the same cell, the H1 polypeptide “rescues” the H2 polypeptide; H2 is processed to its characteristic 50-kDa mature form and is transported to the surface. We conclude that the human ASGP-R is a multichain heterooligomer, probably a trimer of H1 molecules in noncovalent association with one, two, or three H2 molecules, and that the two polypeptides normally interact early in biosynthesis. Images PMID:2919187

SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs.

PubMed

Shi, Jian Xin; Malitsky, Sergey; De Oliveira, Sheron; Branigan, Caroline; Franke, Rochus B; Schreiber, Lukas; Aharoni, Asaph

2011-05-01

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions.

SHINE Transcription Factors Act Redundantly to Pattern the Archetypal Surface of Arabidopsis Flower Organs

PubMed Central

Shi, Jian Xin; Malitsky, Sergey; De Oliveira, Sheron; Branigan, Caroline; Franke, Rochus B.; Schreiber, Lukas; Aharoni, Asaph

2011-01-01

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions. PMID:21637781

Development of processing procedures for advanced silicon solar cells. [antireflection coatings and short circuit currents

NASA Technical Reports Server (NTRS)

Scott-Monck, J. A.; Stella, P. M.; Avery, J. E.

1975-01-01

Ten ohm-cm silicon solar cells, 0.2 mm thick, were produced with short circuit current efficiencies up to thirteen percent and using a combination of recent technical advances. The cells were fabricated in conventional and wraparound contact configurations. Improvement in cell collection efficiency from both the short and long wavelengths region of the solar spectrum was obtained by coupling a shallow junction and an optically transparent antireflection coating with back surface field technology. Both boron diffusion and aluminum alloying techniques were evaluated for forming back surface field cells. The latter method is less complicated and is compatible with wraparound cell processing.

Small organelle, big responsibility: the role of centrosomes in development and disease.

PubMed

Chavali, Pavithra L; Pütz, Monika; Gergely, Fanni

2014-09-05

The centrosome, a key microtubule organizing centre, is composed of centrioles, embedded in a protein-rich matrix. Centrosomes control the internal spatial organization of somatic cells, and as such contribute to cell division, cell polarity and migration. Upon exiting the cell cycle, most cell types in the human body convert their centrioles into basal bodies, which drive the assembly of primary cilia, involved in sensing and signal transduction at the cell surface. Centrosomal genes are targeted by mutations in numerous human developmental disorders, ranging from diseases exclusively affecting brain development, through global growth failure syndromes to diverse pathologies associated with ciliary malfunction. Despite our much-improved understanding of centrosome function in cellular processes, we know remarkably little of its role in the organismal context, especially in mammals. In this review, we examine how centrosome dysfunction impacts on complex physiological processes and speculate on the challenges we face when applying knowledge generated from in vitro and in vivo model systems to human development. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Bioactive Surface Modification of Hydroxyapatite

PubMed Central

Okazaki, Yohei; Hiasa, Kyou; Yasuda, Keisuke; Nogami, Keisuke; Mizumachi, Wataru; Hirata, Isao

2013-01-01

The purpose of this study was to establish an acid-etching procedure for altering the Ca/P ratio of the nanostructured surface of hydroxyapatite (HAP) by using surface chemical and morphological analyses (XPS, XRD, SEM, surface roughness, and wettability) and to evaluate the in vitro response of osteoblast-like cells (MC3T3-E1 cells) to the modified surfaces. This study utilized HAP and HAP treated with 10%, 20%, 30%, 40%, 50%, or 60% phosphoric acid solution for 10 minutes at 25°C, followed by rinsing 3 times with ultrapure water. The 30% phosphoric acid etching process that provided a Ca/P ratio of 1.50, without destruction of the grain boundary of HAP, was selected as a surface-modification procedure. Additionally, HAP treated by the 30% phosphoric acid etching process was stored under dry conditions at 25°C for 12 hours, and the Ca/P ratio approximated to 1.00 accidentally. The initial adhesion, proliferation, and differentiation (alkaline phosphatase (ALP) activity and relative mRNA level for ALP) of MC3T3-E1 cells on the modified surfaces were significantly promoted (P < 0.05 and 0.01). These findings show that the 30% phosphoric acid etching process for the nanostructured HAP surface can alter the Ca/P ratio effectively and may accelerate the initial adhesion, proliferation, and differentiation of MC3T3-E1 cells. PMID:23862150

Concepts for thin-film GaAs concentrator cells. [for solar photovoltaic space power systems

NASA Technical Reports Server (NTRS)

Spitzer, M. B.; Gale, R. P.; Mcclelland, R.; King, B.; Dingle, J.

1989-01-01

The development of advanced GaAs concentrator solar cells, and in particular, the use of CLEFT (cleavage of lateral epitaxial films for transfer) processes for formation of thin-film structures is reported. The use of CLEFT has made possible processing of the back, and cells with back surface grids are discussed. Data on patterned junction development are presented; such junctions are expected to be useful in back surface applications requiring point contacts, grating structures, and interdigitated back contacts. CLEFT concentrator solar cells with grids on the front and back surfaces are reported here; these cells are 4 microns thick and are bonded to glass covers for support. Air mass zero efficiency of 18.8 percent has been obtained for a CLEFT concentrator operating at 18.5 suns.

Chimeric Antigen Receptor Therapy for Cancer

PubMed Central

Barrett, David M.; Singh, Nathan; Porter, David L.; Grupp, Stephan A.; June, Carl H.

2014-01-01

Improved outcomes for patients with cancer hinge on the development of new targeted therapies with acceptable short-term and long-term toxicity. Progress in basic, preclinical, and clinical arenas spanning cellular immunology, synthetic biology, and cell-processing technologies has paved the way for clinical applications of chimeric antigen receptor– based therapies. This new form of targeted immunotherapy merges the exquisite targeting specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence provided by cytotoxic T cells. Although this field is still in its infancy, clinical trials have already shown clinically significant antitumor activity in neuroblastoma, chronic lymphocytic leukemia, and B cell lymphoma, and trials targeting a variety of other adult and pediatric malignancies are under way. Ongoing work is focused on identifying optimal tumor targets and on elucidating and manipulating both cell- and host-associated factors to support expansion and persistence of the genetically engineered cells in vivo. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. PMID:24274181

Systemic surfaceome profiling identifies target antigens for immune-based therapy in subtypes of advanced prostate cancer

PubMed Central

Lee, John K.; Bangayan, Nathanael J.; Chai, Timothy; Smith, Bryan A.; Pariva, Tiffany E.; Yun, Sangwon; Vashisht, Ajay; Zhang, Qingfu; Park, Jung Wook; Corey, Eva; Huang, Jiaoti; Wohlschlegel, James; Witte, Owen N.

2018-01-01

Prostate cancer is a heterogeneous disease composed of divergent molecular and histologic subtypes, including prostate adenocarcinoma (PrAd) and neuroendocrine prostate cancer (NEPC). While PrAd is the major histology in prostate cancer, NEPC can evolve from PrAd as a mechanism of treatment resistance that involves a transition from an epithelial to a neurosecretory cancer phenotype. Cell surface markers are often associated with specific cell lineages and differentiation states in normal development and cancer. Here, we show that PrAd and NEPC can be broadly discriminated by cell-surface profiles based on the analysis of prostate cancer gene expression datasets. To overcome a dependence on predictions of human cell-surface genes and an assumed correlation between mRNA levels and protein expression, we integrated transcriptomic and cell-surface proteomic data generated from a panel of prostate cancer cell lines to nominate cell-surface markers associated with these cancer subtypes. FXYD3 and CEACAM5 were validated as cell-surface antigens enriched in PrAd and NEPC, respectively. Given the lack of effective treatments for NEPC, CEACAM5 appeared to be a promising target for cell-based immunotherapy. As a proof of concept, engineered chimeric antigen receptor T cells targeting CEACAM5 induced antigen-specific cytotoxicity in NEPC cell lines. Our findings demonstrate that the surfaceomes of PrAd and NEPC reflect unique cancer differentiation states and broadly represent vulnerabilities amenable to therapeutic targeting. PMID:29686080

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 quantification, dynamic cell tracking, and migration quantification following varied drug treatments. This system provides a versatile platform to study collective cell migration in high throughput for a broad range of applications.

A novel form of epithelial wound healing of the embryonic epidermis.

PubMed

Armstrong, Margaret T; Turlo, Kirsten; Elges, Chris J; Dayton, Sarah M; Lee, Janet; Armstrong, Peter B

2006-08-01

The embryonic epidermis of amniotes is a two-cell layer sheet with a periderm positioned superficial to the basal cell layer which, itself, attaches apically to the basal surface of the periderm and basally to the basal lamina. The presence of the periderm is essential to maintain the basal layer as a two-dimensional monolayer. Wounds to the epidermis that remove selectively just the periderm are healed by a stacking of the basal layer cells that constitute the wound bed. Basal cell stacking involves the desertion of the basal lamina by many of the cells so as to increase their contact area with other basal layer cells. This suggests that a preferential adhesion to the planar basal lamina is not important for the monolayered organization of the basal layer but, instead, association with inner surface of the planar periderm is the principal process that maintains the basal layer as a monolayer. The conversion of the basal layer from monolayer to multilayer during wound healing diminishes its planar area, resulting in movement of the wound borders toward the center of the wound. This is a novel scenario for wound healing.

Development of high efficiency thin film polycrystalline silicon solar cells using VEST process

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

Ishihara, T.; Arimoto, S.; Morikawa, H.

1998-12-31

Thin film Si solar cell has been developed using Via-hole Etching for the Separation of Thin films (VEST) process. The process is based on SOI technology of zone-melting recrystallization (ZMR) followed by chemical vapor deposition (CVD), separation of thin film, and screen printing. Key points for achieving high efficiency are (1) quality of Si films, (2) back surface emitter (BSE), (3) front surface emitter etch-back process, (4) back surface field (BSF) layer thickness and its resistivity, and (5) defect passivation by hydrogen implantation. As a result of experiments, the authors have achieved 16% efficiency (V{sub oc}:0.589V, J{sub sc}:35.6mA/cm{sup 2}, F,F:0.763)more » with a cell size of 95.8cm{sup 2} and the thickness of 77 {micro}m. It is the highest efficiency ever reported for large area thin film Si solar cells.« less

Post-adsorption process of Yb phosphate nano-particle formation by Saccharomyces cerevisiae

NASA Astrophysics Data System (ADS)

Jiang, MingYu; Ohnuki, Toshihiko; Tanaka, Kazuya; Kozai, Naofumi; Kamiishi, Eigo; Utsunomiya, Satoshi

2012-09-01

In this study, we have investigated the post-adsorption process of ytterbium (Yb) phosphate nano-particle formation by Saccharomyces cerevisiae (yeast). The yeast grown in P-rich medium were exposed to 1.44 × 10-4 mol/L Yb(III) solution for 2-120 h, and 2 months at 25 ± 1 °C at an initial pH of 3, 4, or 5, respectively. Ytterbium concentrations in solutions decreased as a function of exposure time. Field-emission scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy (FESEM), transmission electron microscopy (TEM), and synchrotron-based extended X-ray absorption fine structure (EXAFS) analyses revealed that nano-sized blocky Yb phosphate with an amorphous phase formed on the yeast cells surfaces in the solutions with Yb. These nano-sized precipitates that formed on the cell surfaces remained stable even after 2 months of exposure at 25 ± 1 °C around neutral pHs. The EXAFS data revealed that the chemical state of the accumulated Yb on the cell surfaces changed from the adsorption on both phosphate and carboxyl sites at 30 min to Yb phosphate precipitates at 5 days, indicating the Yb-phosphate precipitation as a major post-adsorption process. In addition, the precipitation of Yb phosphate occurred on cell surfaces during 7 days of exposure in Yb-free solution after 2 h of exposure (short-term Yb adsorption) in Yb solution. These results suggest that the released P from the inside of yeast cells reacted with adsorbed Yb on cell surfaces, resulting in the formation of Yb precipitates, even though no P was added to the exposure solution. In an abiotic system, the EXAFS data showed that the speciation of sorbed Yb on the reference materials, carboxymethyl cellulose and Ln resin, did not change even when the Yb was exposed to P solution, without forming Yb phosphate precipitates. This result strongly suggests that the cell surface of the yeast plays an important role in the Yb-phosphate precipitation process, not only as a carrier of the functional groups but also as a substrate inducing the nucleation of phosphate nanoparticles. Stable nano-sized Yb phosphate precipitates formed on yeast cell surfaces in the present study, which implies that this post-adsorption nano-particle formation process caused by microbial cells should be one of the important processes governing the long-term migration of heavy rare earth elements and presumably trivalent actinides in geological repository.

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.

RAFTK, a novel member of the focal adhesion kinase family, is phosphorylated and associates with signaling molecules upon activation of mature T lymphocytes.

PubMed

Ganju, R K; Hatch, W C; Avraham, H; Ona, M A; Druker, B; Avraham, S; Groopman, J E

1997-03-17

The related adhesion focal tyrosine kinase (RAFTK), a recently discovered member of the focal adhesion kinase family, has previously been reported to participate in signal transduction in neuronal cells, megakaryocytes, and B lymphocytes. We have found that RAFTK is constitutively expressed in human T cells and is rapidly phosphorylated upon the activation of the T cell receptor (TCR). This activation also results in an increase in the autophosphorylation and kinase activity of RAFTK. After its stimulation, there was an increase in the association of the src cytoplasmic tyrosine kinase Fyn and the adapter protein Grb2. This association was mediated through the SH2 domains of Fyn and Grb2. RAFTK also co-immunoprecipitates with the SH2 domain of Lck and with the cytoskeletal protein paxillin through its COOH-terminal proline-rich domain. The tyrosine phosphorylation of RAFTK after T cell receptor-mediated stimulation was reduced by the pretreatment of cells with cytochalasin D, suggesting the role of the cytoskeleton in this process. These observations indicate that RAFTK participates in T cell receptor signaling and may act to link signals from the cell surface to the cytoskeleton and thereby affect the host immune response.

Novel INTeraction of MUC4 and galectin: potential pathobiological implications for metastasis in lethal pancreatic cancer.

PubMed

Senapati, Shantibhusan; Chaturvedi, Pallavi; Chaney, William G; Chakraborty, Subhankar; Gnanapragassam, Vinayaga S; Sasson, Aaron R; Batra, Surinder K

2011-01-15

Several studies have reported aberrant expression of MUC4 in pancreatic cancer (PC), which is associated with tumorigenicity and metastasis. Mechanisms through which MUC4 promote metastasis of PC cells to distant organs are poorly defined. Identification of MUC4-galectin-3 interaction and its effect on the adhesion of cancer cells to endothelial cells were done by immunoprecipitation and cell-cell adhesion assays, respectively. Serum galectin-3 level for normal and PC patients were evaluated through ELISA. In the present study, we have provided clinical evidence that the level of galectin-3 is significantly elevated in the sera of PC patients with metastatic disease compared with patients without metastasis (P = 0.04) and healthy controls (P = 0.00001). Importantly, for the first time, we demonstrate that MUC4 present on the surface of circulating PC cells plays a significant role in the transient and reversible attachment (docking) of circulating tumor cells to the surface of endothelial cells. Further, exogenous galectin-3 at concentrations similar to that found in the sera of PC patients interacts with MUC4 via surface glycans such as T antigens, which results in the clustering of MUC4 on the cell surface and a stronger attachment (locking) of circulating tumor cells to the endothelium. Altogether, these findings suggest that PC cell-associated MUC4 helps in the docking of tumor cells on the endothelial surface. During cancer progression, MUC4-galectin-3 interaction-mediated clustering of MUC4 may expose the surface adhesion molecules, which in turn promotes a stronger attachment (locking) of tumor cells to the endothelial surface. ©2010 AACR.

Silica-gelatin hybrid sol-gel coatings: a proteomic study with biocompatibility implications.

PubMed

Araújo-Gomes, N; Romero-Gavilán, F; Lara-Sáez, I; Elortza, F; Azkargorta, M; Iloro, I; Martínez-Ibañez, M; Martín de Llano, J J; Gurruchaga, M; Goñi, I; Suay, J; Sánchez-Pérez, A M

2018-05-21

Osseointegration, including the foreign body reaction to biomaterials, is an immune-modulated, multifactorial, and complex healing process in which various cells and mediators are involved. The buildup of the osseointegration process is immunological and inflammation-driven, often triggered by the adsorption of proteins on the surfaces of the biomaterials and complement activation. New strategies for improving osseointegration use coatings as vehicles for osteogenic biomolecules delivery from implants. Natural polymers, such as gelatin, can mimic collagen I and enhance the biocompatibility of a material. In this experimental study, two different base sol-gel formulations and their combination with gelatin, were applied as coatings on sandblasted, acid-etched titanium (SAE-Ti) substrates and their biological potential as osteogenic biomaterials was tested. We examined the proteins adsorbed onto each surface and their in vitro and in vivo effects. In vitro results showed an improvement in cell proliferation and mineralization in gelatin-containing samples. In vivo testing showed the presence of a looser connective tissue layer in those coatings with substantially more complement activation proteins adsorbed, especially those containing gelatin. Vitronectin and FETUA, proteins associated with mineralization process, were significantly more adsorbed in gelatin coatings. This article is protected by copyright. All rights reserved.

Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples

NASA Technical Reports Server (NTRS)

Sundaram, Shivshankar; Prabhakarpandian, Balabhaskar; Pant, Kapil; Wang, Yi

2014-01-01

RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads in a shape-optimized chamber. A secondary proprietary feature is in the particular layout integrating these components to perform the desired operation of RNA isolation. Apart from a novel functional capability, advantages of the innovation include reduced or eliminated use of toxic reagents, and operator-independent extraction of RNA.

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.

Receptor revision in CD4 T cells is influenced by follicular helper T cell formation and germinal-center interactions.

PubMed

Higdon, Lauren E; Deets, Katherine A; Friesen, Travis J; Sze, Kai-Yin; Fink, Pamela J

2014-04-15

Peripheral CD4 T cells in Vβ5 transgenic (Tg) C57BL/6J mice undergo tolerance to an endogenous superantigen encoded by mouse mammary tumor virus 8 (Mtv-8) by either deletion or T-cell receptor (TCR) revision. Revision is a process by which surface expression of the Vβ5(+) TCR is down-regulated in response to Mtv-8 and recombination activating genes are expressed to drive rearrangement of the endogenous TCRβ locus, effecting cell rescue through the expression of a newly generated, non-self-reactive TCR. In an effort to identify the microenvironment in which revision takes place, we show here that the proportion of T follicular helper cells (Tfh) and production of high-affinity antibody during a primary response are increased in Vβ5 Tg mice in an Mtv-8-dependent manner. Revising T cells have a Tfh-like surface phenotype and transcription factor profile, with elevated expression of B-cell leukemia/lymphoma 6 (Bcl-6), CXC chemokine receptor 5, programmed death-1, and other Tfh-associated markers. Efficient revision requires Bcl-6 and is inhibited by B lymphocyte-induced maturation protein-1. Revision completes less efficiently in the absence of signaling lymphocytic activation molecule-associated protein although initiation proceeds normally. These data indicate that Tfh formation is required for the initiation of revision and germinal-center interactions for its completion. The germinal center is known to provide a confined space in which B-cell antigen receptors undergo selection. Our data extend the impact of this selective microenvironment into the arena of T cells, suggesting that this fluid structure also provides a regulatory environment in which TCR revision can safely take place.

Record high efficiency of screen-printed silicon aluminum back surface field solar cell: 20.29%

NASA Astrophysics Data System (ADS)

Kim, Ki Hyung; Park, Chang Sub; Doo Lee, Jae; Youb Lim, Jong; Yeon, Je Min; Kim, Il Hwan; Lee, Eun Joo; Cho, Young Hyun

2017-08-01

We have achieved a record high cell efficiency of 20.29% for an industrial 6-in. p-type monocrystalline silicon solar cell with a full-area aluminum back surface field (Al-BSF) by simply modifying the cell structure and optimizing the process with the existing cell production line. The cell efficiency was independently confirmed by the Solar Energy Research Institute of Singapore (SERIS). To increase the cell efficiency, for example, in four busbars, double printing, a lightly doped emitter with a sheet resistance of 90 to 100 Ω/□, and front surface passivation by using silicon oxynitride (SiON) on top of a silicon nitride (SiN x ) antireflection layer were adopted. To optimize front side processing, PC1D simulation was carried out prior to cell fabrication. The resulting efficiency gain is 0.64% compared with that in the reference cells with three busbars, a single antireflection coating layer, and a low-sheet-resistance emitter.

Processes affecting the transport of Cryptosporidium parvum and other persistent pathogens in surface- and ground-waters

NASA Astrophysics Data System (ADS)

Packman, A. I.; Lau, B. L.; Harter, T.; Atwill, E. R.

2007-12-01

Waterborne diseases are transmitted through numerous environmental pathways, and their migration is strongly mediated by interaction with a wide variety of sediments and other natural materials during transport. Here we provide an overview of factors that affect the fate of persistent water-borne pathogens, focusing particularly on the zoonotic pathogen Cryptosporidium parvum as an example. While individual microbial cells are both small and have low specific gravity, suggesting that they should be highly mobile and remain suspended for long periods of time, attachment to a variety of background materials can substantially reduce pathogen mobility. Cryptosporidium oocysts readily associate with both inorganic and organic particles, resulting in the formation of aggregates. This process tends to increase the effective settling velocity of C. parvum in surface waters. Similarly, pathogens readily become associated with the solid matrix during transport in groundwater, resulting in removal by filtration. However, this process is reversible with C. parvum, resulting in a slow long-term release following the initial deposition. Pathogens also become associated with biofilms, which are surface-attached communities of microorganisms in a gelatinous matrix. The presence of biofilms increases the immobilization and retention of Cryptosporidium on solid surfaces. All of these processes influence pathogen transmission in surface waters such as rivers and water-supply canals. In these environments, pathogens can be immobilized by deposition into stable sediment beds by a combination of gravitational sedimentation and advection into pore waters followed by subsurface filtration. Association with background suspended matter tends to increase pathogen deposition by sedimentation, and the presence of benthic (sedimentary) biofilms also tends to increase pathogen retention. For pathogens that remain viable for long periods of time in natural aquatic systems, as is the case with Cryptosporidium and other cyst-and spore-forming organisms, then the sediments and sedimentary biofilms become an environmental reservoir of pathogens. Cysts retained in biofilms appear to be relatively difficult to resuspend, but slow, long-term biological release and high-flow events that mobilize streambed sediments both deliver pathogens into transport.

A Morphological and Cytochemical Study of the Interaction between Paracoccidiodes brasiliensis and Neutrophils

NASA Astrophysics Data System (ADS)

Dias, Maria Fernanda R. G.; Filgueira, Absalom L.; de Souza, Wanderley

2004-04-01

Paracoccidioidomycosis is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. It is the most prevalent systemic mycosis of Latin America and 80% of the reported cases are from Brazil. Because of the great number of neutrophils found in the P. brasiliensis granuloma, studies have been done to evaluate the role of these cells during the development of the infection. Scanning and transmission electron microscopy of thin sections showed that the neutrophils ingest yeast cells through a typical phagocytic process with the formation of pseudopodes. The pseudopodes even disrupt the connection established between the mother and the bud cells. Neutrophils also associate to each other, forming a kind of extracellular vacuole where large yeast cells are encapsulated. Cytochemical studies showed that once P. brasiliensis attaches to the neutrophil surface, it triggers a respiratory burst with release of oxygen-derived products. Attachment also triggers neutrophils' degranulation, with release of endogenous peroxidase localized in cytoplasmic granules. Together, these processes lead to killing of both ingested and extracellular P. brasiliensis.

Chlorine-rich plasma polymer coating for the prevention of attachment of pathogenic fungal cells onto materials surfaces

NASA Astrophysics Data System (ADS)

Lamont-Friedrich, Stephanie J.; Michl, Thomas D.; Giles, Carla; Griesser, Hans J.; Coad, Bryan R.

2016-07-01

The attachment of pathogenic fungal cells onto materials surfaces, which is often followed by biofilm formation, causes adverse consequences in a wide range of areas. Here we have investigated the ability of thin film coatings from chlorinated molecules to deter fungal colonization of solid materials by contact killing of fungal cells reaching the surface of the coating. Coatings were deposited onto various substrate materials via plasma polymerization, which is a substrate-independent process widely used for industrial coating applications, using 1,1,2-trichloroethane as the process vapour. XPS surface analysis showed that the coatings were characterized by a highly chlorinated hydrocarbon polymer nature, with only a very small amount of oxygen incorporated. The activity of these coatings against human fungal pathogens was quantified using a recently developed, modified yeast assay and excellent antifungal activity was observed against Candida albicans and Candida glabrata. Plasma polymer surface coatings derived from chlorinated hydrocarbon molecules may therefore offer a promising solution to preventing yeast and mould biofilm formation on materials surfaces, for applications such as air conditioners, biomedical devices, food processing equipment, and others.

Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

PubMed

Gadelha, Catarina; Zhang, Wenzhu; Chamberlain, James W; Chait, Brian T; Wickstead, Bill; Field, Mark C

2015-07-01

Surface membrane organization and composition is key to cellular function, and membrane proteins serve many essential roles in endocytosis, secretion, and cell recognition. The surface of parasitic organisms, however, is a double-edged sword; this is the primary interface between parasites and their hosts, and those crucial cellular processes must be carried out while avoiding elimination by the host immune defenses. For extracellular African trypanosomes, the surface is partitioned such that all endo- and exocytosis is directed through a specific membrane region, the flagellar pocket, in which it is thought the majority of invariant surface proteins reside. However, very few of these proteins have been identified, severely limiting functional studies, and hampering the development of potential treatments. Here we used an integrated biochemical, proteomic and bioinformatic strategy to identify surface components of the human parasite Trypanosoma brucei. This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival. Molecules with receptor-like properties are almost exclusively parasite-specific, whereas transporter-like proteins are conserved in model organisms. Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket. Moreover, the largest systematic analysis of trypanosome surface molecules to date provides evidence that the cell surface is compartmentalized into three distinct domains with free diffusion of molecules in each, but selective, asymmetric traffic between. This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Method of making a back contacted solar cell

DOEpatents

Gee, J.M.

1995-11-21

A back-contacted solar cell is described having laser-drilled vias connecting the front-surface carrier-collector junction to an electrode grid on the back surface. The structure may also include a rear surface carrier-collector junction connected to the same grid. The substrate is connected to a second grid which is interdigitated with the first. Both grids are configured for easy series connection with neighboring cells. Several processes are disclosed to produce the cell. 2 figs.

Oxidative Processing of Latent Fas in the Endoplasmic Reticulum Controls the Strength of Apoptosis

PubMed Central

Anathy, Vikas; Roberson, Elle; Cunniff, Brian; Nolin, James D.; Hoffman, Sidra; Spiess, Page; Guala, Amy S.; Lahue, Karolyn G.; Goldman, Dylan; Flemer, Stevenson; van der Vliet, Albert; Heintz, Nicholas H.; Budd, Ralph C.; Tew, Kenneth D.

2012-01-01

We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241–252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis. PMID:22751926

Components of the Engulfment Machinery Have Distinct Roles in Corpse Processing

PubMed Central

Meehan, Tracy L.; Joudi, Tony F.; Timmons, Allison K.; Taylor, Jeffrey D.; Habib, Corey S.; Peterson, Jeanne S.; Emmanuel, Shanan; Franc, Nathalie C.; McCall, Kimberly

2016-01-01

Billions of cells die in our bodies on a daily basis and are engulfed by phagocytes. Engulfment, or phagocytosis, can be broken down into five basic steps: attraction of the phagocyte, recognition of the dying cell, internalization, phagosome maturation, and acidification. In this study, we focus on the last two steps, which can collectively be considered corpse processing, in which the engulfed material is degraded. We use the Drosophila ovarian follicle cells as a model for engulfment of apoptotic cells by epithelial cells. We show that engulfed material is processed using the canonical corpse processing pathway involving the small GTPases Rab5 and Rab7. The phagocytic receptor Draper is present on the phagocytic cup and on nascent, phosphatidylinositol 3-phosphate (PI(3)P)- and Rab7-positive phagosomes, whereas integrins are maintained on the cell surface during engulfment. Due to the difference in subcellular localization, we investigated the role of Draper, integrins, and downstream signaling components in corpse processing. We found that some proteins were required for internalization only, while others had defects in corpse processing as well. This suggests that several of the core engulfment proteins are required for distinct steps of engulfment. We also performed double mutant analysis and found that combined loss of draper and αPS3 still resulted in a small number of engulfed vesicles. Therefore, we investigated another known engulfment receptor, Crq. We found that loss of all three receptors did not inhibit engulfment any further, suggesting that Crq does not play a role in engulfment by the follicle cells. A more complete understanding of how the engulfment and corpse processing machinery interact may enable better understanding and treatment of diseases associated with defects in engulfment by epithelial cells. PMID:27347682

Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity.

PubMed

Tauber, Svantje; Lauber, Beatrice A; Paulsen, Katrin; Layer, Liliana E; Lehmann, Martin; Hauschild, Swantje; Shepherd, Naomi R; Polzer, Jennifer; Segerer, Jürgen; Thiel, Cora S; Ullrich, Oliver

2017-01-01

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable "steady state" after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions.

Surface Interactions between Escherichia coli and Hemocytes of the Mediterranean Mussel Mytilus galloprovincialis Lam. Leading to Efficient Bacterial Clearance

PubMed Central

Canesi, Laura; Pruzzo, Carla; Tarsi, Renato; Gallo, Gabriella

2001-01-01

The role of type 1 fimbriae in the interactions between Escherichia coli and Mytilus galloprovincialis Lam. hemocytes was evaluated. The association of fimbriated strain MG155 with hemocyte monolayers at 18°C was 1.5- and 3- to 4-fold greater than the association of unfimbriated mutant AAEC072 in artificial seawater and in hemolymph serum, respectively. Such differences were apparently due to different adhesive properties since MG155 adhered more efficiently than AAEC072 when hemocytes were incubated at 4°C to inhibit the internalization process. Hemolymph serum increased both association and adherence of MG155 two- to threefold but did not affect association and adherence of AAEC072. MG155 was also 1.5- to 1.7-fold more sensitive to killing by hemocytes than AAEC072, as evaluated by the number of culturable bacteria after 60 and 120 min of incubation. The role of type 1 fimbriae in MG155 interactions with hemocytes was confirmed by the inhibitory effect of d-mannose. In in vivo experiments MG155 cells were cleared from circulating hemolymph more rapidly than AAEC072 cells were cleared. These results confirm that surface properties are crucial in influencing bacterial persistence and survival within mussel hemolymph. PMID:11133482

Cellular and Molecular Pathways Leading to External Root Resorption

PubMed Central

Iglesias-Linares, A.; Hartsfield, J.K.

2016-01-01

External apical root resorption during orthodontic treatment implicates specific molecular pathways that orchestrate nonphysiologic cellular activation. To date, a substantial number of in vitro and in vivo molecular, genomic, and proteomic studies have supplied data that provide new insights into root resorption. Recent mechanisms and developments reviewed here include the role of the cellular component—specifically, the balance of CD68+, iNOS+ M1- and CD68+, CD163+ M2-like macrophages associated with root resorption and root surface repair processes linked to the expression of the M1-associated proinflammatory cytokine tumor necrosis factor, inducible nitric oxide synthase, the M1 activator interferon γ, the M2 activator interleukin 4, and M2-associated anti-inflammatory interleukin 10 and arginase I. Insights into the role of mesenchymal dental pulp cells in attenuating dentin resorption in homeostasis are also reviewed. Data on recently deciphered molecular pathways are reviewed at the level of (1) clastic cell adhesion in the external apical root resorption process and the specific role of α/β integrins, osteopontin, and related extracellular matrix proteins; (2) clastic cell fusion and activation by the RANKL/RANK/OPG and ATP-P2RX7-IL1 pathways; and (3) regulatory mechanisms of root resorption repair by cementum at the proteomic and transcriptomic levels. PMID:27811065

Lactate signalling regulates fungal β-glucan masking and immune evasion

PubMed Central

Ballou, Elizabeth R.; Avelar, Gabriela M.; Childers, Delma S.; Mackie, Joanna; Bain, Judith M.; Wagener, Jeanette; Kastora, Stavroula L.; Panea, Mirela D.; Hardison, Sarah E.; Walker, Louise A.; Erwig, Lars P.; Munro, Carol A.; Gow, Neil A.R.; Brown, Gordon D.; MacCallum, Donna M.; Brown, Alistair J.P.

2017-01-01

Summary Paragraph As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno-competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key Pathogen Associated Molecular Pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signaling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell wall related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system. PMID:27941860

Gene Silencing of Human Neuronal Cells for Drug Addiction Therapy using Anisotropic Nanocrystals

PubMed Central

Law, Wing-Cheung; Mahajan, Supriya D.; Kopwitthaya, Atcha; Reynolds, Jessica L.; Liu, Maixian; Liu, Xin; Chen, Guanying; Erogbogbo, Folarin; Vathy, Lisa; Aalinkeel, Ravikumar; Schwartz, Stanley A.; Yong, Ken-Tye; Prasad, Paras N.

2012-01-01

Theranostic platform integrating diagnostic imaging and therapeutic function into a single system has become a new direction of nanoparticle research. In the process of treatment, therapeutic efficacy is monitored. The use of theranostic nanoparticle can add an additional "layer" to keep track on the therapeutic agent such as the pharmacokinetics and biodistribution. In this report, we have developed quantum rod (QR) based formulations for the delivery of small interfering RNAs (siRNAs) to human neuronal cells. PEGlyated QRs with different surface functional groups (amine and maleimide) were designed for selectively down-regulating the dopaminergic signaling pathway which is associated with the drug abuse behavior. We have demonstrated that the DARPP-32 siRNAs were successfully delivered to dopaminergic neuronal (DAN) cells which led to drastic knockdown of specific gene expression by both the electrostatic and covalent bond conjugation regimes. The PEGlyated surface offered high biocompatibilities and negligible cytotoxicities to the QR formulations that may facilitate the in vivo applications of these nanoparticles. PMID:22896771

Anionic lipids and the maintenance of membrane electrostatics in eukaryotes

PubMed Central

Platre, Matthieu Pierre

2017-01-01

ABSTRACT A wide range of signaling processes occurs at the cell surface through the reversible association of proteins from the cytosol to the plasma membrane. Some low abundant lipids are enriched at the membrane of specific compartments and thereby contribute to the identity of cell organelles by acting as biochemical landmarks. Lipids also influence membrane biophysical properties, which emerge as an important feature in specifying cellular territories. Such parameters are crucial for signal transduction and include lipid packing, membrane curvature and electrostatics. In particular, membrane electrostatics specifies the identity of the plasma membrane inner leaflet. Membrane surface charges are carried by anionic phospholipids, however the exact nature of the lipid(s) that powers the plasma membrane electrostatic field varies among eukaryotes and has been hotly debated during the last decade. Herein, we discuss the role of anionic lipids in setting up plasma membrane electrostatics and we compare similarities and differences that were found in different eukaryotic cells. PMID:28102755

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

Finegan, Donal P.; Tjaden, Bernhard; M. M. Heenan, Thomas

Mechanical abuse of lithium-ion batteries is widely used during testing to induce thermal runaway, characterize associated risks, and expose cell and module vulnerabilities. But, the repeatability of puncture or 'nail penetration' tests is a key issue as there is often a high degree of variability in the resulting thermal runaway process. Here, the failure mechanisms of 18650 cells punctured at different locations and orientations are characterized with respect to their internal structural degradation, and both their internal and surface temperature, all of which are monitored in real time. The initiation and propagation of thermal runaway is visualized via high-speed synchrotronmore » X-ray radiography at 2000 frames per second, and the surface and internal temperatures are recorded via infrared imaging and a thermocouple embedded in the tip of the penetrating nail, respectively. The influence of the nail, as well as how and where it penetrates the cell, on the initiation and propagation of thermal runaway is described and the suitability of this test method for representing in-field failures is discussed.« less

The Cryptococcus neoformans Capsule: a Sword and a Shield

PubMed Central

O'Meara, Teresa R.

2012-01-01

Summary: The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell. PMID:22763631

Zinc Oxide Grown by CVD Process as Transparent Contact for Thin Film Solar Cell Applications

NASA Astrophysics Data System (ADS)

Faÿ, S.; Shah, A.

Metalorganic chemical vapor deposition of ZnO films (MOCVD) [1] started to be comprehensively investigated in the 1980s, when thin film industries were looking for ZnO deposition processes especially useful for large-scale coatings at high growth rates. Later on, when TCO for thin film solar cells started to be developed, another advantage of growing TCO films by the CVD process has been highlighted: the surface roughness. Indeed, a large number of studies on CVD ZnO revealed that an as-grown rough surface cn be obtained with this deposition process [2-4]. A rough surface induces a light scattering effect, which can significantly improve light trapping (and therefore current photo-generation) within thin film silicon solar cells. The CVD process, indeed, directly leads to as-grown rough ZnO films without any post-etching step (the latter is often introduced to obtain a rough surface, when working with as-deposited flat sputtered ZnO). This fact could turn out to be a significant advantage when upscaling the manufacturing process for actual commercial production of thin film solar modules. The zinc and oxygen sources for CVD growth of ZnO films are given in Table 6.1.

Effect of surface pre-treatments on biocompatibility of magnesium.

PubMed

Lorenz, Carla; Brunner, Johannes G; Kollmannsberger, Philip; Jaafar, Leila; Fabry, Ben; Virtanen, Sannakaisa

2009-09-01

This study reports the influence of Mg surface passivation on the survival rate of human HeLa cells and mouse fibroblasts in cell culture experiments. Polished samples of commercially pure Mg show high reactivity in the cell culture medium, leading to a pH shift in the alkaline direction, and therefore cell adhesion and survival is strongly impaired. Passivation of the Mg surface in 1M NaOH can strongly enhance cell survival. The best initial cell adhesion is observed for Mg samples incubated in simulated body fluid (M-SBF), which leads to the formation of a biomimetic, amorphous Ca/Mg-phosphate layer with high surface roughness. This surface layer, however, passivates and seals the Mg surface only partially. Subsequent Mg dissolution leads to a significantly stronger pH increase compared to NaOH-passivated samples, which prevents long-term cell survival. These results demonstrate that surface passivation with NaOH and M-SBF together with the associated changes of surface reactivity, chemistry and roughness provide a viable strategy to facilitate cell survival on otherwise non-biocompatible Mg surfaces.

HLA-A11-mediated protection from NK cell-mediated lysis: role of HLA-A11-presented peptides.

PubMed

Gavioli, R; Zhang, Q J; Masucci, M G

1996-08-01

The capacity of MHC class I to protect target cells from NK is well established, but the mechanism by which these molecules influence NK recognition and the physical properties associated with this function remain poorly defined. We have examined this issue using as a model the HLA-A11 allele. HLA-A11 expression correlated with reduced susceptibility to NK and interferon-activated cytotoxicity in transfected sublines of the A11-defective Burkitt's lymphoma WW2-BL and the HLA class I A,B-null C1R cell line. Protection was also achieved by transfection of HLA-A11 in the peptide processing mutant T2 cells line (T2/A11), despite a very low expression of the transfected product at the cell surface. Induction of surface HLA-A11 by culture of T2/A11 cells at 26 degrees C or in the presence of beta 2m did not affect lysis, whereas NK sensitivity was restored by culture in the presence of HLA-All-binding synthetic peptides derived from viral or cellular proteins. Acid treatment rendered T2/A11 and C1R/A11 cells sensitive to lysis, but protection was restored after preincubation with peptide preparations derived from surface stripping of T2/A11 cells. Similar peptide preparations from T2 cells had no effect. The results suggest that NK protection is mediated by HLA-A11 molecules carrying a particular set of peptides that are translocated to the site of MHC class I assembly in the ER in a TAP-independent fashion.

Tracking Internal Temperature and Structural Dynamics during Nail Penetration of Lithium-Ion Cells

DOE PAGES

Finegan, Donal P.; Tjaden, Bernhard; M. M. Heenan, Thomas; ...

2017-10-31

Mechanical abuse of lithium-ion batteries is widely used during testing to induce thermal runaway, characterize associated risks, and expose cell and module vulnerabilities. But, the repeatability of puncture or 'nail penetration' tests is a key issue as there is often a high degree of variability in the resulting thermal runaway process. Here, the failure mechanisms of 18650 cells punctured at different locations and orientations are characterized with respect to their internal structural degradation, and both their internal and surface temperature, all of which are monitored in real time. The initiation and propagation of thermal runaway is visualized via high-speed synchrotronmore » X-ray radiography at 2000 frames per second, and the surface and internal temperatures are recorded via infrared imaging and a thermocouple embedded in the tip of the penetrating nail, respectively. The influence of the nail, as well as how and where it penetrates the cell, on the initiation and propagation of thermal runaway is described and the suitability of this test method for representing in-field failures is discussed.« less

Automated fabrication of back surface field silicon solar cells with screen printed wraparound contacts

NASA Technical Reports Server (NTRS)

Thornhill, J. W.

1977-01-01

The development of a process for fabricating 2 x 4 cm back surface field silicon solar cells having screen printed wraparound contacts is described. This process was specifically designed to be amenable for incorporation into the automated nonvacuum production line. Techniques were developed to permit the use of screen printing for producing improved back surface field structures, wraparound dielectric layers, and wraparound contacts. The optimized process sequence was then used to produce 1852 finished cells. Tests indicated an average conversion efficiency of 11% at AMO and 28 C, with an average degradation of maximum power output of 1.5% after boiling water immersion or thermal shock cycling. Contact adherence was satisfactory after these tests, as well as long term storage at high temperature and high humidity.

Effect of cholesterol depletion on exocytosis of alveolar type II cells.

PubMed

Chintagari, Narendranath Reddy; Jin, Nili; Wang, Pengcheng; Narasaraju, Telugu Akula; Chen, Jiwang; Liu, Lin

2006-06-01

Alveolar epithelial type II cells secrete lung surfactant via exocytosis. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) are implicated in this process. Lipid rafts, the cholesterol- and sphingolipid-rich microdomains, may offer a platform for protein organization on the cell membrane. We tested the hypothesis that lipid rafts organize exocytotic proteins in type II cells and are essential for the fusion of lamellar bodies, the secretory granules of type II cells, with the plasma membrane. The lipid rafts, isolated from type II cells using 1% Triton X-100 and a sucrose gradient centrifugation, contained the lipid raft markers, flotillin-1 and -2, whereas they excluded the nonraft marker, Na+-K+ ATPase. SNAP-23, syntaxin 2, and VAMP-2 were enriched in lipid rafts. When type II cells were depleted of cholesterol, the association of SNAREs with the lipid rafts was disrupted and the formation of fusion pore was inhibited. Furthermore, the cholesterol-depleted plasma membrane had less ability to fuse with lamellar bodies, a process mediated by annexin A2. The secretagogue-stimulated secretion of lung surfactant from type II cells was also reduced by methyl-beta-cyclodextrin. When the raft-associated cell surface protein, CD44, was cross-linked using anti-CD44 antibodies, the CD44 clusters were observed. Syntaxin 2, SNAP-23, and annexin A2 co-localized with the CD44 clusters, which were cholesterol dependent. Our results suggested that lipid rafts may form a functional platform for surfactant secretion in alveolar type II cells, and raft integrity was essential for the fusion between lamellar bodies with the plasma membrane.

Electrophoresis in space.

PubMed

Bauer, J; Hymer, W C; Morrison, D R; Kobayashi, H; Seaman, G V; Weber, G

1999-01-01

Programs for free flow electrophoresis in microgravity over the past 25 years are reviewed. Several studies accomplished during 20 spaceflight missions have demonstrated that sample throughput is significantly higher in microgravity than on the ground. Some studies have shown that resolution is also increased. However, many cell separation trials have fallen victim to difficulties associated with experimenting in the microgravity environment such as microbial contamination, air bubbles in electrophoresis chambers, and inadequate facilities for maintaining cells before and after separation. Recent studies suggest that the charge density of cells at their surface may also be modified in microgravity. If this result is confirmed, a further cellular mechanism of "sensing" the low gravity environment will have been found. Several free fluid electrophoresis devices are now available. Most have been tried at least once in microgravity. Newer units not yet tested in spaceflight have been designed to accommodate problems associated with space processing. The USCEPS device and the Japanese FFEU device are specifically designed for sterile operations, whereas the Octopus device is designed to reduce electroosmotic and electrohydrodynamic effects, which become dominant and detrimental in microgravity. Some of these devices will also separate proteins by zone electrophoresis, isotachophoresis, or isoelectric focusing in a single unit. Separation experiments with standard test particles are useful and necessary for testing and optimizing new space hardware. A cohesive free fluid electrophoresis program in the future will obviously require (1) flight opportunities and funding, (2) identification of suitable cellular and macromolecular candidate samples, and (3) provision of a proper interface of electrophoresis processing equipment with biotechnological facilities--equipment like bioreactors and protein crystal growth chambers. The authors feel that such capabilities will lead to the production of commercially useful quantities of target products and to an accumulation of new knowledge relating to the complexities of electrostatic phenomena at the cell surface.

Inactivation of stressed Escherichia coli O157:H7 cells on the surfaces of rocket salad leaves by chlorine and peroxyacetic acid.

PubMed

Al-Nabulsi, Anas A; Osaili, Tareq M; Obaidat, Heba M; Shaker, Reyad R; Awaisheh, Saddam S; Holley, Richard A

2014-01-01

Because Escherichia coli O157:H7 has been frequently associated with many foodborne outbreaks caused by consumption of leafy greens (lettuce, spinach, and celery), this study investigated the ability of deionized water, chlorine, and peroxyacetic acid to detach or inactivate stressed and unstressed cells of E. coli O157:H7 contaminating the surfaces of rocket salad leaves. E. coli O157:H7 cells stressed by acid, cold, starvation, or NaCl exposure, as well as unstressed cells, were inoculated on the surfaces of rocket salad leaves at 4°C. The effectiveness of two sanitizers (200 ppm of chlorine and 80 ppm of peroxyacetic acid) and deionized water for decontaminating the leaves treated with stressed and unstressed E. coli O157:H7 were evaluated during storage at 10 or 25°C for 0.5, 1, 3, and 7 days. It was found that washing with 80 ppm of peroxyacetic acid was more effective and reduced unstressed and stressed cells of E. coli O157:H7 by about 1 log CFU per leaf on the leaves. There was no apparent difference in the ability of stressed and unstressed cells to survive surface disinfection with the tested agents. Treatments to reduce viable E. coli O157:H7 cells on rocket leaves stored at 25°C were more effective than when used on those stored at 10°C. Washing with peroxyacetic acid or chlorine solution did not ensure the safety of rocket leaves, but such treatments could reduce the likelihood of water-mediated transfer of E. coli O157:H7 during washing and subsequent processing.

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.

Antibacterial and anticancer PDMS surface for mammalian cell growth using the Chinese herb extract paeonol(4-methoxy-2-hydroxyacetophenone)

NASA Astrophysics Data System (ADS)

Jiao, Jiajia; Sun, Lili; Guo, Zaiyu; Hou, Sen; Holyst, Robert; Lu, Yun; Feng, Xizeng

2016-12-01

Polydimethylsiloxane (PDMS) is widely used as a cell culture platform to produce micro- and nano-technology based microdevices. However, the native PDMS surface is not suitable for cell adhesion and is always subject to bacterial pollution and cancer cell invasion. Coating the PDMS surface with antibacterial or anticancer materials often causes considerable harm to the non-cancer mammalian cells on it. We have developed a method to fabricate a biocompatible PDMS surface which not only promotes non-cancer mammalian cell growth but also has antibacterial and anticancer activities, by coating the PDMS surface with a Chinese herb extract, paeonol. Coating changes the wettability and the elemental composition of the PDMS surface. Molecular dynamic simulation indicates that the absorption of paeonol onto the PDMS surface is an energy favourable process. The paeonol-coated PDMS surface exhibits good antibacterial activity against both Gram-positive and Gram-negative bacteria. Moreover considerable antibacterial activity is maintained after the coated surface is rinsed or incubated in water. The coated PDMS surface inhibits bacterial growth on the contact surface and promotes non-cancer mammalian cell growth with low cell toxicity; meanwhile the growth of cancer cells is significantly inhibited. Our study will potentially guide PDMS surface modification approaches to produce biomedical devices.

Discovery of cell surface vimentin targeting mAb for direct disruption of GBM tumor initiating cells.

PubMed

Noh, Hyangsoon; Yan, Jun; Hong, Sungguan; Kong, Ling-Yuan; Gabrusiewicz, Konrad; Xia, Xueqing; Heimberger, Amy B; Li, Shulin

2016-11-01

Intracellular vimentin overexpression has been associated with epithelial-mesenchymal transition, metastasis, invasion, and proliferation, but cell surface vimentin (CSV) is less understood. Furthermore, it remains unknown whether CSV can serve as a therapeutic target in CSV-expressing tumor cells. We found that CSV was present on glioblastoma multiforme (GBM) cancer stem cells and that CSV expression was associated with spheroid formation in those cells. A newly developed monoclonal antibody against CSV, 86C, specifically and significantly induced apoptosis and inhibited spheroid formation in GBM cells in vitro. The addition of 86C to GBM cells in vitro also led to rapid internalization of vimentin and decreased GBM cell viability. These findings were associated with an increase in caspase-3 activity, indicating activation of apoptosis. Finally, treatment with 86C inhibited GBM progression in vivo. In conclusion, CSV-expressing GBM cells have properties of tumor initiating cells, and targeting CSV with the monoclonal antibody 86C is a promising approach in the treatment of GBM.

Bacteriophage-encoded virion-associated enzymes to overcome the carbohydrate barriers during the infection process.

PubMed

Latka, Agnieszka; Maciejewska, Barbara; Majkowska-Skrobek, Grazyna; Briers, Yves; Drulis-Kawa, Zuzanna

2017-04-01

Bacteriophages are bacterial viruses that infect the host after successful receptor recognition and adsorption to the cell surface. The irreversible adherence followed by genome material ejection into host cell cytoplasm must be preceded by the passage of diverse carbohydrate barriers such as capsule polysaccharides (CPSs), O-polysaccharide chains of lipopolysaccharide (LPS) molecules, extracellular polysaccharides (EPSs) forming biofilm matrix, and peptidoglycan (PG) layers. For that purpose, bacteriophages are equipped with various virion-associated carbohydrate active enzymes, termed polysaccharide depolymerases and lysins, that recognize, bind, and degrade the polysaccharide compounds. We discuss the existing diversity in structural locations, variable architectures, enzymatic specificities, and evolutionary aspects of polysaccharide depolymerases and virion-associated lysins (VALs) and illustrate how these aspects can correlate with the host spectrum. In addition, we present methods that can be used for activity determination and the application potential of these enzymes as antibacterials, antivirulence agents, and diagnostic tools.

The Otto Aufranc Award: enhanced biocompatibility of stainless steel implants by titanium coating and microarc oxidation.

PubMed

Lim, Young Wook; Kwon, Soon Yong; Sun, Doo Hoon; Kim, Yong Sik

2011-02-01

Stainless steel is one of the most widely used biomaterials for internal fixation devices, but is not used in cementless arthroplasty implants because a stable oxide layer essential for biocompatibility cannot be formed on the surface. We applied a Ti electron beam coating, to form oxide layer on the stainless steel surface. To form a thicker oxide layer, we used a microarc oxidation process on the surface of Ti coated stainless steel. Modification of the surface using Ti electron beam coating and microarc oxidation could improve the ability of stainless steel implants to osseointegrate. The ability of cells to adhere to grit-blasted, titanium-coated, microarc-oxidated stainless steel in vitro was compared with that of two different types of surface modifications, machined and titanium-coated, and microarc-oxidated. We performed energy-dispersive x-ray spectroscopy and scanning electron microscopy investigations to assess the chemical composition and structure of the stainless steel surfaces and cell morphology. The biologic responses of an osteoblastlike cell line (SaOS-2) were examined by measuring proliferation (cell proliferation assay), differentiation (alkaline phosphatase activity), and attraction ability (cell migration assay). Cell proliferation, alkaline phosphatase activity, migration, and adhesion were increased in the grit-blasted, titanium-coated, microarc-oxidated group compared to the two other groups. Osteoblastlike cells on the grit-blasted, titanium-coated, microarc-oxidated surface were strongly adhered, and proliferated well compared to those on the other surfaces. The surface modifications we used (grit blasting, titanium coating, microarc oxidation) enhanced the biocompatibility (proliferation and migration of osteoblastlike cells) of stainless steel. This process is not unique to stainless steel; it can be applied to many metals to improve their biocompatibility, thus allowing a broad range of materials to be used for cementless implants.

Surface cytologic features on intraocular lenses: can increased biocompatibility have disadvantages?

PubMed

Hollick, E J; Spalton, D J; Ursell, P G

1999-07-01

To compare the anterior surface cytologic features and effect on blood-aqueous barrier of polymethyl methacrylate, silicone, and hydrogel intraocular lens (IOL) implants to give an indication of their biocompatibility. This prospective study was performed at an English-teaching hospital. Ninety eyes were randomized to receive a polymethyl methacrylate, silicone, or hydrogel implant. A standardized surgical protocol was performed by a single surgeon using phacoemulsification. Patients were seen at intervals for 1 year. Measurements of visual acuity, contrast sensitivity, and anterior chamber laser flare and cells were obtained; and an assessment of lens cytologic features using specular microscopy of the anterior IOL surface was performed. Visual acuity and contrast sensitivity were not significantly different among the 3 groups. Hydrogel IOLs were associated with fewer inflammatory cells on their surface than polymethyl methacrylate and silicone IOLs (P<.001), but with significantly more lens epithelial cells (LECs) (P<.001). Patients with hydrogel implants without LECs had greater blood-aqueous barrier breakdown than those with LECs. The hydrogel IOLs were associated with a reduced inflammatory cell reaction but had many more LECs on their anterior surface. Those IOLs associated with increased blood-aqueous barrier damage did not develop LECs. If an IOL is too biocompatible, then it may incite the growth of LECs over its surface, which could have disadvantages.

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.

Genome Wide assessment of Early Osseointegration in Implant-Adherent Cells

NASA Astrophysics Data System (ADS)

Thalji, Ghadeer N.

Objectives: To determine the molecular processes involved in osseointegration. Materials and methods: A structured literature review concerning in vitro and in vivo molecular assessment of osseointegration was performed. A rat and a human model were then used to identify the early molecular processes involved in osseointegration associated with a micro roughened and nanosurface superimposed featured implants. In the rat model, 32 titanium implants with surface topographies exhibiting a micro roughened (AT-II) and nanosurface superimposed featured implants (AT-I) were placed in the tibiae of 8 rats and subsequently harvested at 2 and 4 days after placement. Whereas in the human model, four titanium mini-implants with either a moderately roughened surface (TiOblast) or super-imposed nanoscale topography (Osseospeed) were placed in edentulous sites of eleven systemically healthy subjects and subsequently removed after 3 and 7 days. Total RNA was isolated from cells adherent to retrieved implants. A whole genome microarray using the Affymetrix 1.1 ST Array platform was used to describe the gene expression profiles that were differentially regulated by the implant surfaces. Results: The literature review provided evidence that particular topographic cues can be specifically integrated among the many extracellular signals received by the cell in its signal transduction network. In the rat model, functionally relevant categories related to ossification, skeletal system development, osteoblast differentiation, bone development and biomineral tissue development were upregulated and more prominent at AT-I compared to AT-II. In the human model, there were no significant differences when comparing the two-implant surfaces at each time point. However, the microarray identified several genes that were differentially regulated at day 7 vs. day 3 for both implant surfaces. Functionally relevant categories related to the extracellular matrix, collagen fibril organization and angiogenesis were upregulated at both surfaces. Abundant upregulation of several differential markers of alternative activated macrophages was also observed. The biological processes involved with the inflammatory/immune response gene expression were concomitantly downregulated. Conclusions: The presence of micro-roughened and nanosurface features modulated in vivo bone response. This work confirms previous evaluations and further implicates modulation of the inflammatory/immune responses as a factor affecting the accrual of bone mass shortly after implant placement.

Associative list processing unit

DOEpatents

Hemmert, Karl Scott; Underwood, Keith D.

2013-01-29

An associative list processing unit and method comprising employing a plurality of prioritized cell blocks and permitting inserts to occur in a single clock cycle if all of the cell blocks are not full. Also, an associative list processing unit and method comprising employing a plurality of prioritized cell blocks and using a tree of prioritized multiplexers descending from the plurality of cell blocks.

Nanoparticles-cell association predicted by protein corona fingerprints

NASA Astrophysics Data System (ADS)

Palchetti, S.; Digiacomo, L.; Pozzi, D.; Peruzzi, G.; Micarelli, E.; Mahmoudi, M.; Caracciolo, G.

2016-06-01

In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells.In a physiological environment (e.g., blood and interstitial fluids) nanoparticles (NPs) will bind proteins shaping a ``protein corona'' layer. The long-lived protein layer tightly bound to the NP surface is referred to as the hard corona (HC) and encodes information that controls NP bioactivity (e.g. cellular association, cellular signaling pathways, biodistribution, and toxicity). Decrypting this complex code has become a priority to predict the NP biological outcomes. Here, we use a library of 16 lipid NPs of varying size (Ø ~ 100-250 nm) and surface chemistry (unmodified and PEGylated) to investigate the relationships between NP physicochemical properties (nanoparticle size, aggregation state and surface charge), protein corona fingerprints (PCFs), and NP-cell association. We found out that none of the NPs' physicochemical properties alone was exclusively able to account for association with human cervical cancer cell line (HeLa). For the entire library of NPs, a total of 436 distinct serum proteins were detected. We developed a predictive-validation modeling that provides a means of assessing the relative significance of the identified corona proteins. Interestingly, a minor fraction of the HC, which consists of only 8 PCFs were identified as main promoters of NP association with HeLa cells. Remarkably, identified PCFs have several receptors with high level of expression on the plasma membrane of HeLa cells. Electronic supplementary information (ESI) available: Table S1. Cell viability (%) and cell association of the different nanoparticles used. Table S2. Total number of identified proteins on the different nanoparticles used. Tables S3-S18. Top 25 most abundant corona proteins identified in the protein corona of nanoparticles NP2-NP16 following 1 hour incubation with HP. Table S19. List of descriptors used. Table S20. Potential targets of protein corona fingerprints with its own interaction score (mentha) and the expression median value in Hela cells. Fig. S1 and S2. Effect of exposure to human plasma on size and zeta potential of NPs. Fig. S3. Predictive modeling of nanoparticle-cell association. See DOI: 10.1039/c6nr03898k

The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness.

PubMed

Chen, Hsin-Ying; Chang, Joseph Tung-Chieh; Chien, Kun-Yi; Lee, Yun-Shien; You, Guo-Rung; Cheng, Ann-Joy

2018-01-11

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.

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.

Surface Density of the Hendra G Protein Modulates Hendra F Protein-Promoted Membrane Fusion: Role for Hendra G Protein Trafficking and Degradation

PubMed Central

Whitman, Shannon D.; Dutch, Rebecca Ellis

2007-01-01

Hendra virus, like most paramyxoviruses, requires both a fusion (F) and attachment (G) protein for promotion of cell-cell fusion. Recent studies determined that Hendra F is proteolytically processed by the cellular protease cathepsin L after endocytosis. This unique cathepsin L processing results in a small percentage of Hendra F on the cell surface. To determine how the surface densities of the two Hendra glycoproteins affect fusion promotion, we performed experiments that varied the levels of glycoproteins expressed in transfected cells. Using two different fusion assays, we found a marked increase in fusion when expression of the Hendra G protein was increased, with a 1:1 molar ratio of Hendra F:G on the cell surface resulting in optimal membrane fusion. Our results also showed that Hendra G protein levels are modulated by both more rapid protein turnover and slower protein trafficking than is seen for Hendra F. PMID:17328935

Leptospirosis pulmonary haemorrhage syndrome is associated with linear deposition of immunoglobulin and complement on the alveolar surface.

PubMed

Croda, J; Neto, A N D; Brasil, R A; Pagliari, C; Nicodemo, A C; Duarte, M I S

2010-06-01

Leptospirosis is a zoonotic infection associated with severe diseases such as leptospirosis pulmonary haemorrhage syndrome (LPHS). The cause of pulmonary haemorrhage is unclear. Understanding which mechanisms and processes are involved in LPHS will be important in treatment regimens under development for this life-threatening syndrome. In the present study, we evaluated 30 lung specimens from LPHS patients and seven controls using histology and immunohistochemistry (detection of IgM, IgG, IgA and C3) in order to describe the pathological features associated with this syndrome. Immunoglobulin deposits were detected on the alveolar surface in 18/30 LPHS patients. Three staining patterns were observed for the immunoglobulins and C3 in the lung tissues of LPHS patients: AS, delicate linear staining adjacent to the alveolar surface, which was indicative of a membrane covering the luminal surface of type I and II pneumocyte cells; S, heterogeneous staining which was sporadically distributed along the alveolar septum; and IA, weak, focal intra-alveolar granular staining. Human LPHS is associated with individual and unique histological patterns that differ from those of other causes of pulmonary haemorrhage. In the present study, it was found that the linear deposition of immunoglobulins (IgA, IgG and IgM) and complement on the alveolar surface may play a role in the pathogenesis of pulmonary haemorrhage in human leptospirosis.

Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells.

PubMed

Pinazza, Marica; Ghisi, Margherita; Minuzzo, Sonia; Agnusdei, Valentina; Fossati, Gianluca; Ciminale, Vincenzo; Pezzè, Laura; Ciribilli, Yari; Pilotto, Giorgia; Venturoli, Carolina; Amadori, Alberto; Indraccolo, Stefano

2018-04-12

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.

Energy-independent intracellular gene delivery mediated by polymeric biomimetics of cell-penetrating peptides.

PubMed

Chae, Su Young; Kim, Hyun June; Lee, Min Sang; Jang, Yeon Lim; Lee, Yuhan; Lee, Soo Hyeon; Lee, Kyuri; Kim, Sun Hwa; Kim, Hong Tae; Chi, Sang-Cheol; Park, Tae Gwan; Jeong, Ji Hoon

2011-09-09

Efficient gene transfer into mammalian cells mediated by small molecular amphiphile-polymer conjugates, bile acid-polyethylenimine (BA-PEI), is demonstrated, opening an efficient transport route for genetic materials across the cell membrane. This process occurs without the aid of endocytosis or other energy-consuming processes, thus mimicking macromolecular transduction by cell-penetrating peptides. The exposure of a hydrophilic face of the amphiphilic BA moiety on the surface of BA-PEI/DNA complex that mediates direct contact of the BA molecules to the cell surface seems to play an important role in the endocytosis- and energy-independent internalization process. The new modality of the polymeric biomimetics can be applied to enhanced delivery of macromolecular therapeutics. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Rapid engineering of endothelial cell-lined vascular-like structures in in situ crosslinkable hydrogels.

PubMed

Kageyama, Tatsuto; Kakegawa, Takahiro; Osaki, Tatsuya; Enomoto, Junko; Ito, Taichi; Nittami, Tadashi; Fukuda, Junji

2014-06-01

Fabrication of perfusable vascular networks in vitro is one of the most critical challenges in the advancement of tissue engineering. Because cells consume oxygen and nutrients during the fabrication process, a rapid fabrication approach is necessary to construct cell-dense vital tissues and organs, such as the liver. In this study, we propose a rapid molding process using an in situ crosslinkable hydrogel and electrochemical cell transfer for the fabrication of perfusable vascular structures. The in situ crosslinkable hydrogel was composed of hydrazide-modified gelatin (gelatin-ADH) and aldehyde-modified hyaluronic acid (HA-CHO). By simply mixing these two solutions, the gelation occurred in less than 20 s through the formation of a stable hydrazone bond. To rapidly transfer cells from a culture surface to the hydrogel, we utilized a zwitterionic oligopeptide, which forms a self-assembled molecular layer on a gold surface. Human umbilical vein endothelial cells adhering on a gold surface via the oligopeptide layer were transferred to the hydrogel within 5 min, along with electrochemical desorption of the oligopeptides. This approach was applicable to cylindrical needles 200-700 µm in diameter, resulting in the formation of perfusable microchannels where the internal surface was fully enveloped with the transferred endothelial cells. The entire fabrication process was completed within 10 min, including 20 s for the hydrogel crosslinking and 5 min for the electrochemical cell transfer. This rapid fabrication approach may provide a promising strategy to construct perfusable vasculatures in cell-dense tissue constructs and subsequently allow cells to organize complicated and fully vascularized tissues while preventing hypoxic cell injury.

Surface multiheme c-type cytochromes from Thermincola potens: Implications for dissimilatory metal reduction by Gram-positive bacteria

NASA Astrophysics Data System (ADS)

Carlson, H. K.; Iavarone, A. T.; Gorur, A.; Yeo, B. S.; Tran, R.; Melnyk, R. A.; Mathies, R. A.; Auer, M.; Coates, J. D.

2011-12-01

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they have been shown to be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or the humic substances analog, anthraquinone-2,6-disulfonate (AQDS). The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS and that several MHCs are localized to the cell wall or cell surface of T. potens. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results are the first direct evidence for cell-wall associated cytochromes and MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium.

Development of Low-cost, High Energy-per-unit-area Solar Cell Modules

NASA Technical Reports Server (NTRS)

Jones, G. T.; Chitre, S.; Rhee, S. S.

1978-01-01

The development of two hexagonal solar cell process sequences, a laserscribing process technique for scribing hexagonal and modified hexagonal solar cells, a large through-put diffusion process, and two surface macrostructure processes suitable for large scale production is reported. Experimental analysis was made on automated spin-on anti-reflective coating equipment and high pressure wafer cleaning equipment. Six hexagonal solar cell modules were fabricated. Also covered is a detailed theoretical analysis on the optimum silicon utilization by modified hexagonal solar cells.

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.

Process Development for Automated Solar Cell and Module Production. Task 4: Automated Array Assembly

NASA Technical Reports Server (NTRS)

1979-01-01

A baseline sequence for the manufacture of solar cell modules was specified. Starting with silicon wafers, the process goes through damage etching, texture etching, junction formation, plasma edge etch, aluminum back surface field formation, and screen printed metallization to produce finished solar cells. The cells were then series connected on a ribbon and bonded into a finished glass tedlar module. A number of steps required additional developmental effort to verify technical and economic feasibility. These steps include texture etching, plasma edge etch, aluminum back surface field formation, array layup and interconnect, and module edge sealing and framing.

Effector biology of plant-associated organisms: concepts and perspectives.

PubMed

Win, J; Chaparro-Garcia, A; Belhaj, K; Saunders, D G O; Yoshida, K; Dong, S; Schornack, S; Zipfel, C; Robatzek, S; Hogenhout, S A; Kamoun, S

2012-01-01

Every plant is closely associated with a variety of living organisms. Therefore, deciphering how plants interact with mutualistic and parasitic organisms is essential for a comprehensive understanding of the biology of plants. The field of plant-biotic interactions has recently coalesced around an integrated model. Major classes of molecular players both from plants and their associated organisms have been revealed. These include cell surface and intracellular immune receptors of plants as well as apoplastic and host-cell-translocated (cytoplasmic) effectors of the invading organism. This article focuses on effectors, molecules secreted by plant-associated organisms that alter plant processes. Effectors have emerged as a central class of molecules in our integrated view of plant-microbe interactions. Their study has significantly contributed to advancing our knowledge of plant hormones, plant development, plant receptors, and epigenetics. Many pathogen effectors are extraordinary examples of biological innovation; they include some of the most remarkable proteins known to function inside plant cells. Here, we review some of the key concepts that have emerged from the study of the effectors of plant-associated organisms. In particular, we focus on how effectors function in plant tissues and discuss future perspectives in the field of effector biology.

Coaggregation between Rhodococcus and Acinetobacter strains isolated from the food industry.

PubMed

Møretrø, Trond; Sharifzadeh, Shahab; Langsrud, Solveig; Heir, Even; Rickard, Alexander H

2015-07-01

In this study, coaggregation interactions between Rhodococcus and Acinetobacter strains isolated from food-processing surfaces were characterized. Rhodococcus sp. strain MF3727 formed intrageneric coaggregates with Rhodococcus sp. strain MF3803 and intergeneric coaggregates with 2 strains of Acinetobacter calcoaceticus (MF3293, MF3627). Stronger coaggregation between A. calcoaceticus MF3727 and Rhodococcus sp. MF3293 was observed after growth in batch culture at 30 °C than at 20 °C, after growth in tryptic soy broth than in liquid R2A medium, and between cells in exponential and early stationary phases than cells in late stationary phase. The coaggregation ability of Rhodococcus sp. MF3727 was maintained even after heat and Proteinase K treatment, suggesting its ability to coaggregate was protein independent whereas the coaggregation determinants of the other strains involved proteinaceous cell-surface-associated polymers. Coaggregation was stable at pH 5-9. The mechanisms of coaggregation among Acinetobacter and Rhodococcus strains bare similarity to those displayed by coaggregating bacteria of oral and freshwater origin, with respect to binding between proteinaceous and nonproteinaceous determinants and the effect of environmental factors on coaggregation. Coaggregation may contribute to biofilm formation on industrial food surfaces, protecting bacteria against cleaning and disinfection.

Nanoscale imaging of photocurrent and efficiency in CdTe solar cells

DOE PAGES

Leite, Marina S.; National Inst. of Standards and Technology; Abashin, Maxim; ...

2014-10-15

The local collection characteristics of grain interiors and grain boundaries in thin film CdTe polycrystalline solar cells are investigated using scanning photocurrent microscopy. The carriers are locally generated by light injected through a small aperture (50-300 nm) of a near-field scanning optical microscope in an illumination mode. Possible influence of rough surface topography on light coupling is examined and eliminated by sculpting smooth wedges on the granular CdTe surface. By varying the wavelength of light, nanoscale spatial variations in external quantum efficiency are mapped. We find that the grain boundaries (GBs) are better current collectors than the grain interiors (GIs).more » The increased collection efficiency is caused by two distinct effects associated with the material composition of GBs. First, GBs are charged, and the corresponding built-in field facilitates the separation and the extraction of the photogenerated carriers. Second, the GB regions generate more photocurrent at long wavelength corresponding to the band edge, which can be caused by a smaller local band gap. As a result, resolving carrier collection with nanoscale resolution in solar cell materials is crucial for optimizing the polycrystalline device performance through appropriate thermal processing and passivation of defect and surfaces.« less

Understanding Microbe-Mineral Reactions Using Synchrotron Radiation Fourier Transform Infrared Spectromicroscopy

NASA Astrophysics Data System (ADS)

Kauffman, M. E.; Lehman, R. M.; Martin, M. C.; Bauer, W. F.

2002-12-01

Microorganisms are able to alter their surrounding microenvironment to an extent not predicted by the thermodynamics of the macro-environment chemistry. Microbially induced environmental alterations include weathering, biomineralization and mobilization or immobilization of authegenic metals or contaminants. Microbial colonization of surfaces, followed by biofilm formation, are the first steps in alteration processes. With the exception of iron oxides and iron-reducing bacteria, the fundamentals of how microbes react with various mineral surfaces is not well understood. Synchrotron radiation Fourier transform infrared spectromicroscopy (SR-FTIR) is a non-destructive analytical technique capable of probing, in situ, the microbe-mineral interface. The SR-FTIR beamline 1.4.3, at the Advanced Light Source, Berkeley, CA, has a diffraction-limited spatial resolution of 10 um, is 2-3 orders of magnitude brighter than traditional FTIR, and is not harmful to living samples. Aliquots of pure cultures of Burkholderia cepacia G4 were deposited on four individual mineral surfaces (plagioclase, ilmenite, augite and olivine) and spectra were collected within 20-40 min. Reference spectra were collected from the same pure cultures deposited on gold-coated glass slides. Additionally, reference spectra were collected of commercially available biomolecules deposited on the four individual mineral specimens. The spectra of the bacterial cells on gold and the spectra of the separate biomolecules contained all the relevant peaks documented in the literature. However, the spectra collected from the microbe-mineral interfaces were markedly different from the reference spectra and varied between the four mineral surfaces. Bacterial cells in contact with plagioclase exhibited predominantly absorption bands associated with phosphate groups, while the spectra of olivine and bacterial cells were limited to absorption bands associated with bacterial proteins. Spectra of the same bacterial cells in contact with augite indicated a strong peak attributed to amino acids, specifically tyrosine. The results presented here document the changes in the biogeochemistry of the microbial-mineral interface that can occur within minutes when cells react to various mineral surfaces. These results advance the understanding of how microorganisms impact the natural environment.

Interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in human epithelial cancer cells.

PubMed

Piyush, Tushar; Chacko, Anisha R; Sindrewicz, Paulina; Hilkens, John; Rhodes, Jonathan M; Yu, Lu-Gang

2017-11-01

Epidermal growth factor receptor (EGFR) is an important regulator of epithelial cell growth and survival in normal and cancerous tissues and is a principal therapeutic target for cancer treatment. EGFR is associated in epithelial cells with the heavily glycosylated transmembrane mucin protein MUC1, a natural ligand of galectin-3 that is overexpressed in cancer. This study reveals that the expression of cell surface MUC1 is a critical enhancer of EGF-induced EGFR activation in human breast and colon cancer cells. Both the MUC1 extracellular and intracellular domains are involved in EGFR activation but the predominant influence comes from its extracellular domain. Binding of galectin-3 to the MUC1 extracellular domain induces MUC1 cell surface polarization and increases MUC1-EGFR association. This leads to a rapid increase of EGFR homo-/hetero-dimerization and subsequently increased, and also prolonged, EGFR activation and signalling. This effect requires both the galectin-3 C-terminal carbohydrate recognition domain and its N-terminal ligand multi-merization domain. Thus, interaction of galectin-3 with MUC1 on cell surface promotes EGFR dimerization and activation in epithelial cancer cells. As MUC1 and galectin-3 are both commonly overexpressed in most types of epithelial cancers, their interaction and impact on EGFR activation likely makes important contribution to EGFR-associated tumorigenesis and cancer progression and may also influence the effectiveness of EGFR-targeted cancer therapy.

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.

Advanced Oxidation Protein Products-Modified Albumin Induces Differentiation of RAW264.7 Macrophages into Dendritic-Like Cells Which Is Modulated by Cell Surface Thiols.

PubMed

Garibaldi, Silvano; Barisione, Chiara; Marengo, Barbara; Ameri, Pietro; Brunelli, Claudio; Balbi, Manrico; Ghigliotti, Giorgio

2017-01-10

Local accumulation of Advanced Oxidation Protein Products (AOPP) induces pro-inflammatory and pro-fibrotic processes in kidneys and is an independent predictor of renal fibrosis and of rapid decline of eGFR in patients with chronic kidney disease (CKD). In addition to kidney damage, circulating AOPP may be regarded as mediators of systemic oxidative stress and, in this capacity, they might play a role in the progression of atherosclerotic damage of arterial walls. Atherosclerosis is a chronic inflammatory disease that involves activation of innate and adaptive immunity. Dendritic cells (DCs) are key cells in this process, due to their role in antigen presentation, inflammation resolution and T cell activation. AOPP consist in oxidative modifications of proteins (such as albumin and fibrinogen) that mainly occur through myeloperoxidase (MPO)-derived hypochlorite (HOCl). HOCl modified proteins have been found in atherosclerotic lesions. The oxidizing environment and the shifts in cellular redox equilibrium trigger inflammation, activate immune cells and induce immune responses. Thus, surface thiol groups contribute to the regulation of immune functions. The aims of this work are: (1) to evaluate whether AOPP-proteins induce activation and differentiation of mature macrophages into dendritic cells in vitro; and (2) to define the role of cell surface thiol groups and of free radicals in this process. AOPP-proteins were prepared by in vitro incubation of human serum albumin (HSA) with HOCl. Mouse macrophage-like RAW264.7 were treated with various concentrations of AOPP-HSA with or without the antioxidant N -acetyl cysteine (NAC). Following 48 h of HSA-AOPP treatment, RAW264.7 morphological changes were evaluated by microscopic observation, while markers of dendritic lineage and activation (CD40, CD86, and MHC class II) and allogeneic T cell proliferation were evaluated by flow cytometry. Cell surface thiols were measured by AlexaFluor-maleimide binding, and ROS production was assessed as DCF fluorescence by flow cytometry. HSA-AOPP induced the differentiation of RAW264.7 cells into a dendritic-like phenotype, as shown by morphological changes, by increased CD40, CD86 and MHC class II surface expression and by induction of T cell proliferation. The cell surface thiols dose dependently decreased following HSA-AOPP treatment, while ROS production increased. NAC pre-treatment enhanced the amount of cell surface thiols and prevented their reduction due to treatment with AOPP. Both ROS production and RAW264.7 differentiation into DC-like cells induced by HSA-AOPP were reduced by NAC. Our results highlight that oxidized plasma proteins modulate specific immune responses of macrophages through a process involving changes in the thiol redox equilibrium. We suggest that this mechanism may play a role in determining the rapid progression of the atherosclerotic process observed in CKD patients.

GARP (LRRC32) is essential for the surface expression of latent TGF-beta on platelets and activated FOXP3+ regulatory T cells.

PubMed

Tran, Dat Q; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M

2009-08-11

TGF-beta family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-beta is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFbeta-binding protein (LTBP) to produce a large latent form. Latent TGF-beta is also found on the surface of activated FOXP3(+) regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-beta to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-beta and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-beta expression on activated Tregs and recombinant latent TGF-beta1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-beta on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism.

GARP (LRRC32) is essential for the surface expression of latent TGF-β on platelets and activated FOXP3+ regulatory T cells

PubMed Central

Tran, Dat Q.; Andersson, John; Wang, Rui; Ramsey, Heather; Unutmaz, Derya; Shevach, Ethan M.

2009-01-01

TGF-β family members are highly pleiotropic cytokines with diverse regulatory functions. TGF-β is normally found in the latent form associated with latency-associated peptide (LAP). This latent complex can associate with latent TGFβ-binding protein (LTBP) to produce a large latent form. Latent TGF-β is also found on the surface of activated FOXP3+ regulatory T cells (Tregs), but it is unclear how it is anchored to the cell membrane. We show that GARP or LRRC32, a leucine-rich repeat molecule of unknown function, is critical for tethering TGF-β to the cell surface. We demonstrate that platelets and activated Tregs co-express latent TGF-β and GARP on their membranes. The knockdown of GARP mRNA with siRNA prevented surface latent TGF-β expression on activated Tregs and recombinant latent TGF-β1 is able to bind directly with GARP. Confocal microscopy and immunoprecipitation strongly support their interactions. The role of TGF-β on Tregs appears to have dual functions, both for Treg-mediated suppression and infectious tolerance mechanism. PMID:19651619

Reduction of CD147 surface expression on primary T cells leads to enhanced cell proliferation.

PubMed

Biegler, Brian; Kasinrerk, Watchara

2012-12-01

CD147 is a ubiquitously expressed membrane glycoprotein that has numerous functional associations in health and disease. However, the molecular mechanisms by which CD147 participates in these processes are unclear. Establishing physiologically relevant silencing of CD147 in primary T cells could provide clues essential for elucidating some aspects of CD147 biology. To date, achieving the knockdown of CD147 in primary T cells has remained elusive. Utilizing RNA interference and the Nucleofector transfection system, we were able to reduce the expression of CD147 in primary T cells. Comparison of basic functions, such as proliferation and CD25 expression, were then made between control populations and populations with reduced expression. Up-regulation of CD147 was found upon T-cell activation, indicating a role in T-cell responses. To better understand the possible importance of this up-regulation, we knocked down the expression of CD147 using RNA interference. When compared to control populations the CD147 knockdown populations exhibited increased proliferation. This alteration of cell proliferation, however, was not linked to a change in CD25 expression. We achieved reduction of CD147 surface expression in primary T cells by siRNA-mediated gene silencing. Our results point to CD147 having a possible negative regulatory role in T cell-mediated immune responses.

The mouse neuronal cell surface protein F3: a phosphatidylinositol- anchored member of the immunoglobulin superfamily related to chicken contactin

PubMed Central

1989-01-01

Several members of the Ig superfamily are expressed on neural cells where they participate in surface interactions between cell bodies and processes. Their Ig domains are more closely related to each other than to Ig variable and constant domains and have been grouped into the C2 set. Here, we report the cloning and characterization of another member of this group, the mouse neuronal cell surface antigen F3. The F3 cDNA sequence contains an open reading frame that could encode a 1,020-amino acid protein consisting of a signal sequence, six Ig-like domains of the C2 type, a long premembrane region containing two segments that exhibit sequence similarity to fibronectin type III repeats and a moderately hydrophobic COOH-terminal sequence. The protein does not contain a typical transmembrane segment but appears to be attached to the membrane by a phosphatidylinositol anchor. Antibodies against the F3 protein recognize a prominent 135-kD protein in mouse brain. In fetal brain cultures, they stain the neuronal cell surface and, in cultures maintained in chemically defined medium, most prominently neurites and neurite bundles. The mouse f3 gene maps to band F of chromosome 15. The gene transcripts detected in the brain by F3 cDNA probes are developmentally regulated, the highest amounts being expressed between 1 and 2 wk after birth. The F3 nucleotide and deduced amino acid sequence show striking similarity to the recently published sequence of the chicken neuronal cell surface protein contactin. However, there are important differences between the two molecules. In contrast to F3, contactin has a transmembrane and a cytoplasmic domain. Whereas contactin is insoluble in nonionic detergent and is tightly associated with the cytoskeleton, about equal amounts of F3 distribute between buffer-soluble, nonionic detergent-soluble, and detergent- insoluble fractions. Among other neural cell surface proteins, F3 most resembles the neuronal cell adhesion protein L1, with 25% amino acid identity between their extracellular domains. Based on its structural similarity with known cell adhesion proteins of nervous tissue and with L1 in particular, we propose that F3 mediates cell surface interactions during nervous system development. PMID:2474555

Antigen-Induced but Not Innate Memory CD8 T Cells Express NKG2D and Are Recruited to the Lung Parenchyma upon Viral Infection.

PubMed

Grau, Morgan; Valsesia, Séverine; Mafille, Julien; Djebali, Sophia; Tomkowiak, Martine; Mathieu, Anne-Laure; Laubreton, Daphné; de Bernard, Simon; Jouve, Pierre-Emmanuel; Ventre, Erwan; Buffat, Laurent; Walzer, Thierry; Leverrier, Yann; Marvel, Jacqueline

2018-05-15

The pool of memory-phenotype CD8 T cells is composed of Ag-induced (AI) and cytokine-induced innate (IN) cells. IN cells have been described as having properties similar to those of AI memory cells. However, we found that pathogen-induced AI memory cells can be distinguished in mice from naturally generated IN memory cells by surface expression of NKG2D. Using this marker, we described the increased functionalities of AI and IN memory CD8 T cells compared with naive cells, as shown by comprehensive analysis of cytokine secretion and gene expression. However, AI differed from IN memory CD8 T cells by their capacity to migrate to the lung parenchyma upon inflammation or infection, a process dependent on their expression of ITGA1/CD49a and ITGA4/CD49d integrins. Copyright © 2018 by The American Association of Immunologists, Inc.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes.

PubMed

Balasubramaniam, Muthukumar; Davids, Benem; Addai, Amma B; Pandhare, Jui; Dash, Chandravanu

2017-02-22

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3' ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Characterization of initial events in bacterial surface colonization by two Pseudomonas species using image analysis.

PubMed

Mueller, R F; Characklis, W G; Jones, W L; Sears, J T

1992-05-01

The processes leading to bacterial colonization on solid-water interfaces are adsorption, desorption, growth, and erosion. These processes have been measured individually in situ in a flowing system in real time using image analysis. Four different substrata (copper, silicon, 316 stainless-steel and glass) and 2 different bacterial species (Pseudomonas aeruginosa and Pseudomonas fluorescens) were used in the experiments. The flow was laminar (Re = 1.4) and the shear stress was kept constant during all experiments at 0.75 N m(-2). The surface roughness varied among the substrata from 0.002 microm (for silicon) to 0.015 microm (for copper). Surface free energies varied from 25.1 dynes cm(-1) for silicon to 31.2 dynes cm(-1) for copper. Cell curface hydrophobicity, reported as hydrocarbon partitioning values, ranged from 0.67 for Ps. fluorescens to 0.97 for Ps. aeruginosa.The adsorption rate coefficient varied by as much as a factor of 10 among the combinations of bacterial strain and substratum material, and was positively correlated with surface free energy, the surface roughness of the substratum, and the hydrophobicity of the cells. The probability of desorption decreased with increasing surface free energy and surface roughness of the substratum. Cell growth was inhibited on copper, but replication of cells overlying an initial cell layer was observed with increased exposure time to the cell-containing bulk water. A mathematical model describing cell accumulation on a substratum is presented.

Virion-associated phosphatidylethanolamine promotes TIM1-mediated infection by Ebola, dengue, and West Nile viruses.

PubMed

Richard, Audrey Stéphanie; Zhang, Adam; Park, Sun-Jin; Farzan, Michael; Zong, Min; Choe, Hyeryun

2015-11-24

Phosphatidylserine (PS) receptors contribute to two crucial biological processes: apoptotic clearance and entry of many enveloped viruses. In both cases, they recognize PS exposed on the plasma membrane. Here we demonstrate that phosphatidylethanolamine (PE) is also a ligand for PS receptors and that this phospholipid mediates phagocytosis and viral entry. We show that a subset of PS receptors, including T-cell immunoglobulin (Ig) mucin domain protein 1 (TIM1), efficiently bind PE. We further show that PE is present in the virions of flaviviruses and filoviruses, and that the PE-specific cyclic peptide lantibiotic agent Duramycin efficiently inhibits the entry of West Nile, dengue, and Ebola viruses. The inhibitory effect of Duramycin is specific: it inhibits TIM1-mediated, but not L-SIGN-mediated, virus infection, and it does so by blocking virus attachment to TIM1. We further demonstrate that PE is exposed on the surface of apoptotic cells, and promotes their phagocytic uptake by TIM1-expressing cells. Together, our data show that PE plays a key role in TIM1-mediated virus entry, suggest that disrupting PE association with PS receptors is a promising broad-spectrum antiviral strategy, and deepen our understanding of the process by which apoptotic cells are cleared.

An N-terminal Retention Module Anchors the Giant Adhesin LapA of Pseudomonas fluorescens at the Cell Surface: A Novel Sub-family of Type I Secretion Systems.

PubMed

Smith, T Jarrod; Font, Maria E; Kelly, Carolyn M; Sondermann, Holger; O'Toole, George A

2018-02-05

LapA of Pseudomonas fluorescens Pf0-1 belongs to a diverse family of cell surface associated bacterial adhesins that are secreted via the type-1 secretion system (T1SS). We previously reported that the periplasmic protease LapG cleaves the N-terminus of LapA at a canonical dialanine motif to release the adhesin from the cell surface under conditions unfavorable to biofilm formation, thus decreasing biofilm formation. Here, we characterize LapA as the first type 1 secreted substrate that does not follow the "one-step" rule of T1SS. Rather, a novel N-terminal element, called the retention module (RM), localizes LapA at the cell surface as a secretion intermediate. Our genetic, biochemical, and molecular modeling analysis support a model wherein LapA is tethered to the cell surface through its T1SS outer membrane TolC-like pore, LapE, until LapG cleaves LapA in the periplasm. We further demonstrate this unusual retention strategy is likely conserved among LapA-like proteins, and reveals a new subclass of T1SS ABC transporters involved in transporting this group of surface-associated, LapA-like adhesins. These studies demonstrate a novel cell surface retention strategy used throughout the Proteobacteria and highlight a previously unappreciated flexibility of function for T1SS. Importance. Bacteria have evolved multiple secretion strategies to interact with their environment. For many bacteria, the secretion of cell surface associated adhesins is key for initiating contact with a preferred substratum to facilitate biofilm formation. Our work demonstrates that P. fluorescens uses a previously unrecognized secretion strategy to retain the giant adhesin LapA at its cell surface. Further, we identify likely LapA-like adhesins in various pathogenic and commensal Proteobacteria and provide phylogenetic evidence that these adhesins are secreted by a new subclass of T1SS ABC transporters. Copyright © 2018 American Society for Microbiology.

Attachment of Salmonella strains to a plant cell wall model is modulated by surface characteristics and not by specific carbohydrate interactions.

PubMed

Tan, Michelle Sze-Fan; Moore, Sean C; Tabor, Rico F; Fegan, Narelle; Rahman, Sadequr; Dykes, Gary A

2016-09-15

Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface. We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin. Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils.

Gas-solid carbonation as a possible source of carbonates in cold planetary environments

NASA Astrophysics Data System (ADS)

Garenne, A.; Montes-Hernandez, G.; Beck, P.; Schmitt, B.; Brissaud, O.; Pommerol, A.

2013-02-01

Carbonates are abundant sedimentary minerals at the surface and sub-surface of the Earth and they have been proposed as tracers of liquid water in extraterrestrial environments. Their formation mechanism is since generally associated with aqueous alteration processes. Recently, carbonate minerals have been discovered on Mars' surface by different orbitals or rover missions. In particular, the phoenix mission has measured from 1% to 5% of calcium carbonate (calcite type) within the soil (Smith et al., 2009). These occurrences have been reported in area where the relative humidity is significantly high (Boynton et al., 2009). The small concentration of carbonates suggests an alternative process on mineral grain surfaces (as suggested by Shaheen et al., 2010) than carbonation in aqueous conditions. Such an observation could rather point toward a possible formation mechanism by dust-gas reaction under current Martian conditions. To understand the mechanism of carbonate formation under conditions relevant to current Martian atmosphere and surface, we designed an experimental setup consisting of an infrared microscope coupled to a cryogenic reaction cell (IR-CryoCell setup). Three different mineral precursors of carbonates (Ca and Mg hydroxides, and a hydrated Ca silicate formed from Ca2SiO4), low temperature (from -10 to +30 °C), and reduced CO2 pressure (from 100 to 2000 mbar) were utilized to investigate the mechanism of gas-solid carbonation at mineral surfaces. These mineral materials are crucial precursors to form Ca and Mg carbonates in humid environments (0%

Role of GARP in the activation of latent TGF-β1.

PubMed

Stockis, Julie; Dedobbeleer, Olivier; Lucas, Sophie

2017-09-26

TGF-β1, 2 and 3 cytokines are involved in many cellular processes including cell proliferation, differentiation, migration and survival. Whereas TGF-β2 and 3 play important roles in embryonic development, TGF-β1 is mostly implicated in controlling immune responses after birth. The production of TGF-β1 is a tightly regulated process, occurring mostly at a post-translational level. Virtually all cells produce the latent, inactive form of TGF-β1. In latent TGF-β1, the mature TGF-β1 dimer is non-covalently associated to the Latency Associated Peptide, or LAP, which prevents binding to the TGF-β1 receptor. Activation of the cytokine implies release of mature TGF-β1 from LAP. Only a few cell types activate latent TGF-β1, via mechanisms that are cell type specific. Proteins such as integrins, proteases and thrombospondin-1 activate TGF-β1 in epithelial cells, fibroblasts and dendritic cells. More recently, the protein GARP was shown to be involved in TGF-β1 activation by regulatory T cells (Treg), a subset of CD4 + T lymphocytes specialized in suppression of immune responses. GARP is a transmembrane protein that binds latent-TGF-β1 and tethers it on the Treg surface. The role of GARP was studied mostly in Tregs, and this was recently reviewed in L. Sun, H. Jin and H. Li, Oncotarget, 2016, 7, 42826-42836. However, GARP is also expressed in non-immune cells. This review focuses on the roles of GARP in latent TGF-β1 activation by immune and non-immune cells.

Characterization of the cell surface properties of drinking water pathogens by microbial adhesion to hydrocarbon and electrophoretic mobility measurements.

PubMed

Popovici, Jonathan; White, Colin P; Hoelle, Jill; Kinkle, Brian K; Lytle, Darren A

2014-06-01

The surface characteristics of microbial cells directly influence their mobility and behavior within aqueous environments. The cell surface hydrophobicity (CSH) and electrophoretic mobility (EPM) of microbial cells impact a number of interactions and processes including aggregation, adhesion to surfaces, and stability of the cells within the aqueous environments. These cell characteristics are unique to the bacterial species and are a reflection of the large diversity of surface structures, proteins, and appendages of microorganisms. CSH and EPM of bacterial cells contribute substantially to the effectiveness of drinking water treatment to remove them, and therefore an investigation of these properties will be useful in predicting their removal through drinking water treatment processes and transport through drinking water distribution systems. EPM and CSH measurements of six microbiological pathogen or surrogate species suspended in phosphate-buffered water are reported in this work. Two strains of Vibrio cholerae were hydrophobic, while three strains of Escherichia coli were hydrophilic. Bacillus cereus was categorized as moderately hydrophobic. The strains of E. coli had the highest (most negative) EPM. Based on the measurements, E. coli species is predicted to be most difficult to remove from water while V. cholerae will be the easiest to remove. Copyright © 2014 Elsevier B.V. All rights reserved.

Rat Indwelling Urinary Catheter Model of Candida albicans Biofilm Infection

PubMed Central

Nett, Jeniel E.; Brooks, Erin G.; Cabezas-Olcoz, Jonathan; Sanchez, Hiram; Zarnowski, Robert; Marchillo, Karen

2014-01-01

Indwelling urinary catheters are commonly used in the management of hospitalized patients. Candida can adhere to the device surface and propagate as a biofilm. These Candida biofilm communities differ from free-floating Candida, exhibiting high tolerance to antifungal therapy. The significance of catheter-associated candiduria is often unclear, and treatment may be problematic considering the biofilm drug-resistant phenotype. Here we describe a rodent model for the study of urinary catheter-associated Candida albicans biofilm infection that mimics this common process in patients. In the setting of a functioning, indwelling urinary catheter in a rat, Candida proliferated as a biofilm on the device surface. Characteristic biofilm architecture was observed, including adherent, filamentous cells embedded in an extracellular matrix. Similar to what occurs in human patients, animals with this infection developed candiduria and pyuria. Infection progressed to cystitis, and a biofilmlike covering was observed over the bladder surface. Furthermore, large numbers of C. albicans cells were dispersed into the urine from either the catheter or bladder wall biofilm over the infection period. We successfully utilized the model to test the efficacy of antifungals, analyze transcriptional patterns, and examine the phenotype of a genetic mutant. The model should be useful for future investigations involving the pathogenesis, diagnosis, therapy, prevention, and drug resistance of Candida biofilms in the urinary tract. PMID:25183731

Role of Outer Membrane C-Type Cytochromes MtrC and OmcA in Shewanella Oneidensis MR-1 Cell Production, Accumulation, and Detachment During Respiration on Hematite

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

Mitchell, Andrew C.; Peterson, L.; Reardon, Catherine L.

2012-07-01

Solid phase iron oxides are considered to be important terminal electron acceptors for microbial respiration in many anoxic environments. Besides the knowledge that cells attach to and reduce these substrates, other aspects of surface-associated cell behavior and the related cell surface components that influence cell-mineral interactions are not well understood. In the present study, wild-type cells of the dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 formed thin biofilms one-to-two cell layers in thickness when respiring on natural specular hematite under flow conditions similar to those which exist in aquatic sediments and subsurface environments. The distribution of cells within the biofilm indicatedmore » that direct contact was not required for electron transfer from cells to the mineral surface. Detached biomass in the form of single cells represented >99% of the surface-associated wild-type cell production from respiration on hematite over the biofilm life cycle. A mutant deficient in the outer membrane c35 type cytochrome OmcA, while still able to respire and replicate on hematite, established a lower steady-state cell density on the mineral surface than that of the wild-type strain. A mutant deficient in MtrC, another outer membrane c-type cytochrome, and a mutant deficient in both cytochromes were unable to reduce sufficient amounts of hematite to support detectable growth on the mineral surface. When considered in the context of previous work, the results support a growing body of evidence that the relative importance of OmcA and MtrC to cell respiration and replication depends on the form of iron oxide available as terminal electron acceptor.« less

A monoclonal antibody inhibits calcium accumulation and skeleton formation in cultured embryonic cells of the sea urchin.

PubMed

Carson, D D; Farach, M C; Earles, D S; Decker, G L; Lennarz, W J

1985-06-01

The assembly of the spicules (primitive skeleton) of the sea urchin embryo is being studied in primary mesenchyme cells cultured in vitro. A monoclonal antibody (1223) has been prepared that inhibits the deposition of CaCO3 into the spicules. This antibody reacts with a 130,000 Mr cell-surface protein that is concentrated on the surface of approximately 5% of the cells of dissociated gastrula stage embryos. When primary mesenchyme cells in the embryo or cells cultured in vitro are examined, the 1223 antigen is detected on the surface of the cells and on the extracellular material associated with the spicule. We conclude that the 1223 antibody recognizes a cell-surface protein that plays an essential role in spicule formation.

Manipulating and Monitoring On-Surface Biological Reactions by Light-Triggered Local pH Alterations.

PubMed

Peretz-Soroka, Hagit; Pevzner, Alexander; Davidi, Guy; Naddaka, Vladimir; Kwiat, Moria; Huppert, Dan; Patolsky, Fernando

2015-07-08

Significant research efforts have been dedicated to the integration of biological species with electronic elements to yield smart bioelectronic devices. The integration of DNA, proteins, and whole living cells and tissues with electronic devices has been developed into numerous intriguing applications. In particular, the quantitative detection of biological species and monitoring of biological processes are both critical to numerous areas of medical and life sciences. Nevertheless, most current approaches merely focus on the "monitoring" of chemical processes taking place on the sensing surfaces, and little efforts have been invested in the conception of sensitive devices that can simultaneously "control" and "monitor" chemical and biological reactions by the application of on-surface reversible stimuli. Here, we demonstrate the light-controlled fine modulation of surface pH by the use of photoactive molecularly modified nanomaterials. Through the use of nanowire-based FET devices, we showed the capability of modulating the on-surface pH, by intensity-controlled light stimulus. This allowed us simultaneously and locally to control and monitor pH-sensitive biological reactions on the nanodevices surfaces, such as the local activation and inhibition of proteolytic enzymatic processes, as well as dissociation of antigen-antibody binding interactions. The demonstrated capability of locally modulating the on-surface effective pH, by a light stimuli, may be further applied in the local control of on-surface DNA hybridization/dehybridization processes, activation or inhibition of living cells processes, local switching of cellular function, local photoactivation of neuronal networks with single cell resolution and so forth.

Selective emitter solar cell formation by NH3 plasma nitridation and single diffusion

NASA Astrophysics Data System (ADS)

Wu, Yung-Hsien; Chen, Lun-Lun; Wu, Jia-Rong; Wu, Min-Lin

2010-01-01

A new and simple process for fabricating a selective emitter solar cell has been proposed. Lightly and heavily doped emitters could be concurrently formed after a single POCl3 diffusion step through the selective formation of SiNx, which serves as the diffusion barrier and can be grown by NH3 plasma nitridation of the Si surface. The desired phosphorus depth profile for the lightly and heavily doped region verifies the eligibility of this process. From the electrical characterization, the selective emitter solar cell fabricated by this process manifests a higher absolute conversion efficiency than a conventional one by 0.5%. It is the enhanced response to the short wavelength light and the reduced surface recombination that causes the considerable improvement in conversion efficiency which is beneficial to further hold the competitive advantage for solar cell manufacturers. Most importantly, the proposed process can be fully integrated into the conventional solar cell process in a mass-production laboratory.

Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1.

PubMed

Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

2014-06-01

T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130,000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1-calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.

Early-stage detection of VE-cadherin during endothelial differentiation of human mesenchymal stem cells using SPR biosensor.

PubMed

Fathi, Farzaneh; Rezabakhsh, Aysa; Rahbarghazi, Reza; Rashidi, Mohammad-Reza

2017-10-15

Surface plasmon resonance (SPR) biosensors are most commonly applied for real-time dynamic analysis and measurement of interactions in bio-molecular studies and cell-surface analysis without the need for labeling processes. Up to present, SPR application in stem cell biology and biomedical sciences was underused. Herein, a very simple and sensitive method was developed to evaluate human mesenchymal stem cells trans-differentiation to endothelial lineage of over a period of 14 days based on VE-cadherin biomarker. The SPR signals increased with the increase of the amount of VE-cadherin expression on the cell surface during cell differentiation process. The method was able to detect ≈27 cells permm 2 . No significant effect was observed on the cell viability during the cell attachment to the surface of immune-reactive biochips and during the SPR analysis. Using this highly sensitive SPR method, it was possible to sense the early stage of endothelial differentiation on day 3 in label-free form, whereas flow cytometry and fluorescent microscopy methods were found unable to detect the cell differentiation at the same time. Therefore, the proposed method can rapidly and accurately detect cell differentiation in live cells and label-free manner without any need of cell breakage and has great potential for both diagnostic and experimental approaches. Copyright © 2017. Published by Elsevier B.V.

Facile Synthesis of Conductive Polypyrrole Wrinkle Topographies on Polydimethylsiloxane via a Swelling-Deswelling Process and Their Potential Uses in Tissue Engineering.

PubMed

Aufan, M Rifqi; Sumi, Yang; Kim, Semin; Lee, Jae Young

2015-10-28

Electrically conducting biomaterials have gained great attention in various biomedical studies especially to influence cell and tissue responses. In addition, wrinkling can present a unique topography that can modulate cell-material interactions. In this study, we developed a simple method to create wrinkle topographies of conductive polypyrrole (wPPy) on soft polydimethylsiloxane surfaces via a swelling-deswelling process during and after PPy polymerization and by varying the thickness of the PPy top layers. As a result, various features of wPPy in the range of the nano- and microscales were successfully obtained. In vitro cell culture studies with NIH 3T3 fibroblasts and PC12 neuronal cells indicated that the conductive wrinkle topographies promote cell adhesion and neurite outgrowth of PC12 cells. Our studies help to elucidate the design of the surface coating and patterning of conducting polymers, which will enable us to simultaneously provide topographical and electrical signals to improve cell-surface interactions for potential tissue-engineering applications.

Protein disulfide isomerase and Nox: new partners in redox signaling.

PubMed

Trevelin, Silvia Cellone; Lopes, Lucia Rossetti

2015-01-01

Reactive oxygen species (ROS) contribute to the pathogenesis of cardiovascular disease, including hypertension, atherosclerosis, cardiac hypertrophy, heart failure and restenosis. Thiol proteins and thiol oxidoreductases are key players in cell signaling, and their altered expression and/or activity has been associated with a disrupture in cardiac and vascular homeostasis. Protein disulfide isomerase (PDI) is a thiol oxidoreductase member of the thioredoxin family that has multiple roles in cellular function. Originally discovered in the endoplasmic reticulum (ER), PDI is essential for protein folding. However, it can also be found in the cytosol and closely associated with the surface of platelets, smooth muscle cells, neutrophils and endothelial cells. On the cell surface, PDI is imperative for platelet aggregation and transnitrosation, which are related to thrombosis and control of vascular tone by nitric oxide, respectively. Furthermore, PDI signaling contributes to redox-dependent events such as smooth muscle cell migration induced by PDGF and TNFα-dependent angiogenesis. Studies from our group have shown that intracellular PDI regulates the expression and activity of the NADPH oxidase family of proteins (Nox), which are enzymes dedicated to ROS generation. PDI acts as a new organizer of leukocyte Nox2 by redox dependently associating with p47phox and controlling its recruitment to the plasma membrane, an essential step for assembly of the active enzyme. Such multiple effects of PDI suggest that specific targeting of this oxidoreductase could represent a new approach in the treatment of vascular disease. In this review, we present a novel role for PDI as an adaptor protein involved in redox processes and Nox signaling and propose PDI as a potential therapeutic target in the treatment of atherosclerosis, thrombosis and hypertension.

Hydroxy-Al and cell-surface negativity are responsible for the enhanced sensitivity of Rhodotorula taiwanensis to aluminum by increased medium pH.

PubMed

Zhao, Xue Qiang; Bao, Xue Min; Wang, Chao; Xiao, Zuo Yi; Hu, Zhen Min; Zheng, Chun Li; Shen, Ren Fang

2017-10-01

Aluminum (Al) is ubiquitous and toxic to microbes. High Al 3+ concentration and low pH are two key factors responsible for Al toxicity, but our present results contradict this idea. Here, an Al-tolerant yeast strain Rhodotorula taiwanensis RS1 was incubated in glucose media containing Al with a continuous pH gradient from pH 3.1-4.2. The cells became more sensitive to Al and accumulated more Al when pH increased. Calculations using an electrostatic model Speciation Gouy Chapman Stern indicated that, the increased Al sensitivity of cells was associated with AlOH 2+ and Al(OH) 2 + rather than Al 3+ . The alcian blue (a positively charged dye) adsorption and zeta potential determination of cell surface indicated that, higher pH than 3.1 increased the negative charge and Al adsorption at the cell surface. Taken together, the enhanced sensitivity of R. taiwanensis RS1 to Al from pH 3.1-4.2 was associated with increased hydroxy-Al and cell-surface negativity.

Enhancing wear resistance of working bodies of grinder through lining crushed material

NASA Astrophysics Data System (ADS)

Romanovich, A. A.; Annenko, D. M.; Romanovich, M. A.; Apukhtina, I. V.

2018-03-01

The article presents the analysis of directions of increasing wear resistance of working surfaces of rolls. A technical solution developed at the level of the invention is proposed, which is simple to implement in production conditions and which makes it possible to protect the roll surface from heavy wear due to surfacing of wear-resistant mesh material, cells of which are filling with grinding material in the process of work. Retaining them enables one to protect the roll surface from wear. The paper dwells on conditions of pressing materials in cells of eccentric rolls on the working surface with a grid of rectangular shape. The paper presents an equation for calculation of the cell dimension that provides the lining of the working surface by a mill material with respect to its properties. The article presents results of comparative studies on the grinding process of a press roller grinder (PRG) between rolls with and without a fusion-bonded mesh. It is clarified that the lining of rolls working surface slightly reduces the quality of the grinding, since the material thickness in the cell is small and has a finely divided and compacted structure with high strength.

Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition.

PubMed

Li, Qisheng; Sodroski, Catherine; Lowey, Brianna; Schweitzer, Cameron J; Cha, Helen; Zhang, Fang; Liang, T Jake

2016-07-05

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

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 ranges of pattern sizes having the desired effects on the cellular behaviors.

Hydrophobic pinning with copper nanowhiskers leads to bactericidal properties.

PubMed

Singh, Ajay Vikram; Baylan, Semanur; Park, Byung-Wook; Richter, Gunther; Sitti, Metin

2017-01-01

The considerable morbidity associated with hospitalized patients and clinics in developed countries due to biofilm formation on biomedical implants and surgical instruments is a heavy economic burden. An alternative to chemically treated surfaces for bactericidal activity started emerging from micro/nanoscale topographical cues in the last decade. Here, we demonstrate a putative antibacterial surface using copper nanowhiskers deposited by molecular beam epitaxy. Furthermore, the control of biological response is based on hydrophobic pinning of water droplets in the Wenzel regime, causing mechanical injury and cell death. Scanning electron microscopy images revealed the details of the surface morphology and non-contact mode laser scanning of the surface revealed the microtopography-associated quantitative parameters. Introducing the bacterial culture over nanowhiskers produces mechanical injury to cells, leading to a reduction in cell density over time due to local pinning of culture medium to whisker surfaces. Extended culture to 72 hours to observe biofilm formation revealed biofilm inhibition with scattered microcolonies and significantly reduced biovolume on nanowhiskers. Therefore, surfaces patterned with copper nanowhiskers can serve as potential antibiofilm surfaces. The topography-based antibacterial surfaces introduce a novel prospect in developing mechanoresponsive nanobiomaterials to reduce the risk of medical device biofilm-associated infections, contrary to chemical leaching of copper as a traditional bactericidal agent.

Fuel processing for PEM fuel cells: transport and kinetic issues of system design

NASA Astrophysics Data System (ADS)

Zalc, J. M.; Löffler, D. G.

In light of the distribution and storage issues associated with hydrogen, efficient on-board fuel processing will be a significant factor in the implementation of PEM fuel cells for automotive applications. Here, we apply basic chemical engineering principles to gain insight into the factors that limit performance in each component of a fuel processor. A system consisting of a plate reactor steam reformer, water-gas shift unit, and preferential oxidation reactor is used as a case study. It is found that for a steam reformer based on catalyst-coated foils, mass transfer from the bulk gas to the catalyst surface is the limiting process. The water-gas shift reactor is expected to be the largest component of the fuel processor and is limited by intrinsic catalyst activity, while a successful preferential oxidation unit depends on strict temperature control in order to minimize parasitic hydrogen oxidation. This stepwise approach of sequentially eliminating rate-limiting processes can be used to identify possible means of performance enhancement in a broad range of applications.

Differential regulation of membrane-associated mucins in the human ocular surface epithelium.

PubMed

Hori, Yuichi; Spurr-Michaud, Sandra; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K

2004-01-01

Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.

Corrosion resistance and biological activity of TiO2 implant coatings produced in oxygen-rich environments.

PubMed

Zhang, Rui; Wan, Yi; Ai, Xing; Liu, Zhanqiang; Zhang, Dong

2017-01-01

The physical and chemical properties of bio-titanium alloy implant surfaces play an important role in their corrosion resistance and biological activity. New turning and turning-rolling processes are presented, employing an oxygen-rich environment in order to obtain titanium dioxide layers that can both protect implants from corrosion and also promote cell adhesion. The surface topographies, surface roughnesses and chemical compositions of the sample surfaces were obtained using scanning electron microscopy, a white light interferometer, and the Auger electron spectroscopy, respectively. The corrosion resistance of the samples in a simulated body fluid was determined using electrochemical testing. Biological activity on the samples was also analyzed, using a vitro cell culture system. The results show that compared with titanium oxide layers formed using a turning process in air, the thickness of the titanium oxide layers formed using turning and turning-rolling processes in an oxygen-rich environment increased by 4.6 and 7.3 times, respectively. Using an oxygen-rich atmosphere in the rolling process greatly improves the corrosion resistance of the resulting samples in a simulated body fluid. On samples produced using the turning-rolling process, cells spread quickly and exhibited the best adhesion characteristics.

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.

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.

Stop codons in the hepatitis B surface proteins are enriched during antiviral therapy and are associated with host cell apoptosis

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

Colledge, Danielle; Soppe, Sally; Yuen, Lilly

Premature stop codons in the hepatitis B virus (HBV) surface protein can be associated with nucleos(t)ide analogue resistance due to overlap of the HBV surface and polymerase genes. The aim of this study was to determine the effect of the replication of three common surface stop codon variants on the hepatocyte. Cell lines were transfected with infectious HBV clones encoding surface stop codons rtM204I/sW196*, rtA181T/sW172*, rtV191I/sW182*, and a panel of substitutions in the surface proteins. HBsAg was measured by Western blotting. Proliferation and apoptosis were measured using flow cytometry. All three surface stop codon variants were defective in HBsAg secretion.more » Cells transfected with these variants were less proliferative and had higher levels of apoptosis than those transfected with variants that did not encode surface stop codons. The most cytopathic variant was rtM204I/sW196*. Replication of HBV encoding surface stop codons was toxic to the cell and promoted apoptosis, exacerbating disease progression. - Highlights: •Under normal circumstances, HBV replication is not cytopathic. •Premature stop codons in the HBV surface protein can be selected and enriched during nucleos(t)ide analogue therapy. •Replication of these variants can be cytopathic to the cell and promote apoptosis. •Inadequate antiviral therapy may actually promote disease progression.« less

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting.

PubMed

Juzwa, W; Duber, A; Myszka, K; Białas, W; Czaczyk, K

2016-09-01

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.

Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers

PubMed Central

D’Addio, Suzanne M.; Baldassano, Steven; Shi, Lei; Cheung, Lila; Adamson, Douglas H.; Bruzek, Matthew; Anthony, John E.; Laskin, Debra L.; Sinko, Patrick J.; Prud’homme, Robert K.

2013-01-01

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5 kg mol−1 methoxy-terminated PEG corona. While a 5 kg mol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8 kg mol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associate with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This characterization of nanocarrier uptake and targeting provides promise for optimizing drug delivery to macrophages for TB treatment and establishes a general route for optimizing targeted formulations of nanocarriers for specific delivery at targeted sites. PMID:23419950

Designing perturbative metamaterials from discrete models.

PubMed

Matlack, Kathryn H; Serra-Garcia, Marc; Palermo, Antonio; Huber, Sebastian D; Daraio, Chiara

2018-04-01

Identifying material geometries that lead to metamaterials with desired functionalities presents a challenge for the field. Discrete, or reduced-order, models provide a concise description of complex phenomena, such as negative refraction, or topological surface states; therefore, the combination of geometric building blocks to replicate discrete models presenting the desired features represents a promising approach. However, there is no reliable way to solve such an inverse problem. Here, we introduce 'perturbative metamaterials', a class of metamaterials consisting of weakly interacting unit cells. The weak interaction allows us to associate each element of the discrete model with individual geometric features of the metamaterial, thereby enabling a systematic design process. We demonstrate our approach by designing two-dimensional elastic metamaterials that realize Veselago lenses, zero-dispersion bands and topological surface phonons. While our selected examples are within the mechanical domain, the same design principle can be applied to acoustic, thermal and photonic metamaterials composed of weakly interacting unit cells.

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

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.

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.

Structural and surface property characterization of titanium dioxide nanotubes for orthopedic implants

NASA Astrophysics Data System (ADS)

Shokuhfar, Tolou

This research focused on the to modification of the surface structure of titanium implants with nanostructured morphology of TiO2 nanotubes and studied the interaction of nanotubes with osteoblast cells to understand the parameters that affect the cell growth. The electrical, mechanical, and structural properties of TiO2 nanotubes were characterized to establish a better understanding on the properties of such nanoscale morphological structures. To achieve the objectives of this research work I transformed the titanium and its alloys, either in bulk sheet form, bulk machined form, or thin film deposited on another substrate into a surface of titania nanotubes using a low cost and environmentally friendly process. The process requires only a simple electrolyte, low cost electrode, and a DC power supply. With this simple approach of scalable nanofabrication, a typical result is nanotubes that are each approximately 100nm in diameter and have a wall thickness of about 20nm. By changing the fabrication parameters, independent nanotubes can be fabricated with open volume between them. Titanium in this form is termed onedimensional since electron transport is narrowly confined along the length of the nanotube. My Ph.D. accomplishments have successfully shown that osteoblast cells, the cells that are the precursors to bone, have a strong tendency to attach to the inside and outside of the titanium nanotubes onto which they are grown using their filopodia -- cell's foot used for locomotion -- anchored to titanium nanotubes. In fact it was shown that the cell prefers to find many anchoring sites. These sites are critical for cell locomotion during the first several weeks of maturity and upon calcification as a strongly anchored bone cell. In addition I have shown that such a surface has a greater cell density than a smooth titanium surface. My work also developed a process that uses a focused and controllably rastered ion beam as a nano-scalpel to cut away sections of the osteoblast cells to probe the attachment beneath the main cell body. Ultimately the more rapid growth of osteoblasts, coupled with a stronger cell-surface interface, could provide cost reduction, shorter rehabilitation, and fewer follow-on surgeries due to implant loosening.

High CD49f expression is associated with osteosarcoma tumor progression: a study using patient-derived primary cell cultures.

PubMed

Penfornis, Patrice; Cai, David Z; Harris, Michael R; Walker, Ryan; Licini, David; Fernandes, Joseph D A; Orr, Griffin; Koganti, Tejaswi; Hicks, Chindo; Induru, Spandana; Meyer, Mark S; Khokha, Rama; Barr, Jennifer; Pochampally, Radhika R

2014-08-01

Overall prognosis for osteosarcoma (OS) is poor despite aggressive treatment options. Limited access to primary tumors, technical challenges in processing OS tissues, and the lack of well-characterized primary cell cultures has hindered our ability to fully understand the properties of OS tumor initiation and progression. In this study, we have isolated and characterized cell cultures derived from four central high-grade human OS samples. Furthermore, we used the cell cultures to study the role of CD49f in OS progression. Recent studies have implicated CD49f in stemness and multipotency of both cancer stem cells and mesenchymal stem cells. Therefore, we investigated the role of CD49f in osteosarcomagenesis. First, single cell suspensions of tumor biopsies were subcultured and characterized for cell surface marker expression. Next, we characterized the growth and differentiation properties, sensitivity to chemotherapy drugs, and anchorage-independent growth. Xenograft assays showed that cell populations expressing CD49f(hi) /CD90(lo) cell phenotype produced an aggressive tumor. Multiple lines of evidence demonstrated that inhibiting CD49f decreased the tumor-forming ability. Furthermore, the CD49f(hi) /CD90(lo) cell population is generating more aggressive OS tumor growth and indicating this cell surface marker could be a potential candidate for the isolation of an aggressive cell type in OSs. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Plasma ignition and tuning in different cells of a 1.3 GHz nine-cell superconducting radio frequency cavity: Proof of principle

NASA Astrophysics Data System (ADS)

Tyagi, P. V.; Moss, Andrew; Goudket, Philippe; Pattalwar, Shrikant; Herbert, Joe; Valizadeh, Reza; McIntosh, Peter

2018-06-01

Field emission is one of the critical issues in the superconducting radio frequency (SRF) cavities and can degrade their accelerating gradient during operation. The contamination present at top surface of the SRF cavity is one of the foremost reasons for field emission. Plasma based surface processing can be a viable option to eliminate such surface contaminants and enhance performance of the SRF cavity especially for in-situ applications. These days, 1.3 GHz nine-cell SRF cavity has become baseline standard for many particle accelerators, it is of interest to develop plasma cleaning technique for such SRF cavities. In the development of the plasma processing technique for SRF cavities, the most challenging task is to ignite and tune the plasma in different cells of the SRF cavity. At Daresbury laboratory, UK, we have successfully achieved plasma ignition in different cells of a 1.3 GHz nine-cell SRF cavity. The plasma ignition in different cells of the cavity was accomplished at room temperature towards room temperature plasma cleaning of the SRF cavity surface. Here, we report the successful demonstration of the plasma ignition in different cells of a 1.3 GHz nine-cell SRF cavity.

Surface LAMP-2 Is an Endocytic Receptor That Diverts Antigen Internalized by Human Dendritic Cells into Highly Immunogenic Exosomes.

PubMed

Leone, Dario Armando; Peschel, Andrea; Brown, Markus; Schachner, Helga; Ball, Miriam J; Gyuraszova, Marianna; Salzer-Muhar, Ulrike; Fukuda, Minoru; Vizzardelli, Caterina; Bohle, Barbara; Rees, Andrew J; Kain, Renate

2017-07-15

The lysosome-associated membrane protein (LAMP) family includes the dendritic cell endocytic receptors DC-LAMP and CD68, as well as LAMP-1 and LAMP-2. In this study we identify LAMP-1 (CD107a) and LAMP-2 (CD107b) on the surface of human monocyte-derived dendritic cells (MoDC) and show only LAMP-2 is internalized after ligation by specific Abs, including H4B4, and traffics rapidly but transiently to the MHC class II loading compartment, as does Ag conjugated to H4B4. However, pulsing MoDC with conjugates of primary (keyhole limpet hemocyanin; KLH) and recall (Bet v 1) Ags (H4B4*KLH and H4B4*Bet v 1) induced significantly less CD4 cell proliferation than pulsing with native Ag or Ag conjugated to control mAb (ISO*KLH and ISO*Bet v 1). In H4B4*KLH-pulsed MoDC, the duration of KLH residence in MHC class II loading compartments was significantly reduced, as were surface HLA-DR and DR-bound KLH-derived peptides. Paradoxically, MoDC pulsed with H4B4*KLH, but not the other KLH preparations, induced robust proliferation of CD4 cells separated from them by a transwell membrane, indicating factors in the supernatant were responsible. Furthermore, extracellular vesicles from supernatants of H4B4*KLH-pulsed MoDC contained significantly more HLA-DR and KLH than those purified from control MoDC, and KLH was concentrated specifically in exosomes that were a uniquely effective source of Ag in standard T cell proliferation assays. In summary, we identify LAMP-2 as an endocytic receptor on human MoDC that routes cargo into unusual Ag processing pathways, which reduces surface expression of Ag-derived peptides while selectively enriching Ag within immunogenic exosomes. This novel pathway has implications for the initiation of immune responses both locally and at distant sites. Copyright © 2017 by The American Association of Immunologists, Inc.

Superior in vitro biological response and mechanical properties of an implantable nanostructured biomaterial: Nanohydroxyapatite-silicone rubber composite.

PubMed

Thein-Han, W W; Shah, J; Misra, R D K

2009-09-01

A potential approach to achieving the objective of favorably modulating the biological response of implantable biopolymers combined with good mechanical properties is to consider compounding the biopolymer with a bioactive nanocrystalline ceramic biomimetic material with high surface area. The processing of silicone rubber (SR)-nanohydroxyapatite (nHA) composite involved uniform dispersion of nHA via shear mixing and ultrasonication, followed by compounding at sub-ambient temperature, and high-pressure solidification when the final curing reaction occurs. The high-pressure solidification approach enabled the elastomer to retain the high elongation of SR even in the presence of the reinforcement material, nHA. The biological response of the nanostructured composite in terms of initial cell attachment, cell viability and proliferation was consistently greater on SR-5wt.% nHA composite surface compared to pure SR. Furthermore, in the nanocomposite, cell spreading, morphology and density were distinctly different from that of pure SR. Pre-osteoblasts grown on SR-nHA were well spread, flat, large in size with a rough cell surface, and appeared as a group. In contrast, these features were less pronounced in SR (e.g. smooth cell surface, not well spread). Interestingly, an immunofluorescence study illustrated distinct fibronectin expression level, and stronger vinculin focal adhesion contacts associated with abundant actin stress fibers in pre-osteoblasts grown on the nanocomposite compared to SR, implying enhanced cell-substrate interaction. This finding was consistent with the total protein content and SDS-PAGE analysis. The study leads us to believe that further increase in nHA content in the SR matrix beyond 5wt.% will encourage even greater cellular response. The integration of cellular and molecular biology with materials science and engineering described herein provides a direction for the development of a new generation of nanostructured materials.

Receptor-mediated cell mechanosensing

PubMed Central

Chen, Yunfeng; Ju, Lining; Rushdi, Muaz; Ge, Chenghao; Zhu, Cheng

2017-01-01

Mechanosensing describes the ability of a cell to sense mechanical cues of its microenvironment, including not only all components of force, stress, and strain but also substrate rigidity, topology, and adhesiveness. This ability is crucial for the cell to respond to the surrounding mechanical cues and adapt to the changing environment. Examples of responses and adaptation include (de)activation, proliferation/apoptosis, and (de)differentiation. Receptor-mediated cell mechanosensing is a multistep process that is initiated by binding of cell surface receptors to their ligands on the extracellular matrix or the surface of adjacent cells. Mechanical cues are presented by the ligand and received by the receptor at the binding interface; but their transmission over space and time and their conversion into biochemical signals may involve other domains and additional molecules. In this review, a four-step model is described for the receptor-mediated cell mechanosensing process. Platelet glycoprotein Ib, T-cell receptor, and integrins are used as examples to illustrate the key concepts and players in this process. PMID:28954860

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.

Low-temperature and conventional scanning electron microscopy of human urothelial neoplasms.

PubMed

Hopkins, D M; Morris, J A; Oates, K; Huddart, H; Staff, W G

1989-05-01

The appearance of neoplastic human urothelium viewed by low-temperature scanning electron microscopy (LTSEM) and conventional scanning electron microscopy (CSEM) was compared. Fixed, dehydrated neoplastic cells viewed by CSEM had well-defined, often raised cell junctions; no intercellular gaps; and varying degrees of pleomorphic surface microvilli. The frozen hydrated material viewed by LTSEM, however, was quite different. The cells had a flat or dimpled surface, but no microvilli. There were labyrinthine lateral processes which interdigitated with those of adjacent cells and outlined large intercellular gaps. The process of fixation and dehydration will inevitably distort cell contours and on theoretical grounds, the images of frozen hydrated material should more closely resemble the in vivo appearance.

Cell Surface Translocation of Annexin A2 Facilitates Glutamate-induced Extracellular Proteolysis*

PubMed Central

Valapala, Mallika; Maji, Sayantan; Borejdo, Julian; Vishwanatha, Jamboor K.

2014-01-01

Glutamate-induced elevation in intracellular Ca2+ has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca2+-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca2+ imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca2+ influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes. PMID:24742684

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.

Fuel cell plates with improved arrangement of process channels for enhanced pressure drop across the plates

DOEpatents

Spurrier, Francis R.; Pierce, Bill L.; Wright, Maynard K.

1986-01-01

A plate for a fuel cell has an arrangement of ribs defining an improved configuration of process gas channels and slots on a surface of the plate which provide a modified serpentine gas flow pattern across the plate surface. The channels are generally linear and arranged parallel to one another while the spaced slots allow cross channel flow of process gas in a staggered fashion which creates a plurality of generally mini-serpentine flow paths extending transverse to the longitudinal gas flow along the channels. Adjacent pairs of the channels are interconnected to one another in flow communication. Also, a bipolar plate has the aforementioned process gas channel configuration on one surface and another configuration on the opposite surface. In the other configuration, there are not slots and the gas flow channels have a generally serpentine configuration.

Biofilms’ Role in Planktonic Cell Proliferation

PubMed Central

Bester, Elanna; Wolfaardt, Gideon M.; Aznaveh, Nahid B.; Greener, Jesse

2013-01-01

The detachment of single cells from biofilms is an intrinsic part of this surface-associated mode of bacterial existence. Pseudomonas sp. strain CT07gfp biofilms, cultivated in microfluidic channels under continuous flow conditions, were subjected to a range of liquid shear stresses (9.42 mPa to 320 mPa). The number of detached planktonic cells was quantified from the effluent at 24-h intervals, while average biofilm thickness and biofilm surface area were determined by confocal laser scanning microscopy and image analysis. Biofilm accumulation proceeded at the highest applied shear stress, while similar rates of planktonic cell detachment was maintained for biofilms of the same age subjected to the range of average shear rates. The conventional view of liquid-mediated shear leading to the passive erosion of single cells from the biofilm surface, disregards the active contribution of attached cell metabolism and growth to the observed detachment rates. As a complement to the conventional conceptual biofilm models, the existence of a biofilm surface-associated zone of planktonic cell proliferation is proposed to highlight the need to expand the traditional perception of biofilms as promoting microbial survival, to include the potential of biofilms to contribute to microbial proliferation. PMID:24201127

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant

PubMed Central

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-01-01

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration. PMID:28772704

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant.

PubMed

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-03-26

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration.

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.

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

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

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex.

PubMed

Wise, Jillian F; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K; Tao, Rong-Hua; Sabichi, Anita L; Ao, Xue; Maeng, Hoyoung; Samaniego, Felipe

2013-06-06

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL-Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target.

Nucleolin inhibits Fas ligand binding and suppresses Fas-mediated apoptosis in vivo via a surface nucleolin-Fas complex

PubMed Central

Wise, Jillian F.; Berkova, Zuzana; Mathur, Rohit; Zhu, Haifeng; Braun, Frank K.; Tao, Rong-Hua; Sabichi, Anita L.; Ao, Xue; Maeng, Hoyoung

2013-01-01

Resistance to Fas-mediated apoptosis is associated with poor cancer outcomes and chemoresistance. To elucidate potential mechanisms of defective Fas signaling, we screened primary lymphoma cell extracts for Fas-associated proteins that would have the potential to regulate Fas signaling. An activation-resistant Fas complex selectively included nucleolin. We confirmed the presence of nucleolin-Fas complexes in B-cell lymphoma cells and primary tissues, and the absence of such complexes in B-lymphocytes from healthy donors. RNA-binding domain 4 and the glycine/arginine-rich domain of nucleolin were essential for its association with Fas. Nucleolin colocalized with Fas on the surface of B-cell lymphoma cells. Nucleolin knockdown sensitized BJAB cells to Fas ligand (FasL)-induced and Fas agonistic antibody-induced apoptosis through enhanced binding, suggesting that nucleolin blocks the FasL–Fas interaction. Mice transfected with nucleolin were protected from the lethal effects of agonistic anti-mouse Fas antibody (Jo2) and had lower rates of hepatocyte apoptosis, compared with vector and a non-Fas-binding mutant of nucleolin. Our results show that cell surface nucleolin binds Fas, inhibits ligand binding, and thus prevents induction of Fas-mediated apoptosis in B-cell lymphomas and may serve as a new therapeutic target. PMID:23599269

Role of gangliosides in active immunotherapy with melanoma vaccine.

PubMed

Ravindranath, M H; Morton, D L

1991-01-01

Among various tumor associated cell surface antigens, gangliosides, the glycosphingolipids that contain sialic acids, offer a variety of epitopes, some of which are preferentially expressed on melanoma cells. These surface components of the bilayered lipid membrane of tumor cells are the targets of active immunotherapy with melanoma vaccine. Purified gangliosides in aqueous solution form micelles and, at high density, form lactones. Their antigenic expression (physical conformation and orientation) on the cell surface is governed by the nature of the sphingosine and the fatty acids they contain. Evidence is accruing to show that the nature of the fatty acid moiety of gangliosides differs in normal and neoplastic cells. Gangliosides per se are not immunogenic and require extrinsic adjuvanticity. Preparation of a melanoma cell vaccine for active immunotherapy requires an understanding of the ganglioside profile of melanoma, the ganglioside-associated heterogeneity of melanoma, and the role of shed melanoma gangliosides in the immunosuppression of cell mediated and humoral immunity. In addition, the role of some of the anti-ganglioside antibodies in the elimination of shed gangliosides, the cytotoxic killing of tumor cells, as well as in the down-regulation of lymphocyte functions must be considered in the formulation of vaccine. Different strategies for augmenting the immunogenicity of melanoma associated gangliosides with melanoma vaccine are evaluated.

Surface multiheme c-type cytochromes from Thermincola potens and implications for respiratory metal reduction by Gram-positive bacteria

PubMed Central

Carlson, Hans K.; Iavarone, Anthony T.; Gorur, Amita; Yeo, Boon Siang; Tran, Rosalie; Melnyk, Ryan A.; Mathies, Richard A.; Auer, Manfred; Coates, John D.

2012-01-01

Almost nothing is known about the mechanisms of dissimilatory metal reduction by Gram-positive bacteria, although they may be the dominant species in some environments. Thermincola potens strain JR was isolated from the anode of a microbial fuel cell inoculated with anaerobic digester sludge and operated at 55 °C. Preliminary characterization revealed that T. potens coupled acetate oxidation to the reduction of hydrous ferric oxides (HFO) or anthraquinone-2,6-disulfonate (AQDS), an analog of the redox active components of humic substances. The genome of T. potens was recently sequenced, and the abundance of multiheme c-type cytochromes (MHCs) is unusual for a Gram-positive bacterium. We present evidence from trypsin-shaving LC-MS/MS experiments and surface-enhanced Raman spectroscopy (SERS) that indicates the expression of a number of MHCs during T. potens growth on either HFO or AQDS, and that several MHCs are localized to the cell wall or cell surface. Furthermore, one of the MHCs can be extracted from cells with low pH or denaturants, suggesting a loose association with the cell wall or cell surface. Electron microscopy does not reveal an S-layer, and the precipitation of silver metal on the cell surface is inhibited by cyanide, supporting the involvement of surface-localized redox-active heme proteins in dissimilatory metal reduction. These results provide unique direct evidence for cell wall-associated cytochromes and support MHC involvement in conducting electrons across the cell envelope of a Gram-positive bacterium. PMID:22307634

Penicillium purpurogenum cultures under ethanol-induced stress and its correlation with fungal adhesion and biodegrading ability.

PubMed

Gomaa, Ola M; Husseiny, Sherif M; Abd El Kareem, Hussein; Talaat, Riham

2016-10-01

Fungi are known to be affected by external environmental stimuli, resulting in different stress response effects, which in turn could be used to enhance its biodegrading ability. In a previous study, ethanol was used to manipulate cell-cell and cell-surface interaction to prevent cell loss and maximize the usage of Penicillium purpurogenum cells in the media, a correlation was drawn between ethanol oxidative stress, surface-bound proteins and fungal adhesion. The present study focuses on a more detailed study of the effect of ethanol on the same fungus. The results show that the presence of Yap1p gene and the detection of an oxidized form of glutathione (GSSG) suggest that a stress response might be involved in the adhesion process. The process of adhesion could be described as a signaling process and it is affected by the germ tube formation as an initial step in adhesion. Protein profile showed polymorphism in surface-bound proteins for cultures amended with ethanol when compared to control cultures. Ethanol also affected the DNA polymorphic profile of DNA, rendering the fungus genetically variable. P. purpurogenum produced phenol oxidase enzyme and could be used to degrade total phenols in olive mill waste water without the formation of biofilm on the surface of the containers.

Quantification of confocal images of biofilms grown on irregular surfaces

PubMed Central

Ross, Stacy Sommerfeld; Tu, Mai Han; Falsetta, Megan L.; Ketterer, Margaret R.; Kiedrowski, Megan R.; Horswill, Alexander R.; Apicella, Michael A.; Reinhardt, Joseph M.; Fiegel, Jennifer

2014-01-01

Bacterial biofilms grow on many types of surfaces, including flat surfaces such as glass and metal and irregular surfaces such as rocks, biological tissues and polymers. While laser scanning confocal microscopy can provide high-resolution images of biofilms grown on any surface, quantification of biofilm-associated bacteria is currently limited to bacteria grown on flat surfaces. This can limit researchers studying irregular surfaces to qualitative analysis or quantification of only the total bacteria in an image. In this work, we introduce a new algorithm called modified connected volume filtration (MCVF) to quantify bacteria grown on top of an irregular surface that is fluorescently labeled or reflective. Using the MCVF algorithm, two new quantification parameters are introduced. The modified substratum coverage parameter enables quantification of the connected-biofilm bacteria on top of the surface and on the imaging substratum. The utility of MCVF and the modified substratum coverage parameter were shown with Pseudomonas aeruginosa and Staphylococcus aureus biofilms grown on human airway epithelial cells. A second parameter, the percent association, provides quantified data on the colocalization of the bacteria with a labeled component, including bacteria within a labeled tissue. The utility of quantifying the bacteria associated with the cell cytoplasm was demonstrated with Neisseria gonorrhoeae biofilms grown on cervical epithelial cells. This algorithm provides more flexibility and quantitative ability to researchers studying biofilms grown on a variety of irregular substrata. PMID:24632515

In situ characterization and analysis of Salmonella biofilm formation under meat processing environments using a combined microscopic and spectroscopic approach.

PubMed

Wang, Huhu; Ding, Shijie; Wang, Guangyu; Xu, Xinglian; Zhou, Guanghong

2013-11-01

Salmonella biofilm on food-contact surfaces present on food processing facilities may serve as a source of cross-contamination. In our work, biofilm formation by multi-strains of meat-borne Salmonella incubated at 20 °C, as well as the composition and distribution of extracellular polymeric substances (EPS), were investigated in situ by combining confocal laser scanning microscopy (CLSM), scanning electron microscope (SEM), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and Raman spectroscopy. A standard laboratory culture medium (tryptic soy broth, TSB) was used and compared with an actual meat substrate (meat thawing-loss broth, MTLB). The results indicated that Salmonella grown in both media were able to form biofilms on stainless steel surfaces via building a three-dimensional structure with multilayers of cells. Although the number of biofilm cells grown in MTLB was less than that in TSB, the cell numbers in MTLB was adequate to form a steady and mature biofilm. Salmonella grown in MTLB showed "cloud-shaped" morphology in the mature biofilm, whereas when grown in TSB appeared "reticular-shaped". The ATR-FTIR and Raman analysis revealed a completely different chemical composition between biofilms and the corresponding planktonic cells, and some important differences in biofilms grown in MTLB and in TSB. Importantly, our findings suggested that the progress towards a mature Salmonella biofilm on stainless steel surfaces may be associated with the production of the EPS matrix, mainly consisting of polysaccharides and proteins, which may serve as useful markers of biofilm formation. Our work indicated that a combination of these non-destructive techniques provided new insights into the formation of Salmonella biofilm matrix. © 2013.

The impact of N- and O-glycosylation on the functions of Glut-1 transporter in human thyroid anaplastic cells.

PubMed

Samih, Nezha; Hovsepian, Sonia; Notel, Frédéric; Prorok, Maëlle; Zattara-Cannoni, Hélène; Mathieu, Sylvie; Lombardo, Dominique; Fayet, Guy; El-Battari, Assou

2003-04-07

It has been previously shown that glucose transporter Glut-1 expression was detectable by immunostaining in tissue sections from anaplastic carcinoma, but not in normal thyroid tissue. Using human thyroid anaplastic carcinoma cells, we studied the mechanism by which Glut-1 molecules are translocated from the endoplasmic reticulum to the cell surface. The contribution of N- and O-linked glycans for the translocation and activity of Glut-1 transporter is emphasized. The inhibition of N-glycosylation with tunicamycin (TM) led to a 50% decrease in glucose transport while glycosylated and unglycosylated forms of Glut-1 were found at the cell surface. However, the inhibition of N-linked oligosaccharide processing with deoxymannojirimycin (dMJ) and swainsonine (SW) influenced neither the intracellular trafficking nor the activity of the transporter. On the other hand, Glut-1 bound to the O-linked glycan-specific lectin jacalin and the O-glycosylation inhibitor benzyl-N-acetylgalactosamine dramatically inhibited glucose transport. These results show that O- and N-linked oligosaccharides arbored by Glut-1 are essential for glucose transport in anaplastic carcinoma cells. The quantitative and qualitative alterations of Glut-1 glycosylation and the increase in glucose transport are associated with the anaplastic phenotype of human thyroid cells.

Attachment of Escherichia coli O157:H7 grown in tryptic soy broth and nutrient broth to apple and lettuce surfaces as related to cell hydrophobicity, surface charge, and capsule production.

PubMed

Hassan, A N; Frank, J F

2004-10-01

This study investigated the effect of growth in tryptic soy broth (TSB) and nutrient broth (NB) on the ability Escherichia coli O157:H7 to attach to lettuce and apple surfaces. In addition, cell surface hydrophobicity, charge and capsule production were determined on cells grown in these media. Cells grown in NB attached less to lettuce and apple surfaces than did those grown in TSB. TSB, but not NB, supported capsule production by E. coli O157:H7. Cells grown in TSB were more hydrophilic than those grown in NB. No difference was found in the electrokinetic properties of cells grown in these media. Electrostatic and hydrophobic interactions and surface proteins did not appear to play an important role in the attachment of E. coli O157:H7 to these surfaces. Of the factors studied, only capsule production was associated with attachment ability. Copyright 2003 Elsevier B.V.

Single-Cell RNA Sequencing of Human T Cells.

PubMed

Villani, Alexandra-Chloé; Shekhar, Karthik

2017-01-01

Understanding how populations of human T cells leverage cellular heterogeneity, plasticity, and diversity to achieve a wide range of functional flexibility, particularly during dynamic processes such as development, differentiation, and antigenic response, is a core challenge that is well suited for single-cell analysis. Hypothesis-free evaluation of cellular states and subpopulations by transcriptional profiling of single T cells can identify relationships that may be obscured by targeted approaches such as FACS sorting on cell-surface antigens, or bulk expression analysis. While this approach is relevant to all cell types, it is of particular interest in the study of T cells for which classical phenotypic criteria are now viewed as insufficient for distinguishing different T cell subtypes and transitional states, and defining the changes associated with dysfunctional T cell states in autoimmunity and tumor-related exhaustion. This unit describes a protocol to generate single-cell transcriptomic libraries of human blood CD4 + and CD8 + T cells, and also introduces the basic bioinformatic steps to process the resulting sequence data for further computational analysis. We show how cellular subpopulations can be identified from transcriptional data, and derive characteristic gene expression signatures that distinguish these states. We believe single-cell RNA-seq is a powerful technique to study the cellular heterogeneity in complex tissues, a paradigm that will be of great value for the immune system.

Endocytosis of Nanoscale Systems for Cancer Treatments.

PubMed

Chen, Kai; Li, Xue; Zhu, Hongyan; Gong, Qiyong; Luo, Kui

2017-04-28

Advances of nanoscale systems for cancer treatment have been involved in enabling highly regulated site-specific localization to sub cellular organelles hidden beneath cell membranes. Thus far, the cellular entry of these nanoscale systems has been not fully understood. Endocytosisis a form of active transport in which cell transports elected extracellular molecules (such as proteins, viruses, micro-organisms and nanoscale systems) are allowed into cell interiors by engulfing them in an energy-dependent process. This process appears at the plasma membrane surface and contains internalization of the cell membrane as well as the membrane proteins and lipids of cell. There are multiform pathways of endocytosis for nanoscale systems. Further comprehension for the mechanisms of endocytosis is achieved with a combination of efficient genetic manipulations, cell dynamic imaging, and chemical endocytosis inhibitors. This review provides an account of various endocytic pathways, itemizes current methods to study endocytosis of nanoscale systems, discusses some factors associated with cellular uptake for nanoscale systems and introduces the trafficking behavior for nanoscale systems with active targeting. An insight into the endocytosis mechanism is urgent and significant for developing safe and efficient nanoscale systems for cancer diagnosis and therapy. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

New insights into the roles of megalin/LRP2 and the regulation of its functional expression.

PubMed

Marzolo, María-Paz; Farfán, Pamela

2011-01-01

Since the discovery of the low-density lipoprotein receptor (LDLR) and its association with familial hypercholesterolemia in the early 1980s, a family of structurally related proteins has been discovered that has apolipoprotein E as a common ligand, and the broad functions of its members have been described. LRP2, or megalin, is a member of the LDLR family and was initially called gp330. Megalin is an endocytic receptor expressed on the apical surface of several epithelial cells that internalizes a variety of ligands including nutrients, hormones and their carrier proteins, signaling molecules, morphogens, and extracellular matrix proteins. Once internalized, these ligands are directed to the lysosomal degradation pathway or transported by transcytosis from one side of the cell to the opposite membrane. The availability of megalin at the cell surface is controlled by several regulatory mechanisms, including the phosphorylation of its cytoplasmic domain by GSK3, the proteolysis of the extracellular domain at the cell surface (shedding), the subsequent intramembrane proteolysis of the transmembrane domain by the gamma-secretase complex, and exosome secretion. Based on the important roles of its ligands and its tissue expression pattern, megalin has been recognized as an important component of many pathological conditions, including diabetic nephropathy, Lowe syndrome, Dent disease, Alzheimer's disease (AD) and gallstone disease. In addition, the expression of megalin and some of its ligands in the central and peripheral nervous system suggests a role for this receptor in neural regeneration processes. Despite its obvious importance, the regulation of megalin expression is poorly understood. In this review, we describe the functions of megalin and its association with certain pathological conditions as well as the current understanding of the mechanisms that underlie the control of megalin expression.

A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation

NASA Astrophysics Data System (ADS)

Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

2016-10-01

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2kBT, in the range of previously proposed rough parts of landscapes models during dissociation.

A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation

PubMed Central

Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

2016-01-01

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2kBT, in the range of previously proposed rough parts of landscapes models during dissociation. PMID:27731375

A Rough Energy Landscape to Describe Surface-Linked Antibody and Antigen Bond Formation.

PubMed

Limozin, Laurent; Bongrand, Pierre; Robert, Philippe

2016-10-12

Antibodies and B cell receptors often bind their antigen at cell-cell interface while both molecular species are surface-bound, which impacts bond kinetics and function. Despite the description of complex energy landscapes for dissociation kinetics which may also result in significantly different association kinetics, surface-bound molecule (2D) association kinetics usually remain described by an on-rate due to crossing of a single free energy barrier, and few experimental works have measured association kinetics under conditions implying force and two-dimensional relative ligand-receptor motion. We use a new laminar flow chamber to measure 2D bond formation with systematic variation of the distribution of encounter durations between antigen and antibody, in a range from 0.1 to 10 ms. Under physiologically relevant forces, 2D association is 100-fold slower than 3D association as studied by surface plasmon resonance assays. Supported by brownian dynamics simulations, our results show that a minimal encounter duration is required for 2D association; an energy landscape featuring a rough initial part might be a reasonable way of accounting for this. By systematically varying the temperature of our experiments, we evaluate roughness at 2k B T, in the range of previously proposed rough parts of landscapes models during dissociation.

Laser-assisted selection and passaging of human pluripotent stem cell colonies.

PubMed

Terstegge, Stefanie; Rath, Barbara H; Laufenberg, Iris; Limbach, Nina; Buchstaller, Andrea; Schütze, Karin; Brüstle, Oliver

2009-09-10

The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6+/-8.7% of the cells remained viable as compared to 88.6+/-1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8+/-4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9+/-3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.

New Roles of the Primary Cilium in Autophagy

PubMed Central

Ávalos, Yenniffer; Peña-Oyarzun, Daniel; Budini, Mauricio

2017-01-01

The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies. PMID:28913352

New Roles of the Primary Cilium in Autophagy.

PubMed

Ávalos, Yenniffer; Peña-Oyarzun, Daniel; Budini, Mauricio; Morselli, Eugenia; Criollo, Alfredo

2017-01-01

The primary cilium is a nonmotile organelle that emanates from the surface of multiple cell types and receives signals from the environment to regulate intracellular signaling pathways. The presence of cilia, as well as their length, is important for proper cell function; shortened, elongated, or absent cilia are associated with pathological conditions. Interestingly, it has recently been shown that the molecular machinery involved in autophagy, the process of recycling of intracellular material to maintain cellular and tissue homeostasis, participates in ciliogenesis. Cilium-dependent signaling is necessary for autophagosome formation and, conversely, autophagy regulates both ciliogenesis and cilium length by degrading specific ciliary proteins. Here, we will discuss the relationship that exists between the two processes at the cellular and molecular level, highlighting what is known about the effects of ciliary dysfunction in the control of energy homeostasis in some ciliopathies.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis.

PubMed

Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

2017-05-01

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick‑end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers.

Correlation of the expression of YY1 and Fas cell surface death receptor with apoptosis of peripheral blood mononuclear cells, and the development of multiple organ dysfunction in children with sepsis

PubMed Central

Reséndiz-Martínez, Judith; Asbun-Bojalil, Juan; Huerta-Yepez, Sara; Vega, Mario

2017-01-01

Multiple organ dysfunction (MOD) is a lethal complication in children with sepsis. Apoptosis of several cell types is involved in this process, and it is associated with increased Fas cell surface death receptor (Fas) expression. As YY1 transcription factor (YY1) negatively regulates the expression of Fas in cancer models, and is associated with the clinical outcome, it may be important in MOD. The present study aimed to determine the association between the expression of Fas, YY1 and apoptosis in children with sepsis, and its association with MOD, these factors were analyzed in 30 pediatric patients that had been diagnosed with sepsis. Peripheral blood mononuclear cells were purified from patients, and YY1 and Fas protein expression was assessed by immunocytochemistry. Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling. Sepsis was monitored using clinical parameters, pediatric logistic organ dysfunction (PELOD) score and the pediatric mortality index. The results demonstrated that Fas expression was directly correlated with apoptosis levels and the expression of YY1 was inversely correlated with apoptosis levels. Patients with high levels of apoptosis exhibited increased disease severity and poor clinical outcome. Notably, the findings of the present study demonstrated that there were higher survival rates in patients with high YY1 expression, compared with those with low YY1 expression. Additionally, patients with MOD exhibited lower proportions of apoptotic cells compared with sepsis patients without MOD. Furthermore, the PELOD score was positively correlated with Fas and inversely correlated with YY1 expression. Finally, high apoptosis and low YY1 expression were prognostic factors associated with poor survival rates. These data suggested that YY1 may be important for apoptosis induction via the regulation of Fas during sepsis. Therefore, Fas may be a potential therapeutic target to prevent MOD through regulation of YY1 expression. Furthermore, YY1 and Fas expression in PBMCs may be used to as prognostic markers. PMID:28447715

Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

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

Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert

2012-09-17

The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pHmore » 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.« less

RhoA-Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography Dependent.

PubMed

Ogino, Yoichiro; Liang, Ruiwei; Mendonça, Daniela B S; Mendonça, Gustavo; Nagasawa, Masako; Koyano, Kiyoshi; Cooper, Lyndon F

2016-03-01

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho-family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA-mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and -mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano-scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. © 2015 Wiley Periodicals, Inc.

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

Physicochemical changes of microbe and solid surface properties during biofilm formation

NASA Astrophysics Data System (ADS)

Sfaelou, Stavroula; Vakros, John; Manariotis, Ioannis D.; Karapanagioti, Hrissi K.

2013-04-01

Cell immobilization is a promising biotechnology process. For example, entrapment of bacteria cells on synthetic polymeric matrices such as biocarriers is widely used for wastewater treatment because they have strong mechanical strength and durability in contrast to natural polymers. This method is based on the formation of biofilm on the surface of the used carriers and combines two different processes; attached and suspended biomass in a hybrid system. Previous studies have shown that immobilized cell systems have the potential to degrade toxic chemicals faster than conventional wastewater treatment systems because high densities of specialized microorganisms are used in immobilized cell systems. The present study elucidates the surface charge and properties of activated sludge and their role in the formation of biofilm. This information can be used for the optimization of the formation of biofilms as well as for the study of the transport of microorganisms in different environments. The two types of biocarriers that were used in this study are polyvinyl alcohol (PVA)-gel beads and Moving Bed Biofilm Reactor (MBBR) carriers. The sludge samples that were investigated were taken from the aeration tank of the wastewater treatment plant of University of Patras (Greece). Measurements of the surface charge of the sludge, the biocarriers and the formed biofilm, were performed using potentiometric mass titrations with different kinds of electrolytes (e.g. NaCl, NaNO3) and at pH ranging from 3 to 11. The determination of pzc and surface charge of activated sludge and biocarriers is significant, because it can provide new valuable informations about the interaction mechanisms and the formation of biofilms. In each case, the point of zero charge (pzc) was identified as the common intersection point of the potentiometric curve of the blank solution of the electrolyte with the corresponding curves of each material. The pzc value for the biofilm was 6.1 to 6.7 and 6.6 to 6.9 for PVA gel and MBBR, respectively. These values differ both from the pzc values found for PVA biocarriers (pzc = 9.4; no pzc value was obtained for MBBR as expected based on its hydrophobic nature and the absence of surface groups with acid-base behavior) and the pzc value of activated sludge (activated sludge mixed liquor: pzc = 8.0 to 8.2, solid activated sludge: pzc = 7.2 to 7.3). These results lead us to the conclusion that the formed biofilms have different acid-base behavior and properties in relation to the activated sludge and the biocarriers. This fact is in accordance to previous studies, where biofilm-associated cells can be differentiated from their suspended counterparts due to the generation of an extracellular polymeric substance (EPS) matrix. One other possible explanation is that the complicated processes of the biofilm formation can alter the distribution of different cells in the sludge compared with the cell distribution in the suspended unsupported sludge.

Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions.

PubMed

Shrivastava, Amulya Nidhi; Aperia, Anita; Melki, Ronald; Triller, Antoine

2017-07-05

Several neurodegenerative disorders, such as Alzheimer's and Parkinson's disease, are characterized by prominent loss of synapses and neurons associated with the presence of abnormally structured or misfolded protein assemblies. Cell-to-cell transfer of misfolded proteins has been proposed for the intra-cerebral propagation of these diseases. When released, misfolded proteins diffuse in the 3D extracellular space before binding to the plasma membrane of neighboring cells, where they diffuse on a 2D plane. This reduction in diffusion dimension and the cell surface molecular crowding promote deleterious interactions with native membrane proteins, favoring clustering and further aggregation of misfolded protein assemblies. These processes open up new avenues for therapeutics development targeting the initial interactions of deleterious proteins with the plasma membrane or the subsequent pathological signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of rainfall and surface flow on chemical diffusion from soil to runoff water

USDA-ARS?s Scientific Manuscript database

Although basic processes of diffusion and convection have been used to quantify chemical transport from soil to surface runoff, there are little research results actually showing how these processes were affected by rainfall and surface flow. We developed a laboratory flow cell and a sequence of exp...

Type I collagen-induced YAP nuclear expression promotes primary cilia growth and contributes to cell migration in confluent mouse embryo fibroblast 3T3-L1 cells.

PubMed

Xu, Qian; Liu, Xiaoling; Liu, Weiwei; Hayashi, Toshihiko; Yamato, Masayuki; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Ikejima, Takashi

2018-05-30

The extracellular matrix (ECM) is a major biomechanical environment for all cells in vivo, and tightly controls wound healing and cancer progression. Type I collagen (Col I) is the most abundant component in ECM and plays an essential role for cell motility control and migration beyond structural support. Our previous results showed that Col I increased the length of primary cilia and the expression of primary cilia-associated proteins in 3T3-L1 cells. The Hippo/YAP pathway serves as a major integrator of cell surface-mediated signals and regulates key processes for the development and maintenance of tissue functions. In this study, we investigated the role of Hippo/YAP signaling in primary cilia growth of cells cultured on Col I-coated plate, as well as the potential link between primary cilia and migration. At 2-day post-confluence, YAP localization in the nucleus was dramatically increased when the cells were cultured on Col I-coated plate, accompanied by cilia growth. YAP inhibitor verteporfin repressed the growth of primary cilia as well as the expressions of ciliogenesis-associated proteins in confluent 3T3-L1 cells cultured on Col I-coated plate. Moreover, knockdown of either YAP or IFT88, one of the ciliogenesis-associated proteins, reversed the migration of confluent 3T3-L1 cells promoted by Col I-coating. In conclusion, activation of YAP pathway by Col I-coating of culture plate for confluent 3T3-L1 cells is positively associated with the primary cilia growth, which eventually results in promoted migration.

Surface Expression of Precursor N-cadherin Promotes Tumor Cell Invasion12

PubMed Central

Maret, Deborah; Gruzglin, Eugenia; Sadr, Mohamad Seyed; Siu, Vincent; Shan, Weisong; Koch, Alexander W; Seidah, Nabil G; Del Maestro, Rolando F; Colman, David R

2010-01-01

The expression of N-cadherin (NCAD) has been shown to correlate with increased tumor cell motility and metastasis. However, NCAD-mediated adhesion is a robust phenomenon and therefore seems to be inconsistent with the “release” from intercellular adhesion required for invasion. We show that in the most invasive melanoma and brain tumor cells, altered posttranslational processing results in abundant nonadhesive precursor N-cadherin (proNCAD) at the cell surface, although total NCAD levels remain constant. We demonstrate that aberrantly processed proNCAD promotes cell migration and invasion in vitro. Furthermore, in human tumor specimens, we find high levels of proNCAD as well, supporting an overall conclusion that proNCAD and mature NCAD coexist on these tumor cell surfaces and that it is the ratio between these functionally antagonistic moieties that directly correlates with invasion potential. Our work provides insight into what may be a widespread mechanism for invasion and metastasis and challenges the current dogma of the functional roles played by classic cadherins in tumor progression. PMID:21170270

[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.

Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

PubMed Central

1979-01-01

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity. PMID:512629

Proteomic Analysis of Serum Opsonins Impacting Biodistribution and Cellular Association of Porous Silicon Microparticles

PubMed Central

Serda, Rita E.; Blanco, Elvin; Mack, Aaron; Stafford, Susan J.; Amra, Sarah; Li, Qingpo; van de Ven, Anne L.; Tanaka, Takemi; Torchilin, Vladimir P.; Wiktorowicz, John E.; Ferrari, Mauro

2014-01-01

Mass transport of drug delivery vehicles is guided by particle properties, such as shape, composition and surface chemistry, as well as biomolecules and serum proteins that adsorb to the particle surface. In an attempt to identify serum proteins influencing cellular associations and biodistribution of intravascularly injected particles, we used two dimensional gel electrophoresis and mass spectrometry to identify proteins eluted from the surface of cationic and anionic silicon microparticles. Cationic microparticles displayed a 25-fold greater abundance of Ig light chain variable region, fibrinogen, and complement component 1 compared to their anionic counterparts. The anionic-surface favored equal accumulation of microparticles in the liver and spleen, while cationic-surfaces favored preferential accumulation in the spleen. Immunohistochemistry supported macrophage internalization of both anionic and cationic silicon microparticles in the liver, as well as evidence of association of cationic microparticles with hepatic endothelial cells. Furthermore, scanning electron micrographs supported cellular competition for cationic microparticles by endothelial cells and macrophages. Despite high macrophage content in the lungs and tumor, microparticle uptake by these cells was minimal, supporting differences in the repertoire of surface receptors expressed by tissue-specific macrophages. In summary, particle surface chemistry drives selective binding of serum components impacting cellular interactions and biodistribution. PMID:21303614

Spectroellipsometric, AFM and XPS probing of stainless steel surfaces subjected to biological influences

NASA Astrophysics Data System (ADS)

Vinnichenko, M.; Chevolleau, Th; Pham, M. T.; Poperenko, L.; Maitz, M. F.

2002-11-01

Surface modification of austenitic stainless steel (SS) 316L after incubation in growing cell cultures and cell-free media as control has been studied. The following treatments were applied: mouse fibrosarcoma cells L929 for 3 and 7 days, polymorphonuclear neutrophils for 3 and 7 days and human osteosarcoma cells SAOS-2 for 7 and 14 days. Cells were enzymatically removed in all cases. The modified surfaces were probed in comparison with untreated ones by means of spectroscopic ellipsometry (SE), X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM). XPS shows the appearance of the peak of bonded nitrogen at 400.5 eV characteristic for adsorbed proteins on the surface for each type of cells and for the cell-free medium. Migration of Ni in the adsorbed layer is observed in all cases for samples after the cell cultures. The protein layer thickness is ellipsometrically determined to be within 2.5-6.0 nm for all treated samples with parameterization of its optical constants in Cauchy approach. The study showed that for such biological treatments of the SS the protein layer adsorption is the dominating process in the first 2 weeks, which could play a role in the process of corrosion by complex forming properties with metal ions.

The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

PubMed Central

Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

2015-01-01

TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

In-depth investigation of spin-on doped solar cells with thermally grown oxide passivation

NASA Astrophysics Data System (ADS)

Ahmad, Samir Mahmmod; Cheow, Siu Leong; Ludin, Norasikin A.; Sopian, K.; Zaidi, Saleem H.

Solar cell industrial manufacturing, based largely on proven semiconductor processing technologies supported by significant advancements in automation, has reached a plateau in terms of cost and efficiency. However, solar cell manufacturing cost (dollar/watt) is still substantially higher than fossil fuels. The route to lowering cost may not lie with continuing automation and economies of scale. Alternate fabrication processes with lower cost and environmental-sustainability coupled with self-reliance, simplicity, and affordability may lead to price compatibility with carbon-based fuels. In this paper, a custom-designed formulation of phosphoric acid has been investigated, for n-type doping in p-type substrates, as a function of concentration and drive-in temperature. For post-diffusion surface passivation and anti-reflection, thermally-grown oxide films in 50-150-nm thickness were grown. These fabrication methods facilitate process simplicity, reduced costs, and environmental sustainability by elimination of poisonous chemicals and toxic gases (POCl3, SiH4, NH3). Simultaneous fire-through contact formation process based on screen-printed front surface Ag and back surface through thermally grown oxide films was optimized as a function of the peak temperature in conveyor belt furnace. Highest efficiency solar cells fabricated exhibited efficiency of ∼13%. Analysis of results based on internal quantum efficiency and minority carried measurements reveals three contributing factors: high front surface recombination, low minority carrier lifetime, and higher reflection. Solar cell simulations based on PC1D showed that, with improved passivation, lower reflection, and high lifetimes, efficiency can be enhanced to match with commercially-produced PECVD SiN-coated solar cells.

Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces – A Novel Method of Enhancing Flow-Resistant Cell Adhesion

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; McCracken, Katherine E.; Riley, Mark R.

2014-01-01

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear. PMID:23225491

Molecular cloning and characterization of a tumor-associated, growth-related, and time-keeping hydroquinone (NADH) oxidase (tNOX) of the HeLa cell surface

NASA Technical Reports Server (NTRS)

Chueh, Pin-Ju; Kim, Chinpal; Cho, NaMi; Morre, Dorothy M.; Morre, D. James

2002-01-01

NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit prion-like properties. The two enzymatic activities alternate to generate a regular period length of about 24 min. Here we report the expression, cloning, and characterization of a tumor-associated NADH oxidase (tNOX). The cDNA sequence of 1830 bp is located on gene Xq25-26 with an open reading frame encoding 610 amino acids. The activities of the bacterially expressed tNOX oscillate with a period length of 22 min as is characteristic of tNOX activities in situ. The activities are inhibited completely by capsaicin, which represents a defining characteristic of tNOX activity. Functional motifs identified by site-directed mutagenesis within the C-terminal portion of the tNOX protein corresponding to the processed plasma membrane-associated form include quinone (capsaicin), copper and adenine nucleotide binding domains, and two cysteines essential for catalytic activity. Four of the six cysteine to alanine replacements retained enzymatic activity, but the period lengths of the oscillations were increased. A single protein with two alternating enzymatic activities indicative of a time-keeping function is unprecedented in the biochemical literature.

A novel electroless method to prepare a platinum electrocatalyst on diamond for fuel cell applications

NASA Astrophysics Data System (ADS)

Lyu, Xiao; Hu, Jingping; Foord, John S.; Wang, Qiang

2013-11-01

A novel electroless deposition method was demonstrated to prepare a platinum electrocatalyst on boron doped diamond (BDD) substrates without the need for pre-activation. This green method addresses the uniformity and particle size issues associated with electrodeposition and circumvents the pre-activation procedure which is necessary for conventional electroless deposition. The inert BDD substrate formed a galvanic couple with an iron wire, to overcome the activation barrier associated with conventional electroless deposition on diamond, leading to the formation of Pt nanoparticles on the electrode surface in a galvanic process coupled to a chemical process. When sodium hypophosphite was employed as the reducing agent to drive the electroless reaction Pt deposits which were contaminated with iron and phosphorus resulted. In contrast, the reducing agent ascorbic acid gave rise to high purity Pt nanoparticles. Optimal deposition conditions with respect to bath temperature, pH value and stabilizing additives are identified. Using this approach, high purity and uniformly distributed platinum nanoparticles are obtained on the diamond electrode surface, which demonstrate a high electrochemical activity towards methanol oxidation.

CVD-Based Valence-Mending Passivation for Crystalline-Si Solar Cells

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

Tao, Meng

2015-03-01

The objective of this project is to investigate a new surface passivation technique, valence-mending passivation, for its applications in crystalline-Si solar cells to achieve significant efficiency improvement and cost reduction. As the enabling technique, the project includes the development of chemical vapor deposition recipes to passivate textured Si(100) and multicrystalline-Si surfaces by sulfur and the characterization of the passivated Si surfaces, including thermal stability, Schottky barrier height, contact resistance and surface recombination. One important application is to replace the Ag finger electrode in Si cells with Al to reduce cost, by ~$0.1/Wp, and allow terawatt-scale deployment of crystalline-Si solar cells.more » These all-Al Si cells require a low-temperature metallization process for the Al electrode, to be compatible with valence-mending passivation and to prevent Al diffusion into n-type Si. Another application is to explore valence-mending passivation of grain boundaries in multicrystalline Si by diffusing sulfur into grain boundaries, to reduce the efficiency gas between monocrystalline-Si solar cells and multicrystalline-Si cells. The major accomplishments of this project include: 1) Demonstration of chemical vapor deposition processes for valence-mending passivation of both monocrystalline Si(100) and multicrystalline Si surfaces. Record Schottky barriers have been demonstrated, with the new record-low barrier of less than 0.08 eV between Al and sulfur-passivated n-type Si(100) and the new record-high barrier of 1.14 eV between Al and sulfur-passivated p-type Si(100). On the textured p-type monocrystalline Si(100) surface, the highest barrier with Al is 0.85 eV by valence-mending passivation. 2) Demonstration of a low-temperature metallization process for Al in crystalline-Si solar cells. The new metallization process is based on electroplating of Al in a room-temperature ionic liquid. The resistivity of the electroplated Al is ~7×10–6 ohm-cm, similar to that of screen-printed Ag. 3) Demonstration of two all-Al, Ag-free Si solar cells, with an electroplated Al front electrode and a screen-printed Al back electrode. One cell is an industrial p-type front-emitter cell, and the other is an n-type back-emitter cell. The efficiency of the p-type cell is close to 15%. This is an industrial cell and its efficiency is capped at ~18%. 4) Demonstration of grain boundary passivation by both hydrogen and sulfur using hydrogen sulfide (H2S). When the new grain boundary passivation is combined with Al2O3 surface passivation and post-annealing, the minority carrier lifetime in the p-type multicrystalline Si samples shows a significant improvement up to 68 fold. 5) In a side project, a simple green process is developed which is capable of recycling over 90% of the Si material in end-of-life crystalline-Si solar cells. The recycled Si meets the specifications for solar-grade Si and can be used as a new poly-Si feedstock for ingot growth.« less

Study of process variables associated with manufacturing hermetically-sealed nickel-cadmium cells

NASA Technical Reports Server (NTRS)

Miller, L.

1974-01-01

A two year study of the major process variables associated with the manufacturing process for sealed, nickel-cadmium, areospace cells is summarized. Effort was directed toward identifying the major process variables associated with a manufacturing process, experimentally assessing each variable's effect, and imposing the necessary changes (optimization) and controls for the critical process variables to improve results and uniformity. A critical process variable associated with the sintered nickel plaque manufacturing process was identified as the manual forming operation. Critical process variables identified with the positive electrode impregnation/polarization process were impregnation solution temperature, free acid content, vacuum impregnation, and sintered plaque strength. Positive and negative electrodes were identified as a major source of carbonate contamination in sealed cells.

In situ monitoring of biomolecular processes in living systems using surface-enhanced Raman scattering

NASA Astrophysics Data System (ADS)

Altunbek, Mine; Kelestemur, Seda; Culha, Mustafa

2015-12-01

Surface-enhanced Raman scattering (SERS) continues to strive to gather molecular level information from dynamic biological systems. It is our ongoing effort to utilize the technique for understanding of the biomolecular processes in living systems such as eukaryotic and prokaryotic cells. In this study, the technique is investigated to identify cell death mechanisms in 2D and 3D in vitro cell culture models, which is a very important process in tissue engineering and pharmaceutical applications. Second, in situ biofilm formation monitoring is investigated to understand how microorganisms respond to the environmental stimuli, which inferred information can be used to interfere with biofilm formation and fight against their pathogenic activity.

Physical association and functional interaction between beta1 integrin and CD98 on human T lymphocytes

NASA Technical Reports Server (NTRS)

Miyamoto, Yuko J.; Mitchell, Jason S.; McIntyre, Bradley W.

2003-01-01

CD98 is a cell surface protein previously characterized as a cell activation marker, an amino acid transporter, and has recently been implicated in integrin-related functions. Integrins are cell surface proteins, important for homotypic cell aggregation, cell adhesion, and coactivation of T lymphocytes. We have previously shown that the anti-CD98 mAb 80A10, when coimmobilized with anti-CD3 mAb OKT3, is able to mediate human T cell coactivation that is inhibited by anti-beta1 integrin specific mAb 18D3. These results indicated a functional association of CD98 and beta1 integrin signaling but left open the question of a physical association. We now show the induction of homotypic aggregation through CD98 among human T cells and this aggregation was inhibited by anti-beta1 integrin mAb. Therefore, CD98-dependent lymphocyte proliferation and adhesion may involve integrins. Competitive binding assays and fluorescence colocalization analysis suggested that CD98 and beta1 integrin were physically associated. Differential extraction techniques and immunoprecipitations provided the first evidence that the alpha4beta1 integrin and CD98 are specifically associated on human T lymphocytes.

Alkane-grown Beauveria bassiana produce mycelial pellets displaying peroxisome proliferation, oxidative stress, and cell surface alterations.

PubMed

Huarte-Bonnet, Carla; Paixão, Flávia R S; Ponce, Juan C; Santana, Marianela; Prieto, Eduardo D; Pedrini, Nicolás

2018-06-01

The entomopathogenic fungus Beauveria bassiana is able to grow on insect cuticle hydrocarbons, inducing alkane assimilation pathways and concomitantly increasing virulence against insect hosts. In this study, we describe some physiological and molecular processes implicated in growth, nutritional stress response, and cellular alterations found in alkane-grown fungi. The fungal cytology was investigated using light and transmission electron microscopy while the surface topography was examined using atomic force microscopy. Additionally, the expression pattern of several genes associated with oxidative stress, peroxisome biogenesis, and hydrophobicity were analysed by qPCR. We found a novel type of growth in alkane-cultured B. bassiana similar to mycelial pellets described in other alkane-free fungi, which were able to produce viable conidia and to be pathogenic against larvae of the beetles Tenebrio molitor and Tribolium castaneum. Mycelial pellets were formed by hyphae cumulates with high peroxidase activity, exhibiting peroxisome proliferation and an apparent surface thickening. Alkane-grown conidia appeared to be more hydrophobic and cell surfaces displayed different topography than glucose-grown cells. We also found a significant induction in several genes encoding for peroxins, catalases, superoxide dismutases, and hydrophobins. These results show that both morphological and metabolic changes are triggered in mycelial pellets derived from alkane-grown B. bassiana. Copyright © 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Impact of environmental factors on the culturability and viability of Listeria monocytogenes under conditions encountered in food processing plants.

PubMed

Overney, Anaïs; Jacques-André-Coquin, Joséphine; Ng, Patricia; Carpentier, Brigitte; Guillier, Laurent; Firmesse, Olivier

2017-03-06

The ability of Listeria monocytogenes to adhere to and persist on surfaces for months or even years may be responsible for its transmission from contaminated surfaces to food products. Hence the necessity to find effective means to prevent the establishment of L. monocytogenes in food processing environments. The aim of this study was to assess, through a fractional experimental design, the environmental factors that could affect the survival of L. monocytogenes cells on surfaces to thereby prevent the persistence of this pathogen in conditions mimicking those encountered in food processing plants: culture with smoked salmon juice or meat exudate, use of two materials with different hygiene status, biofilm of L. monocytogenes in pure-culture or dual-culture with a Pseudomonas fluorescens strain, application of a drying step after cleaning and disinfection (C&D) and comparison of two strains of L. monocytogenes. Bacterial survival was assessed by culture, qPCR to quantify total cells, and propidium monoazide coupled with qPCR to quantify viable cells and highlight viable but non-culturable (VBNC) cells. Our results showed that failure to apply C&D causes cell persistence on surfaces. Moreover, the sanitation procedure leads only to a loss of culturability and appearance of VBNC populations. However, an additional daily drying step after C&D optimises the effectiveness of these procedures to reduce culturable populations. Our results reinforce the importance to use molecular tools to monitor viable pathogens in food processing plants to avoid underestimating the amounts of cells using only methods based on cell culture. Copyright © 2017 Elsevier B.V. All rights reserved.

Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

PubMed

Anastasia, Luigi; Holguera, Javier; Bianchi, Anna; D'Avila, Francesca; Papini, Nadia; Tringali, Cristina; Monti, Eugenio; Villar, Enrique; Venerando, Bruno; Muñoz-Barroso, Isabel; Tettamanti, Guido

2008-03-01

The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

MEMBRANE-TYPE 1 MATRIX METALLOPROTEINASE DOWNREGULATES FIBROBLAST GROWTH FACTOR-2 BINDING TO THE CELL SURFACE AND INTRACELLULAR SIGNALING

PubMed Central

Tassone, Evelyne; Valacca, Cristina; Mignatti, Paolo

2014-01-01

Membrane-type 1 matrix metalloproteinase (MT1-MMP, MMP-14), a transmembrane proteinase with an extracellular catalytic domain and a short cytoplasmic tail, degrades extracellular matrix components and controls diverse cell functions through proteolytic and non-proteolytic interactions with extracellular, intracellular and transmembrane proteins. Here we show that in tumor cells MT1-MMP downregulates fibroblast growth factor-2 (FGF-2) signaling by reducing the amount of FGF-2 bound to the cell surface with high and low affinity. FGF-2 induces weaker activation of ERK1/2 MAP kinase in MT1-MMP expressing cells than in cells devoid of MT1-MMP. This effect is abolished in cells that express proteolytically inactive MT1-MMP but persists in cells expressing MT1-MMP mutants devoid of hemopexin-like or cytoplasmic domain, showing that FGF-2 signaling is downregulated by MT1-MMP proteolytic activity. MT1-MMP expression results in downregulation of FGFR-1 and -4, and in decreased amount of cell surface-associated FGF-2. In addition, MT1-MMP strongly reduces the amount of FGF-2 bound to the cell surface with low affinity. Because FGF-2 association with low-affinity binding sites is a prerequisite for binding to its high-affinity receptors, downregulation of low-affinity binding to the cell surface results in decreased FGF-2 signaling. Consistent with this conclusion, FGF-2 induction of tumor cell migration and invasion in vitro is stronger in cells devoid of MT1-MMP than in MT1-MMP expressing cells. Thus, MT1-MMP controls FGF-2 signaling by a proteolytic mechanism that decreases the cell’s biological response to FGF-2. PMID:24986796

Nanotopography Promotes Pancreatic Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

PubMed

Kim, Jong Hyun; Kim, Hyung Woo; Cha, Kyoung Je; Han, Jiyou; Jang, Yu Jin; Kim, Dong Sung; Kim, Jong-Hoon

2016-03-22

Although previous studies suggest that nanotopographical features influence properties and behaviors of stem cells, only a few studies have attempted to derive clinically useful somatic cells from human pluripotent stem cells using nanopatterned surfaces. In the present study, we report that polystyrene nanopore-patterned surfaces significantly promote the pancreatic differentiation of human embryonic and induced pluripotent stem cells. We compared different diameters of nanopores and showed that 200 nm nanopore-patterned surfaces highly upregulated the expression of PDX1, a critical transcription factor for pancreatic development, leading to an approximately 3-fold increase in the percentage of differentiating PDX1(+) pancreatic progenitors compared with control flat surfaces. Furthermore, in the presence of biochemical factors, 200 nm nanopore-patterned surfaces profoundly enhanced the derivation of pancreatic endocrine cells producing insulin, glucagon, or somatostatin. We also demonstrate that nanopore-patterned surface-induced upregulation of PDX1 is associated with downregulation of TAZ, suggesting the potential role of TAZ in nanopore-patterned surface-mediated mechanotransduction. Our study suggests that appropriate cytokine treatments combined with nanotopographical stimulation could be a powerful tool for deriving a high purity of desired cells from human pluripotent stem cells.

Sensor to detect endothelialization on an active coronary stent

PubMed Central

2010-01-01

Background A serious complication with drug-eluting coronary stents is late thrombosis, caused by exposed stent struts not covered by endothelial cells in the healing process. Real-time detection of this healing process could guide physicians for more individualized anti-platelet therapy. Here we present work towards developing a sensor to detect this healing process. Sensors on several stent struts could give information about the heterogeneity of healing across the stent. Methods A piezoelectric microcantilever was insulated with parylene and demonstrated as an endothelialization detector for incorporation within an active coronary stent. After initial characterization, endothelial cells were plated onto the cantilever surface. After they attached to the surface, they caused an increase in mass, and thus a decrease in the resonant frequencies of the cantilever. This shift was then detected electrically with an LCR meter. The self-sensing, self-actuating cantilever does not require an external, optical detection system, thus allowing for implanted applications. Results A cell density of 1300 cells/mm2 on the cantilever surface is detected. Conclusions We have developed a self-actuating, self-sensing device for detecting the presence of endothelial cells on a surface. The device is biocompatible and functions reliably in ionic liquids, making it appropriate for implantable applications. This sensor can be placed along the struts of a coronary stent to detect when the struts have been covered with a layer of endothelial cells and are no longer available surfaces for clot formation. Anti-platelet therapy can be adjusted in real-time with respect to a patient's level of healing and hemorrhaging risks. PMID:21050471

Enhancement of Electrical Properties of Nanostructured Polysilicon Layers Through Hydrogen Passivation.

PubMed

Zhou, D; Xu, T; Lambert, Y; Cristini-Robbe; Stiévenard, D

2015-12-01

The light absorption of polysilicon planar junctions can be improved using nanostructured top surfaces due to their enhanced light harvesting properties. Nevertheless, associated with the higher surface, the roughness caused by plasma etching and defects located at the grain boundary in polysilicon, the concentration of the recombination centers increases, leading to electrical performance deterioration. In this work, we demonstrate that wet oxidation combined with hydrogen passivation using SiN(x):H are the key technological processes to significantly decrease the surface recombination and improve the electrical properties of nanostructured n(+)-i-p junctions. Nanostructured surface is fabricated by nanosphere lithography in a low-cost and controllable approach. Furthermore, it has been demonstrated that the successive annealing of silicon nitride films has significant effect on the passivation quality, resulting in some improvements on the efficiency of the Si nanostructure-based solar cell device.

Pu Anion Exchange Process Intensification

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

Taylor-Pashow, Kathryn M. L.

This research is focused on improving the efficiency of the anion exchange process for purifying plutonium. While initially focused on plutonium, the technology could also be applied to other ion-exchange processes. Work in FY17 focused on the improvement and optimization of porous foam columns that were initially developed in FY16. These foam columns were surface functionalized with poly(4-vinylpyridine) (PVP) to provide the Pu specific anion-exchange sites. Two different polymerization methods were explored for maximizing the surface functionalization with the PVP. The open-celled polymeric foams have large open pores and large surface areas available for sorption. The fluid passes through themore » large open pores of this material, allowing convection to be the dominant mechanism by which mass transport takes place. These materials generally have very low densities, open-celled structures with high cell interconnectivity, small cell sizes, uniform cell size distributions, and high structural integrity. These porous foam columns provide advantages over the typical porous resin beads by eliminating the slow diffusion through resin beads, making the anion-exchange sites easily accessible on the foam surfaces. The best performing samples exceeded the Pu capacity of the commercially available resin, and also offered the advantage of sharper elution profiles, resulting in a more concentrated product, with less loss of material to the dilute heads and tails cuts. An alternate approach to improving the efficiency of this process was also explored through the development of a microchannel array system for performing the anion exchange.« less

Modeling cell adhesion and proliferation: a cellular-automata based approach.

PubMed

Vivas, J; Garzón-Alvarado, D; Cerrolaza, M

Cell adhesion is a process that involves the interaction between the cell membrane and another surface, either a cell or a substrate. Unlike experimental tests, computer models can simulate processes and study the result of experiments in a shorter time and lower costs. One of the tools used to simulate biological processes is the cellular automata, which is a dynamic system that is discrete both in space and time. This work describes a computer model based on cellular automata for the adhesion process and cell proliferation to predict the behavior of a cell population in suspension and adhered to a substrate. The values of the simulated system were obtained through experimental tests on fibroblast monolayer cultures. The results allow us to estimate the cells settling time in culture as well as the adhesion and proliferation time. The change in the cells morphology as the adhesion over the contact surface progress was also observed. The formation of the initial link between cell and the substrate of the adhesion was observed after 100 min where the cell on the substrate retains its spherical morphology during the simulation. The cellular automata model developed is, however, a simplified representation of the steps in the adhesion process and the subsequent proliferation. A combined framework of experimental and computational simulation based on cellular automata was proposed to represent the fibroblast adhesion on substrates and changes in a macro-scale observed in the cell during the adhesion process. The approach showed to be simple and efficient.

Correlation between surface carbon concentration and adhesive strength at the Si cell/EVA interface in a PV module

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

Dhere, N.G.; Wollam, M.E.; Gadre, K.S.

1997-12-31

Silicon solar cell/EVA composite is being studied with an objective to further improve the manufacturing technology of PV modules. Sample extraction and adhesion strength measurement process has been modified. Silicon and EVA samples were extracted from solar cells of new and field-deployed modules. Optical microscopy, SEM, and AES of samples from new modules revealed EVA islands covering most of the silicon cell surface indicating a cohesive failure. A good correlation was observed between the adhesive strength and surface concentration of carbon. A low carbon concentration which indicated less EVA clinging to cell surface always resulted in low adhesive strengths. Themore » correlation provides a simple technique for inferring properties of EVA.« less

In vitro systems for the study of microtubule-based cell polarity in fission yeast.

PubMed

Taberner, Núria; Lof, Andries; Roth, Sophie; Lamers, Dimitry; Zeijlemaker, Hans; Dogterom, Marileen

2015-01-01

Establishment of cell polarity is essential for processes such as growth and division. In fission yeast, as well as other species, polarity factors travel at the ends of microtubules to cortical sites where they associate with the membrane and subsequently maintain a polarized activity pattern despite their ability to diffuse in the membrane. In this chapter we present methods to establish an in vitro system that captures the essential features of this process. This bottom-up approach allows us to identify the minimal molecular requirements for microtubule-based cell polarity. We employ microfabrication techniques combined with surface functionalization to create rigid chambers with affinity for proteins, as well as microfluidic techniques to create and shape emulsion droplets with functionalized lipid boundaries. Preliminary results are shown demonstrating that a properly organized microtubule cytoskeleton can be confined to these confined spaces, and proteins traveling at the ends of growing microtubules can be delivered to their boundaries. Copyright © 2015 Elsevier Inc. All rights reserved.

Suppressing the formation of lipid raft-associated Rac1/PI3K/Akt signaling complexes by curcumin inhibits SDF-1α-induced invasion of human esophageal carcinoma cells.

PubMed

Lin, Meng-Liang; Lu, Yao-Cheng; Chen, Hung-Yi; Lee, Chuan-Chun; Chung, Jing-Gung; Chen, Shih-Shun

2014-05-01

Stromal cell-derived factor-1α (SDF-1α) is a ligand for C-X-C chemokine receptor type 4 (CXCR4), which contributes to the metastasis of cancer cells by promoting cell migration. Here, we show that the SDF-1α/CXCR4 axis can significantly increase invasion of esophageal carcinoma (EC) cells. We accomplished this by examining the effects of CXCR4 knockdown as well as treatment with a CXCR4-neutralizing antibody and the CXCR4-specific inhibitor AMD3100. Curcumin suppressed SDF-1α-induced cell invasion and matrix metalloproteinase-2 (MMP-2) promoter activity, cell surface localization of CXCR4 at lipid rafts, and lipid raft-associated ras-related C3 botulinum toxin substrate 1 (Rac1)/phosphatidylinositol 3-kinase (PI3K) p85α/Akt signaling. Curcumin inhibited SDF-1α-induced cell invasion by suppressing the Rac1-PI3K signaling complex at lipid rafts but did not abrogate lipid raft formation. We further demonstrate that the attenuation of lipid raft-associated Rac1 activity by curcumin was critical for the inhibition of SDF-1α-induced PI3K/Akt/NF-κB activation, cell surface localization of CXCR4 at lipid rafts, MMP-2 promoter activity, and cell invasion. Collectively, our results indicate that curcumin inhibits SDF-1α-induced EC cell invasion by suppressing the formation of the lipid raft-associated Rac1-PI3K-Akt signaling complex, the localization of CXCR4 with lipid rafts at the cell surface, and MMP-2 promoter activity, likely through the inhibition of Rac1 activity. © 2012 Wiley Periodicals, Inc.

The Respiratory Syncytial Virus Phosphoprotein, Matrix Protein, and Fusion Protein Carboxy-Terminal Domain Drive Efficient Filamentous Virus-Like Particle Formation

PubMed Central

Meshram, Chetan D.; Baviskar, Pradyumna S.; Ognibene, Cherie M.

2016-01-01

ABSTRACT Virus-like particles (VLPs) are attractive as a vaccine concept. For human respiratory syncytial virus (hRSV), VLP assembly is poorly understood and appears inefficient. Hence, hRSV antigens are often incorporated into foreign VLP systems to generate anti-RSV vaccine candidates. To better understand the assembly, and ultimately to enable efficient production, of authentic hRSV VLPs, we examined the associated requirements and mechanisms. In a previous analysis in HEp-2 cells, the nucleoprotein (N), phosphoprotein (P), matrix protein (M), and fusion protein (F) were required for formation of filamentous VLPs, which, similar to those of wild-type virus, were associated with the cell surface. Using fluorescence and electron microscopy combined with immunogold labeling, we examined the surfaces of transfected HEp-2 cells and further dissected the process of filamentous VLP formation. Our results show that N is not required. Coexpression of P plus M plus F, but not P plus M, M plus F, or P plus F, induced both viral protein coalescence and formation of filamentous VLPs that resembled wild-type virions. Despite suboptimal coalescence in the absence of P, the M and F proteins, when coexpressed, formed cell surface-associated filaments with abnormal morphology, appearing longer and thinner than wild-type virions. For F, only the carboxy terminus (Fstem) was required, and addition of foreign protein sequences to Fstem allowed incorporation into VLPs. Together, the data show that P, M, and the F carboxy terminus are sufficient for robust viral protein coalescence and filamentous VLP formation and suggest that M-F interaction drives viral filament formation, with P acting as a type of cofactor facilitating the process and exerting control over particle morphology. IMPORTANCE hRSV is responsible for >100,000 deaths in children worldwide, and a vaccine is not available. Among the potential anti-hRSV approaches are virus-like particle (VLP) vaccines, which, based on resemblance to virus or viral components, can induce protective immunity. For hRSV, few reports are available concerning authentic VLP production or testing, in large part because VLP production is inefficient and the mechanisms underlying particle assembly are poorly understood. Here, we took advantage of the cell-associated nature of RSV particles and used high-resolution microscopy analyses to examine the viral proteins required for formation of wild-type-virus-resembling VLPs, the contributions of these proteins to morphology, and the domains involved in incorporation of the antigenically important viral F protein. The results provide new insights that will facilitate future production of hRSV VLPs with defined shapes and compositions and may translate into improved manufacture of live-attenuated hRSV vaccines. PMID:27654298

Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

PubMed

Mumcuoglu, Didem; Sardan Ekiz, Melis; Gunay, Gokhan; Tekinay, Turgay; Tekinay, Ayse B; Guler, Mustafa O

2016-05-11

Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

Adhesions of extracellular surface-layer associated proteins in Lactobacillus M5-L and Q8-L.

PubMed

Zhang, Yingchun; Xiang, Xinling; Lu, Qianhui; Zhang, Lanwei; Ma, Fang; Wang, Linlin

2016-02-01

Surface-layer associated proteins (SLAP) that envelop Lactobacillus paracasei ssp. paracasei M5-L and Lactobacillus casei Q8-L cell surfaces are involved in the adherence of these strain to the human intestinal cell line HT-29. To further elucidate some of the properties of these proteins, we assessed the yields and expressions of SLAP under different incubation conditions. An efficient and selective extraction of SLAP was obtained when cells of Lactobacillus were treated with 5 M LiCl at 37°C in aerobic conditions. The SLAP of Lactobacillus M5-L and Q8-L in cell extracts were visualized by SDS-PAGE and identified by Western blotting with sulfo-N-hydroxysuccinimide-biotin-labeled HT-29 cells as adhesion proteins. Atomic force microscopy contact imaging revealed that Lactobacillus strains M5-L and Q8-L normally display a smooth, homogeneous surface, whereas the surfaces of M5-L and Q8-L treated with 5 M LiCl were rough and more heterogeneous. Analysis of adhesion forces revealed that the initial adhesion forces of 1.41 and 1.28 nN obtained for normal Lactobacillus M5-L and Q8-L strains, respectively, decreased to 0.70 and 0.48 nN, respectively, following 5 M LiCl treatment. Finally, the dominant 45-kDa protein bands of Lactobacillus Q8-L and Lactobacillus M5-L were identified as elongation factor Tu and surface antigen, respectively, by liquid chromatography-tandem mass spectrometry. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Static allometry of unicellular green algae: scaling of cellular surface area and volume in the genus Micrasterias (Desmidiales).

PubMed

Neustupa, J

2016-02-01

The surface area-to-volume ratio of cells is one of the key factors affecting fundamental biological processes and, thus, fitness of unicellular organisms. One of the general models for allometric increase in surface-to-volume scaling involves fractal-like elaboration of cellular surfaces. However, specific data illustrating this pattern in natural populations of the unicellular organisms have not previously been available. This study shows that unicellular green algae of the genus Micrasterias (Desmidiales) have positive allometric surface-to-volume scaling caused by changes in morphology of individual species, especially in the degree of cell lobulation. This allometric pattern was also detected within most of the cultured and natural populations analysed. Values of the allometric S:V scaling within individual populations were closely correlated to the phylogenetic structure of the clade. In addition, they were related to species-specific cellular morphology. Individual populations differed in their allometric patterns, and their position in the allometric space was strongly correlated with the degree of allometric S:V scaling. This result illustrates that allometric shape patterns are an important correlate of the capacity of individual populations to compensate for increases in their cell volumes by increasing the surface area. However, variation in allometric patterns was not associated with phylogenetic structure. This indicates that the position of the populations in the allometric space was not evolutionarily conserved and might be influenced by environmental factors. © 2015 European Society For Evolutionary Biology. Journal of Evolutionary Biology © 2015 European Society For Evolutionary Biology.

Triangle Geometry Processing for Surface Modeling and Cartesian Grid Generation

NASA Technical Reports Server (NTRS)

Aftosmis, Michael J. (Inventor); Melton, John E. (Inventor); Berger, Marsha J. (Inventor)

2002-01-01

Cartesian mesh generation is accomplished for component based geometries, by intersecting components subject to mesh generation to extract wetted surfaces with a geometry engine using adaptive precision arithmetic in a system which automatically breaks ties with respect to geometric degeneracies. During volume mesh generation, intersected surface triangulations are received to enable mesh generation with cell division of an initially coarse grid. The hexagonal cells are resolved, preserving the ability to directionally divide cells which are locally well aligned.

Triangle geometry processing for surface modeling and cartesian grid generation

DOEpatents

Aftosmis, Michael J [San Mateo, CA; Melton, John E [Hollister, CA; Berger, Marsha J [New York, NY

2002-09-03

Cartesian mesh generation is accomplished for component based geometries, by intersecting components subject to mesh generation to extract wetted surfaces with a geometry engine using adaptive precision arithmetic in a system which automatically breaks ties with respect to geometric degeneracies. During volume mesh generation, intersected surface triangulations are received to enable mesh generation with cell division of an initially coarse grid. The hexagonal cells are resolved, preserving the ability to directionally divide cells which are locally well aligned.

Invariant Chain Complexes and Clusters as Platforms for MIF Signaling

PubMed Central

Lindner, Robert

2017-01-01

Invariant chain (Ii/CD74) has been identified as a surface receptor for migration inhibitory factor (MIF). Most cells that express Ii also synthesize major histocompatibility complex class II (MHC II) molecules, which depend on Ii as a chaperone and a targeting factor. The assembly of nonameric complexes consisting of one Ii trimer and three MHC II molecules (each of which is a heterodimer) has been regarded as a prerequisite for efficient delivery to the cell surface. Due to rapid endocytosis, however, only low levels of Ii-MHC II complexes are displayed on the cell surface of professional antigen presenting cells and very little free Ii trimers. The association of Ii and MHC II has been reported to block the interaction with MIF, thus questioning the role of surface Ii as a receptor for MIF on MHC II-expressing cells. Recent work offers a potential solution to this conundrum: Many Ii-complexes at the cell surface appear to be under-saturated with MHC II, leaving unoccupied Ii subunits as potential binding sites for MIF. Some of this work also sheds light on novel aspects of signal transduction by Ii-bound MIF in B-lymphocytes: membrane raft association of Ii-MHC II complexes enables MIF to target Ii-MHC II to antigen-clustered B-cell-receptors (BCR) and to foster BCR-driven signaling and intracellular trafficking. PMID:28208600

The Otto Aufranc Award: Enhanced Biocompatibility of Stainless Steel Implants by Titanium Coating and Microarc Oxidation

PubMed Central

Lim, Young Wook; Kwon, Soon Yong; Sun, Doo Hoon

2010-01-01

Background Stainless steel is one of the most widely used biomaterials for internal fixation devices, but is not used in cementless arthroplasty implants because a stable oxide layer essential for biocompatibility cannot be formed on the surface. We applied a Ti electron beam coating, to form oxide layer on the stainless steel surface. To form a thicker oxide layer, we used a microarc oxidation process on the surface of Ti coated stainless steel. Modification of the surface using Ti electron beam coating and microarc oxidation could improve the ability of stainless steel implants to osseointegrate. Questions/purposes The ability of cells to adhere to grit-blasted, titanium-coated, microarc-oxidated stainless steel in vitro was compared with that of two different types of surface modifications, machined and titanium-coated, and microarc-oxidated. Methods We performed energy-dispersive x-ray spectroscopy and scanning electron microscopy investigations to assess the chemical composition and structure of the stainless steel surfaces and cell morphology. The biologic responses of an osteoblastlike cell line (SaOS-2) were examined by measuring proliferation (cell proliferation assay), differentiation (alkaline phosphatase activity), and attraction ability (cell migration assay). Results Cell proliferation, alkaline phosphatase activity, migration, and adhesion were increased in the grit-blasted, titanium-coated, microarc-oxidated group compared to the two other groups. Osteoblastlike cells on the grit-blasted, titanium-coated, microarc-oxidated surface were strongly adhered, and proliferated well compared to those on the other surfaces. Conclusions The surface modifications we used (grit blasting, titanium coating, microarc oxidation) enhanced the biocompatibility (proliferation and migration of osteoblastlike cells) of stainless steel. Clinical Relevance This process is not unique to stainless steel; it can be applied to many metals to improve their biocompatibility, thus allowing a broad range of materials to be used for cementless implants. PMID:20936386

The effect of mesenchymal stem cells delivered via hydrogel-based tissue engineered periosteum on bone allograft healing

PubMed Central

Hoffman, Michael D.; Xie, Chao; Zhang, Xinping; Benoit, Danielle S.W.

2013-01-01

Allografts remain the clinical “gold standard” for treatment of critical sized bone defects despite minimal engraftment and ~60% long-term failure rates. Therefore, the development of strategies to improve allograft healing and integration are necessary. The periosteum and its associated stem cell population, which are lacking in allografts, coordinate autograft healing. Herein we utilized hydrolytically degradable hydrogels to transplant and localize mesenchymal stem cells (MSCs) to allograft surfaces, creating a periosteum mimetic, termed a ‘tissue engineered periosteum’. Our results demonstrated that this tissue engineering approach resulted in increased graft vascularization (~2.4-fold), endochondral bone formation (~2.8-fold), and biomechanical strength (1.8-fold), as compared to untreated allografts, over 16 weeks of healing. Despite this enhancement in healing, the process of endochondral ossification was delayed compared to autografts, requiring further modifications for this approach to be clinically acceptable. However, this bottom-up biomaterials approach, the engineered periosteum, can be augmented with alternative cell types, matrix cues, growth factors, and/or other small molecule drugs to expedite the process of ossification. PMID:23958029

Impaired respiration elicits SrrAB-dependent programmed cell lysis and biofilm formation in Staphylococcus aureus

PubMed Central

Mashruwala, Ameya A; van de Guchte, Adriana; Boyd, Jeffrey M

2017-01-01

Biofilms are communities of microorganisms attached to a surface or each other. Biofilm-associated cells are the etiologic agents of recurrent Staphylococcus aureus infections. Infected human tissues are hypoxic or anoxic. S. aureus increases biofilm formation in response to hypoxia, but how this occurs is unknown. In the current study we report that oxygen influences biofilm formation in its capacity as a terminal electron acceptor for cellular respiration. Genetic, physiological, or chemical inhibition of respiratory processes elicited increased biofilm formation. Impaired respiration led to increased cell lysis via divergent regulation of two processes: increased expression of the AtlA murein hydrolase and decreased expression of wall-teichoic acids. The AltA-dependent release of cytosolic DNA contributed to increased biofilm formation. Further, cell lysis and biofilm formation were governed by the SrrAB two-component regulatory system. Data presented support a model wherein SrrAB-dependent biofilm formation occurs in response to the accumulation of reduced menaquinone. DOI: http://dx.doi.org/10.7554/eLife.23845.001 PMID:28221135

Ultrastructural study on the follicle-associated epithelium of nasal-associated lymphoid tissue in specific pathogen-free (SPF) and conventional environment-adapted (SPF-CV) rats

PubMed Central

JEONG, KWANG IL; SUZUKI, HODAKA; NAKAYAMA, HIROYUKI; DOI, KUNIO

2000-01-01

Membranous (M) cells in follicle-associated epithelium (FAE) play an important role in the mucosal immunity through transport of a variety of foreign antigens to the underlying mucosa-associated lymphoid tissue (MALT). We aimed to investigate the ultrastructure of M cells in the FAE covering nasal-associated lymphoid tissue (NALT) both in specific pathogen-free (SPF) rats and in conventional environment-adapted (SPF-CV) rats aged 8–38 wk. In NALT of both SPF and SPF-CV rats, FAE included the nonciliated microvillous cell, which appears to be an analogue of M cell previously described in other MALT. In SPF rats, M cells increased in number only slightly with age, and they maintained morphological uniformity irrespective of age. In SPF-CV rats, M cells selectively increased in number resulting in prominent expansion of FAE surface area in parallel with the duration of maintenance in a conventional environment. In addition, M cells in SPF-CV rats showed heterogeneity in their surface morphology such as the length and number of microvilli and cell surface area and outline. In addition, the FAE was stratified by various subtypes of M cells, which were characterised by several subcellular alterations including the presence of many keratin filaments, homogeneous dark bodies and extensive cytoplasmic interfoliation with wide intercellular spaces filled with amorphous proteinaceous material. These characteristics of M cells in SPF-CV rat were intimately related with a preferential influx of immunocompetent cells into the FAE, which was not seen or was very rare in SPF rats irrespective of age. The results suggest the possibility that NALT may effectively carry out the mucosal immune response against antigenic stimuli of different magnitude through the unique dynamics of M cells which seem to be influenced by the infiltration of immunocompetent cells. PMID:10853966

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.

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

PubMed

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

Surface tension and modeling of cellular intercalation during zebrafish gastrulation.

PubMed

Calmelet, Colette; Sepich, Diane

2010-04-01

In this paper we discuss a model of zebrafish embryo notochord development based on the effect of surface tension of cells at the boundaries. We study the process of interaction of mesodermal cells at the boundaries due to adhesion and cortical tension, resulting in cellular intercalation. From in vivo experiments, we obtain cell outlines of time-lapse images of cell movements during zebrafish embryo development. Using Cellular Potts Model, we calculate the total surface energy of the system of cells at different time intervals at cell contacts. We analyze the variations of total energy depending on nature of cell contacts. We demonstrate that our model can be viable by calculating the total surface energy value for experimentally observed configurations of cells and showing that in our model these configurations correspond to a decrease in total energy values in both two and three dimensions.

Coplanar back contacts for thin silicon solar cells

NASA Technical Reports Server (NTRS)

Thornhill, J. W.; Sipperly, W. E.

1980-01-01

A process for fabricating 2 to 3 mil wraparound solar cells was formulated. Sample thin wraparound cells were fabricated using this process. The process used a reinforced perimeter construction to reduce the breakage that occurs during handling of the wafers. A retracting piston post was designed and fabricated to help minimize the breakage that occurs during the screen printing process. Two alternative methods of applying the aluminum back surface field were investigated. In addition to the standard screen printed back surface field, both spin-on and evaporated aluminum techniques were researched. Neither spin-on nor evaporated aluminum made any noticeable improvement over the screen printing technique. A fine screen mesh was chosen for the application of the aluminum paste back surface field. The optimum time and temperature for firing the aluminum turned out to be thirty seconds at 850 C. The development work on the dielectric included looking at three dielectrics for the wraparound application. Transene 1000, Thick Film Systems 1126RCB and an in house formulation 61-2-2A were all tested. Cells with pre-dielectric thickness of 3.0-0-3.5 mils using Transene 1000 as the wraparound dielectric and the procedure outlined above showed an average efficiency of 10.7 percent. Thinner cells were fabricated, but had an unacceptable yield and efficiency.

A comparison of human and porcine acellularized dermis: interactions with human fibroblasts in vitro.

PubMed

Armour, Alexis D; Fish, Joel S; Woodhouse, Kimberly A; Semple, John L

2006-03-01

Dermal substitutes derived from xenograft materials require elaborate processing at a considerable cost. Acellularized porcine dermis is a readily available material associated with minimal immunogenicity. The objective of this study was to evaluate acellularized pig dermis as a scaffold for human fibroblasts. In vitro methods were used to evaluate fibroblast adherence, proliferation, and migration on pig acellularized dermal matrix. Acellular human dermis was used as a control. Pig acellularized dermal matrix was found to be inferior to human acellularized dermal matrix as a scaffold for human fibroblasts. Significantly more samples of human acellularized dermal matrix (83 percent, n = 24; p < 0.05) demonstrated fibroblast infiltration below the cell-seeded surface than pig acellularized dermal matrix (31 percent, n = 49). Significantly more (p < 0.05) fibroblasts infiltrated below the surface of human acellularized dermal matrix (mean, 1072 +/- 80 cells per section; n = 16 samples) than pig acellularized dermal matrix (mean, 301 +/- 48 cells per section; n = 16 samples). Fibroblasts migrated significantly less (p < 0.05) distance from the cell-seeded pig acellularized dermal matrix surface than in the human acellularized dermal matrix (78.8 percent versus 38.3 percent cells within 150 mum from the surface, respectively; n = 5). Fibroblasts proliferated more rapidly (p < 0.05) on pig acellularized dermal matrix (n = 9) than on the human acellularized dermal matrix (7.4-fold increase in cell number versus 1.8-fold increase, respectively; n = 9 for human acellularized dermal matrix). There was no difference between the two materials with respect to fibroblast adherence (8120 versus 7436 average adherent cells per section, for pig and human acellularized dermal matrix, respectively; n = 20 in each group; p > 0.05). Preliminary findings suggest that substantial differences may exist between human fibroblast behavior in cell-matrix interactions of porcine and human acellularized dermis.

Application of response surface methodology to maximize the productivity of scalable automated human embryonic stem cell manufacture.

PubMed

Ratcliffe, Elizabeth; Hourd, Paul; Guijarro-Leach, Juan; Rayment, Erin; Williams, David J; Thomas, Robert J

2013-01-01

Commercial regenerative medicine will require large quantities of clinical-specification human cells. The cost and quality of manufacture is notoriously difficult to control due to highly complex processes with poorly defined tolerances. As a step to overcome this, we aimed to demonstrate the use of 'quality-by-design' tools to define the operating space for economic passage of a scalable human embryonic stem cell production method with minimal cell loss. Design of experiments response surface methodology was applied to generate empirical models to predict optimal operating conditions for a unit of manufacture of a previously developed automatable and scalable human embryonic stem cell production method. Two models were defined to predict cell yield and cell recovery rate postpassage, in terms of the predictor variables of media volume, cell seeding density, media exchange and length of passage. Predicted operating conditions for maximized productivity were successfully validated. Such 'quality-by-design' type approaches to process design and optimization will be essential to reduce the risk of product failure and patient harm, and to build regulatory confidence in cell therapy manufacturing processes.

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

PubMed

Rotter, V; Boss, M A; Baltimore, D

1981-04-01

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

MHC class I antigen presentation and implications for developing a new generation of therapeutic vaccines.

PubMed

Comber, Joseph D; Philip, Ramila

2014-05-01

Major histocompatibility complex class I (MHC-I) presented peptide epitopes provide a 'window' into the changes occurring in a cell. Conventionally, these peptides are generated by proteolysis of endogenously synthesized proteins in the cytosol, loaded onto MHC-I molecules, and presented on the cell surface for surveillance by CD8(+) T cells. MHC-I restricted processing and presentation alerts the immune system to any infectious or tumorigenic processes unfolding intracellularly and provides potential targets for a cytotoxic T cell response. Therefore, therapeutic vaccines based on MHC-I presented peptide epitopes could, theoretically, induce CD8(+) T cell responses that have tangible clinical impacts on tumor eradication and patient survival. Three major methods have been used to identify MHC-I restricted epitopes for inclusion in peptide-based vaccines for cancer: genetic, motif prediction and, more recently, immunoproteomic analysis. Although the first two methods are capable of identifying T cell stimulatory epitopes, these have significant disadvantages and may not accurately represent epitopes presented by a tumor cell. In contrast, immunoproteomic methods can overcome these disadvantages and identify naturally processed and presented tumor associated epitopes that induce more clinically relevant tumor specific cytotoxic T cell responses. In this review, we discuss the importance of using the naturally presented MHC-I peptide repertoire in formulating peptide vaccines, the recent application of peptide-based vaccines in a variety of cancers, and highlight the pros and cons of the current state of peptide vaccines.

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

Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

PubMed Central

2012-01-01

Background Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells. Results The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively. Conclusions The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing. PMID:23039947

Evaluation of the ion implantation process for production of solar cells from silicon sheet materials

NASA Technical Reports Server (NTRS)

Spitzer, M. B.

1983-01-01

The objective of this program is the investigation and evaluation of the capabilities of the ion implantation process for the production of photovoltaic cells from a variety of present-day, state-of-the-art, low-cost silicon sheet materials. Task 1 of the program concerns application of ion implantation and furnace annealing to fabrication of cells made from dendritic web silicon. Task 2 comprises the application of ion implantation and pulsed electron beam annealing (PEBA) to cells made from SEMIX, SILSO, heat-exchanger-method (HEM), edge-defined film-fed growth (EFG) and Czochralski (CZ) silicon. The goals of Task 1 comprise an investigation of implantation and anneal processes applied to dendritic web. A further goal is the evaluation of surface passivation and back surface reflector formation. In this way, processes yielding the very highest efficiency can be evaluated. Task 2 seeks to evaluate the use of PEBA for various sheet materials. A comparison of PEBA to thermal annealing will be made for a variety of ion implantation processes.

Clinical implications of mast cell involvement in allergic conjunctivitis.

PubMed

Elieh Ali Komi, D; Rambasek, T; Bielory, L

2018-03-01

The conjunctiva is a common site for the allergic inflammatory response due to it being highly vascularized, having constant exposure to environmental pollutants and allergenic pollens and having a unique conjunctival associated lymphoid tissue. The primary morbidity of anterior surface conjunctival disorders that include allergic conjunctivitis and tear film disorders is associated with its high frequency of involvement rather than its severity, although the more chronic forms can involve the cornea and lead to sight-threatening conditions. Ocular allergy is associated with IgE-mediated mast cell activation in conjunctival tissue leading to the release of preformed mediators including histamine and proteases and subsequent de novo formation of lipid-derived mediators and cytokines that trigger a cascade of cellular and molecular events leading to extensive migration and infiltration of inflammatory cells to the ocular surface. The trafficking of neutrophils, eosinophils, and lymphocytes to the ocular surface is due to establishing various chemokine gradients (mainly CCL11, CCL24, CCL5, MCP-3, and MCP-4), cell surface expression of adhesion molecules (such as VCAM-1 the ligand for VLA-4), and leukocyte adhesion to vascular endothelium. The release of preformed mediators underlies the acute ocular surface response while the secondary influx of inflammatory cells leading to the recruitment and activation of eosinophils and the subsequent activation of Th2 and Th1 lymphocytes at the level of the conjunctiva reflects the late-phase reaction. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Influence of non-adherent yeast cells on electrical characteristics of diamond-based field-effect transistors

NASA Astrophysics Data System (ADS)

Procházka, Václav; Cifra, Michal; Kulha, Pavel; Ižák, Tibor; Rezek, Bohuslav; Kromka, Alexander

2017-02-01

Diamond thin films provide unique features as substrates for cell cultures and as bio-electronic sensors. Here we employ solution-gated field effect transistors (SGFET) based on nanocrystalline diamond thin films with H-terminated surface which exhibits the sub-surface p-type conductive channel. We study an influence of yeast cells (Saccharomyces cerevisiae) on electrical characteristics of the diamond SGFETs. Two different cell culture solutions (sucrose and yeast peptone dextrose-YPD) are used, with and without the cells. We have found that transfer characteristics of the SGFETs exhibit a negative shift of the gate voltage by -26 mV and -42 mV for sucrose and YPD with cells in comparison to blank solutions without the cells. This effect is attributed to a local pH change in close vicinity of the H-terminated diamond surface due to metabolic processes of the yeast cells. The pH sensitivity of the diamond-based SGFETs, the role of cell and protein adhesion on the gate surface and the role of negative surface charge of yeast cells on the SGFETs electrical characteristics are discussed as well.

Segmentation of whole cells and cell nuclei from 3-D optical microscope images using dynamic programming.

PubMed

McCullough, D P; Gudla, P R; Harris, B S; Collins, J A; Meaburn, K J; Nakaya, M A; Yamaguchi, T P; Misteli, T; Lockett, S J

2008-05-01

Communications between cells in large part drive tissue development and function, as well as disease-related processes such as tumorigenesis. Understanding the mechanistic bases of these processes necessitates quantifying specific molecules in adjacent cells or cell nuclei of intact tissue. However, a major restriction on such analyses is the lack of an efficient method that correctly segments each object (cell or nucleus) from 3-D images of an intact tissue specimen. We report a highly reliable and accurate semi-automatic algorithmic method for segmenting fluorescence-labeled cells or nuclei from 3-D tissue images. Segmentation begins with semi-automatic, 2-D object delineation in a user-selected plane, using dynamic programming (DP) to locate the border with an accumulated intensity per unit length greater that any other possible border around the same object. Then the two surfaces of the object in planes above and below the selected plane are found using an algorithm that combines DP and combinatorial searching. Following segmentation, any perceived errors can be interactively corrected. Segmentation accuracy is not significantly affected by intermittent labeling of object surfaces, diffuse surfaces, or spurious signals away from surfaces. The unique strength of the segmentation method was demonstrated on a variety of biological tissue samples where all cells, including irregularly shaped cells, were accurately segmented based on visual inspection.

Fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy.

PubMed

Carson, Johnny L

2014-09-11

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Extracellular Enzymes Facilitate Electron Uptake in Biocorrosion and Bioelectrosynthesis

PubMed Central

Deutzmann, Jörg S.; Sahin, Merve

2015-01-01

ABSTRACT Direct, mediator-free transfer of electrons between a microbial cell and a solid phase in its surrounding environment has been suggested to be a widespread and ecologically significant process. The high rates of microbial electron uptake observed during microbially influenced corrosion of iron [Fe(0)] and during microbial electrosynthesis have been considered support for a direct electron uptake in these microbial processes. However, the underlying molecular mechanisms of direct electron uptake are unknown. We investigated the electron uptake characteristics of the Fe(0)-corroding and electromethanogenic archaeon Methanococcus maripaludis and discovered that free, surface-associated redox enzymes, such as hydrogenases and presumably formate dehydrogenases, are sufficient to mediate an apparent direct electron uptake. In genetic and biochemical experiments, we showed that these enzymes, which are released from cells during routine culturing, catalyze the formation of H2 or formate when sorbed to an appropriate redox-active surface. These low-molecular-weight products are rapidly consumed by M. maripaludis cells when present, thereby preventing their accumulation to any appreciable or even detectable level. Rates of H2 and formate formation by cell-free spent culture medium were sufficient to explain the observed rates of methane formation from Fe(0) and cathode-derived electrons by wild-type M. maripaludis as well as by a mutant strain carrying deletions in all catabolic hydrogenases. Our data collectively show that cell-derived free enzymes can mimic direct extracellular electron transfer during Fe(0) corrosion and microbial electrosynthesis and may represent an ecologically important but so far overlooked mechanism in biological electron transfer. PMID:25900658

Genetic susceptibility to type 1 diabetes in the intracellular pathway of antigen processing - a subject review and cross-study comparison.

PubMed

Sia, Charles; Weinem, Michael

2005-01-01

Ligand binding grooves of MHC class I molecules are able to load a panel of endogenous peptides of varying length and sequence derived from self or foreign origin to activate or deactivate cytotoxic CD8(+) T cells. Peptides are assembled with class I molecules by pathways that are either dependent or independent of transport by ABC proteins (TAP) and degradation in the immunoproteasome by its subunits LMP2 and LMP7. Those peptides that require TAP and LMP treatment appear to be subject to control and optimization by TAP for proper customizing and efficient presentation. Therefore, allelic variations in the coding sequences of TAP and LMP were suspected for a long time to be responsible for improper antigen processing, interruption of self-peptide presentation and reduced cell surface expression of MHC class I molecules resulting in the activation of autoreactive CD8(+) T cells. In this article we reviewed the controversial findings regarding the role of TAP and LMP genes in autoimmune diabetes and reevaluated data of eleven separate studies in a cross-study analysis by genotype and HLA haplotype matching. We could confirm previous results by showing that TAP2*651-A/F and TAP2*687-A/A are significantly associated with disease, independently of linkage disequilibrium (LD). LMP2-R/H surprisingly seems to be primarily disease-conferring although a weak association with DR4 serotypes can be observed. Our analysis also suggests that LMP7-B/B, TAP1-A/A and TAP2*687-A/B are the protective genotypes and that these associations are not secondary to LD with DRB1. Consequently, intracellular antigen processing associated with TAP- and proteasome-dependent pathways seems to be a critical element in T cell selection for the retention of a balanced immunity.

Traffic jam hypothesis: Relationship between endocytic dysfunction and Alzheimer's disease.

PubMed

Kimura, Nobuyuki; Yanagisawa, Katsuhiko

2017-07-08

Membrane trafficking pathways, like the endocytic pathway, carry out fundamental cellular processes that are essential for normal functioning. One such process is regulation of cell surface receptor signaling. A growing body of evidence suggests that β-amyloid protein (Aβ) plays a key role in Alzheimer's disease (AD) pathogenesis. Cleavage of Aβ from its precursor, β-amyloid precursor protein (APP), occurs through the endocytic pathway in neuronal cells. In early-stage AD, intraneuronal accumulation of abnormally enlarged endosomes is common, indicating that endosome trafficking is disrupted. Strikingly, genome-wide association studies reveal that several endocytosis-related genes are associated with AD onset. Also, recent studies demonstrate that alteration in endocytosis induces not only Aβ pathology but also the propagation of tau protein pathology, another key pathological feature of AD. Endocytic dysfunction can disrupt neuronal physiological functions, such as synaptic vesicle transport and neurotransmitter release. Thus, "traffic jams" in the endocytic pathway may be involved in AD pathogenesis and may serve as a novel target for the development of new therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

PubMed Central

Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

2015-01-01

Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

Effects of rutin on the physicochemical properties of skin fibroblasts membrane disruption following UV radiation.

PubMed

Dobrzyńska, Izabela; Gęgotek, Agnieszka; Gajko, Ewelina; Skrzydlewska, Elżbieta; Figaszewski, Zbigniew A

2018-02-25

Human skin provides the body's first line of defense against physical and environmental assaults. This study sought to determine how rutin affects the membrane electrical properties, sialic acid content, and lipid peroxidation levels of fibroblast membranes after disruption by ultraviolet (UV) radiation. Changes in cell function may affect the basal electrical surface properties of cell membranes, and changes can be detected by electrokinetic measurements. The charge density of the fibroblast membrane surface was measured as a function of pH. A four-component equilibrium model was used to describe the interaction between ions in solution and ions on the membrane surface. Agreement was found between experimental and theoretical charge variation curves of fibroblast cells between pH 2.5 and 8. Sialic acid content was determined by Svennerholm's resorcinol method, and lipid peroxidation was estimated by measuring the malondialdehyde level. Compared to untreated cells, ultraviolet A (UVA)- or ultraviolet B (UVB)-treated skin cell membranes exhibited higher concentrations of acidic functional groups and higher average association constants with hydroxyl ions, but lower average association constants with hydrogen ions. Moreover, our results showed that UVA and UVB radiation is associated with increased levels of sialic acid and lipid peroxidation products in fibroblasts. Rutin protected cells from some deleterious UV-associated membrane changes, including changes in electrical properties, oxidative state, and biological functions. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Forced interaction of cell surface proteins with Derlin-1 in the endoplasmic reticulum is sufficient to induce their dislocation into the cytosol for degradation.

PubMed

Cho, Sunglim; Lee, Miriam; Jun, Youngsoo

2013-01-11

Aberrantly folded proteins in the endoplasmic reticulum (ER) are rapidly removed into the cytosol for degradation by the proteasome via an evolutionarily conserved process termed ER-associated protein degradation (ERAD). ERAD of a subset of proteins requires Derlin-1 for dislocation into the cytosol; however, the molecular function of Derlin-1 remains unclear. Human cytomegalovirus US11 exploits Derlin-1-dependent ERAD to degrade major histocompatibility complex class I (MHC-I) molecules for immune evasion. Because US11 binds to both MHC-I molecules and Derlin-1 via its luminal and transmembrane domains (TMDs), respectively, the major role of US11 has been proposed to simply be delivery of MHC-I molecules to Derlin-1. Here, we directly tested this proposal by generating a hybrid MHC-I molecule, which contains the US11 TMD, and thus can associate with Derlin-1 in the absence of US11. Intriguingly, this MHC-I hybrid was rapidly degraded in a Derlin-1- and proteasome-dependent manner. Similarly, the vesicular stomatitis virus G protein, otherwise expressed at the cell surface, was degraded via Derlin-1-dependent ERAD when its TMD was replaced with that of US11. Thus, forced interaction of cell surface proteins with Derlin-1 is sufficient to induce their degradation via ERAD. Taken together, these results suggest that the main role of US11 is to recruit MHC-I molecules to Derlin-1, which then mediates the dislocation of MHC-I molecules into the cytosol for degradation. Copyright © 2012 Elsevier Inc. All rights reserved.