Ion beam surface treatment: A new capability for rapid melt and resolidification of surfaces

International Nuclear Information System (INIS)

Stinnett, R.W.; McIntyre, D.C.; Buchheit, R.G.; Greenly, J.B.; Thompson, M.O.

1994-01-01

The emerging capability to produce high average power (5--250 kW) pulsed ion beams at 0.2--2 MeV energies is enabling us to develop a new, commercial-scale thermal surface treatment technology called Ion Beam Surface Treatment (IBEST). This technique uses high energy, pulsed (≤100 ns) ion beams to directly deposit energy in the top 2--20 micrometers of the surface of any material. Depth of treatment is controllable by varying the ion energy and species. Deposition of the energy with short pulses in a thin surface layer allows melting of the layer with relatively small energies and allows rapid cooling of the melted layer by thermal diffusion into the underlying substrate. Typical cooling rates of this process (10 9 10 10 K/sec) cause rapid resolidification, resulting in production of non-equilibrium microstructures (nano-crystalline and metastable phases) that have significantly improved corrosion, wear, and hardness properties. We have conducted IBEST feasibility experiments with results confirming surface hardening, nanocrystaline grain formation, metal surface polishing, controlled melt of ceramic surfaces, and surface cleaning

Application of various surface passivation layers in solar cells

International Nuclear Information System (INIS)

Lee, Ji Youn; Lee, Soo Hong

2004-01-01

In this work, we have used different techniques for surface passivation: conventional thermal oxidation (CTO), rapid thermal oxidation (RTO), and plasma-enhanced chemical vapour deposition (PECVD). The surface passivation qualities of eight different single and combined double layers have been investigated both on phosphorus non-diffused p-type Float Zone (FZ) silicon wafers and on diffused emitters (100 Ω/□ and 40 Ω/□). CTO/SiN 1 passivates very well not only on a non-diffused surface (τ eff = 1361 μs) but also on an emitter (τ eff = 414 μs). However, we concluded that RTO/SiN 1 and RTO/SiN 2 stacks were more suitable than CTO/SiN stacks for surface passivation in solar cells since those stacks had relatively good passivation qualities and suitable optical reflections. RTO/SiN 1 for rear-surface passivation and RTO/SiN 2 for front-surface passivation were applied to the fabrication of solar cells. We achieved efficiencies of 18.5 % and 18.8 % on 0.5 Ω-cm (FZ) silicon with planar and textured front surfaces, respectively. An excellent open circuit voltage (V oc ) of 675.6 mV was obtained for the planar cell.

Rapid and sensitive phenotypic marker detection on breast cancer cells using surface-enhanced Raman scattering (SERS) imaging.

Science.gov (United States)

Lee, Sangyeop; Chon, Hyangah; Lee, Jiyoung; Ko, Juhui; Chung, Bong Hyun; Lim, Dong Woo; Choo, Jaebum

2014-01-15

We report a surface-enhanced Raman scattering (SERS)-based cellular imaging technique to detect and quantify breast cancer phenotypic markers expressed on cell surfaces. This technique involves the synthesis of SERS nano tags consisting of silica-encapsulated hollow gold nanospheres (SEHGNs) conjugated with specific antibodies. Hollow gold nanospheres (HGNs) enhance SERS signal intensity of individual particles by localizing surface electromagnetic fields through pinholes in the hollow particle structures. This capacity to enhance imaging at the level of single molecules permits the use of HGNs to detect specific biological markers expressed in living cancer cells. In addition, silica encapsulation greatly enhances the stability of nanoparticles. Here we applied a SERS-based imaging technique using SEHGNs in the multiplex imaging of three breast cancer cell phenotypes. Expression of epidermal growth factor (EGF), ErbB2, and insulin-like growth factor-1 (IGF-1) receptors were assessed in the MDA-MB-468, KPL4 and SK-BR-3 human breast cancer cell lines. SERS imaging technology described here can be used to test the phenotype of a cancer cell and quantify proteins expressed on the cell surface simultaneously. Based on results, this technique may enable an earlier diagnosis of breast cancer than is currently possible and offer guidance in treatment. © 2013 Elsevier B.V. All rights reserved.

Beneficial Autoimmunity at Body Surfaces – Immune Surveillance and Rapid Type 2 Immunity Regulate Tissue Homeostasis and Cancer

Science.gov (United States)

Dalessandri, Tim; Strid, Jessica

2014-01-01

Epithelial cells (ECs) line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation, or toxins cause activation of ECs with release of cytokines and chemokines as well as alterations in the expression of cell-surface ligands. Such display of epithelial stress is rapidly sensed by tissue-resident immunocytes, which can directly interact with self-moieties on ECs and initiate both local and systemic immune responses. ECs are thus key drivers of immune surveillance at body surface tissues. However, ECs have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here, we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis. PMID:25101088

A Rapid Culture Technique Produces Functional Dendritic-Like Cells from Human Acute Myeloid Leukemia Cell Lines

Directory of Open Access Journals (Sweden)

Jian Ning

2011-01-01

Full Text Available Most anti-cancer immunotherapeutic strategies involving dendritic cells (DC as vaccines rely upon the adoptive transfer of DC loaded with exogenous tumour-peptides. This study utilized human acute myeloid leukemia (AML cells as progenitors from which functional dendritic-like antigen presenting cells (DLC were generated, that constitutively express tumour antigens for recognition by CD8+ T cells. DLC were generated from AML cell lines KG-1 and MUTZ-3 using rapid culture techniques and appropriate cytokines. DLC were evaluated for their cell-surface phenotype, antigen uptake and ability to stimulate allogeneic responder cell proliferation, and production of IFN-γ; compared with DC derived from normal human PBMC donors. KG-1 and MUTZ-3 DLC increased expression of CD80, CD83, CD86, and HLA-DR, and MUTZ-3 DLC downregulated CD14 and expressed CD1a. Importantly, both KG-1 and MUTZ-3-derived DLC promoted proliferation of allogeneic responder cells more efficiently than unmodified cells; neither cells incorporated FITC-labeled dextran, but both stimulated IFN-γ production from responding allogeneic CD8+ T cells. Control DC produced from PBMC using the FastDC culture also expressed high levels of critical cell surface ligands and demonstrated good APC function. This paper indicates that functional DLC can be cultured from the AML cell lines KG-1 and MUTZ-3, and FastDC culture generates functional KG-1 DLC.

Nanostructured Surfaces to Target and Kill Circulating Tumor Cells While Repelling Leukocytes

Directory of Open Access Journals (Sweden)

Michael J. Mitchell

2012-01-01

Full Text Available Hematogenous metastasis, the process of cancer cell migration from a primary to distal location via the bloodstream, typically leads to a poor patient prognosis. Selectin proteins hold promise in delivering drug-containing nanocarriers to circulating tumor cells (CTCs in the bloodstream, due to their rapid, force-dependent binding kinetics. However, it is challenging to deliver such nanocarriers while avoiding toxic effects on healthy blood cells, as many possess ligands that adhesively interact with selectins. Herein, we describe a nanostructured surface to capture flowing cancer cells, while preventing human neutrophil adhesion. Microtube surfaces with immobilized halloysite nanotubes (HNTs and E-selectin functionalized liposomal doxorubicin (ES-PEG L-DXR significantly increased the number of breast adenocarcinoma MCF7 cells captured from flow, yet also significantly reduced the number of captured neutrophils. Neutrophils firmly adhered and projected pseudopods on surfaces coated only with liposomes, while neutrophils adherent to HNT-liposome surfaces maintained a round morphology. Perfusion of both MCF7 cells and neutrophils resulted in primarily cancer cell adhesion to the HNT-liposome surface, and induced significant cancer cell death. This work demonstrates that nanostructured surfaces consisting of HNTs and ES-PEG L-DXR can increase CTC recruitment for chemotherapeutic delivery, while also preventing healthy cell adhesion and uptake of therapeutic intended for CTCs.

Ultrathin Oxide Passivation Layer by Rapid Thermal Oxidation for the Silicon Heterojunction Solar Cell Applications

OpenAIRE

Lee, Youngseok; Oh, Woongkyo; Dao, Vinh Ai; Hussain, Shahzada Qamar; Yi, Junsin

2012-01-01

It is difficult to deposit extremely thin a-Si:H layer in heterojunction with intrinsic thin layer (HIT) solar cell due to thermal damage and tough process control. This study aims to understand oxide passivation mechanism of silicon surface using rapid thermal oxidation (RTO) process by examining surface effective lifetime and surface recombination velocity. The presence of thin insulating a-Si:H layer is the key to get high Voc by lowering the leakage current (I0) which improves the efficie...

Interaction of KSHV with Host Cell Surface Receptors and Cell Entry

Directory of Open Access Journals (Sweden)

Mohanan Valiya Veettil

2014-10-01

Full Text Available Virus entry is a complex process characterized by a sequence of events. Since the discovery of KSHV in 1994, tremendous progress has been made in our understanding of KSHV entry into its in vitro target cells. KSHV entry is a complex multistep process involving viral envelope glycoproteins and several cell surface molecules that is utilized by KSHV for its attachment and entry. KSHV has a broad cell tropism and the attachment and receptor engagement on target cells have an important role in determining the cell type-specific mode of entry. KSHV utilizes heparan sulfate, integrins and EphrinA2 molecules as receptors which results in the activation of host cell pre-existing signal pathways that facilitate the subsequent cascade of events resulting in the rapid entry of virus particles, trafficking towards the nucleus followed by viral and host gene expression. KSHV enters human fibroblast cells by dynamin dependant clathrin mediated endocytosis and by dynamin independent macropinocytosis in dermal endothelial cells. Once internalized into endosomes, fusion of the viral envelope with the endosomal membranes in an acidification dependent manner results in the release of capsids which subsequently reaches the nuclear pore vicinity leading to the delivery of viral DNA into the nucleus. In this review, we discuss the principal mechanisms that enable KSHV to interact with the host cell surface receptors as well as the mechanisms that are required to modulate cell signaling machinery for a successful entry.

The effects of surface treatments on rapid chloride permeability tests

KAUST Repository

Yoon, Seyoon

2012-08-01

Surface treatments are commonly applied to improve the chloride resistance of concrete structures exposed to saline environments. Information on chloride ingress to surface-treated concrete is mostly provided by application of the rapid chloride permeability test (RCPT); this test is short in duration and provides rapid results. This study presents a numerical formulation, based on the extended Nernst-Plank/Poisson (NPP) equation, to model the effect of the surface treatment on a sample tested by RCPT. Predictions of the model are compared to experimental measurements. The simulations show that the results from RCPT, in terms of ionic profiles and measurement of the electric field, are dependent on the effectiveness of surface treatments. During RCPT, highly effective surface treatments cause both cations and anions to flocculate at the interface between the surface treatment and the concrete, creating a local electric field. Our numerical model includes these phenomena and presents a methodology to obtain more accurate diffusivities of the surface-treated- concrete from RCPT. © 2012 Elsevier B.V. All rights reserved.

The effects of surface treatments on rapid chloride permeability tests

KAUST Repository

Yoon, Seyoon; Oh, Sang-gyun; Ha, Juyoung; Monteiro, Paulo M.

2012-01-01

Surface treatments are commonly applied to improve the chloride resistance of concrete structures exposed to saline environments. Information on chloride ingress to surface-treated concrete is mostly provided by application of the rapid chloride permeability test (RCPT); this test is short in duration and provides rapid results. This study presents a numerical formulation, based on the extended Nernst-Plank/Poisson (NPP) equation, to model the effect of the surface treatment on a sample tested by RCPT. Predictions of the model are compared to experimental measurements. The simulations show that the results from RCPT, in terms of ionic profiles and measurement of the electric field, are dependent on the effectiveness of surface treatments. During RCPT, highly effective surface treatments cause both cations and anions to flocculate at the interface between the surface treatment and the concrete, creating a local electric field. Our numerical model includes these phenomena and presents a methodology to obtain more accurate diffusivities of the surface-treated- concrete from RCPT. © 2012 Elsevier B.V. All rights reserved.

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

KAUST Repository

So, Hongyun; Lee, Kunwoo; Murthy, Niren; Pisano, Albert P

2014-01-01

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

All-in-one nanowire-decorated multifunctional membrane for rapid cell lysis and direct DNA isolation.

KAUST Repository

So, Hongyun

2014-11-24

This paper describes a handheld device that uses an all-in-one membrane for continuous mechanical cell lysis and rapid DNA isolation without the assistance of power sources, lysis reagents, and routine centrifugation. This nanowire-decorated multifunctional membrane was fabricated to isolate DNA by selective adsorption to silica surface immediately after disruption of nucleus membranes by ultrasharp tips of nanowires for a rapid cell lysis, and it can be directly assembled with commercial syringe filter holders. The membrane was fabricated by photoelectrochemical etching to create microchannel arrays followed by hydrothermal synthesis of nanowires and deposition of silica. The proposed membrane successfully purifies high-quality DNA within 5 min, whereas a commercial purification kit needs more than an hour.

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

Directory of Open Access Journals (Sweden)

Xiuchun Ge

2010-07-01

Full Text Available Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species.We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity.The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Pooled protein immunization for identification of cell surface antigens in Streptococcus sanguinis.

Science.gov (United States)

Ge, Xiuchun; Kitten, Todd; Munro, Cindy L; Conrad, Daniel H; Xu, Ping

2010-07-26

Available bacterial genomes provide opportunities for screening vaccines by reverse vaccinology. Efficient identification of surface antigens is required to reduce time and animal cost in this technology. We developed an approach to identify surface antigens rapidly in Streptococcus sanguinis, a common infective endocarditis causative species. We applied bioinformatics for antigen prediction and pooled antigens for immunization. Forty-seven surface-exposed proteins including 28 lipoproteins and 19 cell wall-anchored proteins were chosen based on computer algorithms and comparative genomic analyses. Eight proteins among these candidates and 2 other proteins were pooled together to immunize rabbits. The antiserum reacted strongly with each protein and with S. sanguinis whole cells. Affinity chromatography was used to purify the antibodies to 9 of the antigen pool components. Competitive ELISA and FACS results indicated that these 9 proteins were exposed on S. sanguinis cell surfaces. The purified antibodies had demonstrable opsonic activity. The results indicate that immunization with pooled proteins, in combination with affinity purification, and comprehensive immunological assays may facilitate cell surface antigen identification to combat infectious diseases.

Characteristics of Turbulent Airflow Deduced from Rapid Surface Thermal Fluctuations: An Infrared Surface Anemometer

Science.gov (United States)

Aminzadeh, Milad; Breitenstein, Daniel; Or, Dani

2017-12-01

The intermittent nature of turbulent airflow interacting with the surface is readily observable in fluctuations of the surface temperature resulting from the thermal imprints of eddies sweeping the surface. Rapid infrared thermography has recently been used to quantify characteristics of the near-surface turbulent airflow interacting with the evaporating surfaces. We aim to extend this technique by using single-point rapid infrared measurements to quantify properties of a turbulent flow, including surface exchange processes, with a view towards the development of an infrared surface anemometer. The parameters for the surface-eddy renewal (α and β ) are inferred from infrared measurements of a single-point on the surface of a heat plate placed in a wind tunnel with prescribed wind speeds and constant mean temperatures of the surface. Thermally-deduced parameters are in agreement with values obtained from standard three-dimensional ultrasonic anemometer measurements close to the plate surface (e.g., α = 3 and β = 1/26 (ms)^{-1} for the infrared, and α = 3 and β = 1/19 (ms)^{-1} for the sonic-anemometer measurements). The infrared-based turbulence parameters provide new insights into the role of surface temperature and buoyancy on the inherent characteristics of interacting eddies. The link between the eddy-spectrum shape parameter α and the infrared window size representing the infrared field of view is investigated. The results resemble the effect of the sampling height above the ground in sonic anemometer measurements, which enables the detection of larger eddies with higher values of α . The physical basis and tests of the proposed method support the potential for remote quantification of the near-surface momentum field, as well as scalar-flux measurements in the immediate vicinity of the surface.

Design and rapid prototyping of DLC coated fractal surfaces for tissue engineering applications

International Nuclear Information System (INIS)

Diaz-Lantada, A; Mosquera, A; Endrino, J L; Lafont, P

2010-01-01

Several medical devices (both implantable and for in vitro diagnosis) benefit greatly from having microtextured surfaces that help to improve and promote phenomena such as osteointegracion and cell / tissue growth on the surface of a device. Normally, the use of abrasives or chemical attacks are employed for obtaining such surface microtextures, however, it is sometimes difficult to precisely control the final surface characteristics (porosity, roughness, among others) and consequently the related biological aspects. In this work, we propose an alternative process based on the use of fractal surface models for designing special surfaces, which helps controlling the desired contact properties (from the design stage) in multiple applications within biomedical engineering, especially regarding tissue engineering tasks. Manufacturing can be directly accomplished by means of rapid prototyping technologies. This method supposes a focus change from a conventional 'top-down' to a more versatile 'bottom-up' approach. Finally, in order to improve the possible biological response, the surfaces of the designed devices were coated with hydrogen-free amorphous carbon (a-C) thin films, known to be highly biocompatible materials. The films were deposited at room temperature using the vacuum filter cathodic arc technique. Our first prototypes have helped verify the viability of the approach and to validate the design, manufacturing and coating processes.

Design and rapid prototyping of DLC coated fractal surfaces for tissue engineering applications

Energy Technology Data Exchange (ETDEWEB)

Diaz-Lantada, A; Mosquera, A; Endrino, J L; Lafont, P, E-mail: adiaz@etsii.upm.es

2010-11-01

Several medical devices (both implantable and for in vitro diagnosis) benefit greatly from having microtextured surfaces that help to improve and promote phenomena such as osteointegracion and cell / tissue growth on the surface of a device. Normally, the use of abrasives or chemical attacks are employed for obtaining such surface microtextures, however, it is sometimes difficult to precisely control the final surface characteristics (porosity, roughness, among others) and consequently the related biological aspects. In this work, we propose an alternative process based on the use of fractal surface models for designing special surfaces, which helps controlling the desired contact properties (from the design stage) in multiple applications within biomedical engineering, especially regarding tissue engineering tasks. Manufacturing can be directly accomplished by means of rapid prototyping technologies. This method supposes a focus change from a conventional 'top-down' to a more versatile 'bottom-up' approach. Finally, in order to improve the possible biological response, the surfaces of the designed devices were coated with hydrogen-free amorphous carbon (a-C) thin films, known to be highly biocompatible materials. The films were deposited at room temperature using the vacuum filter cathodic arc technique. Our first prototypes have helped verify the viability of the approach and to validate the design, manufacturing and coating processes.

The effect of cerium valence states at cerium oxide nanoparticle surfaces on cell proliferation

KAUST Repository

Naganuma, Tamaki

2014-05-01

Understanding and controlling cell proliferation on biomaterial surfaces is critical for scaffold/artificial-niche design in tissue engineering. The mechanism by which underlying integrin ligates with functionalized biomaterials to induce cell proliferation is still not completely understood. In this study, poly-l-lactide (PL) scaffold surfaces were functionalized using layers of cerium oxide nanoparticles (CNPs), which have recently attracted attention for use in therapeutic application due to their catalytic ability of Ce4+ and Ce3+ sites. To isolate the influence of Ce valance states of CNPs on cell proliferation, human mesenchymal stem cells (hMSCs) and osteoblast-like cells (MG63) were cultured on the PL/CNP surfaces with dominant Ce4+ and Ce3+ regions. Despite cell type (hMSCs and MG63 cells), different surface features of Ce4+ and Ce3+ regions clearly promoted and inhibited cell spreading, migration and adhesion behavior, resulting in rapid and slow cell proliferation, respectively. Cell proliferation results of various modified CNPs with different surface charge and hydrophobicity/hydrophilicity, indicate that Ce valence states closely correlated with the specific cell morphologies and cell-material interactions that trigger cell proliferation. This finding suggests that the cell-material interactions, which influence cell proliferation, may be controlled by introduction of metal elements with different valence states onto the biomaterial surface. © 2014 Elsevier Ltd.

Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors

DEFF Research Database (Denmark)

Isa, Adiba; Nehlin, Jan; Sabir, Hardee Jawad

2010-01-01

HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C...... at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either...... undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced...

Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering

Science.gov (United States)

Rogozhnikov, Dmitry; O'Brien, Paul J.; Elahipanah, Sina; Yousaf, Muhammad N.

2016-12-01

There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

A vertically aligned carbon nanotube-based impedance sensing biosensor for rapid and high sensitive detection of cancer cells.

Science.gov (United States)

Abdolahad, Mohammad; Taghinejad, Mohammad; Taghinejad, Hossein; Janmaleki, Mohsen; Mohajerzadeh, Shams

2012-03-21

A novel vertically aligned carbon nanotube based electrical cell impedance sensing biosensor (CNT-ECIS) was demonstrated for the first time as a more rapid, sensitive and specific device for the detection of cancer cells. This biosensor is based on the fast entrapment of cancer cells on vertically aligned carbon nanotube arrays and leads to mechanical and electrical interactions between CNT tips and entrapped cell membranes, changing the impedance of the biosensor. CNT-ECIS was fabricated through a photolithography process on Ni/SiO(2)/Si layers. Carbon nanotube arrays have been grown on 9 nm thick patterned Ni microelectrodes by DC-PECVD. SW48 colon cancer cells were passed over the surface of CNT covered electrodes to be specifically entrapped on elastic nanotube beams. CNT arrays act as both adhesive and conductive agents and impedance changes occurred as fast as 30 s (for whole entrapment and signaling processes). CNT-ECIS detected the cancer cells with the concentration as low as 4000 cells cm(-2) on its surface and a sensitivity of 1.7 × 10(-3)Ω cm(2). Time and cell efficiency factor (TEF and CEF) parameters were defined which describe the sensor's rapidness and resolution, respectively. TEF and CEF of CNT-ECIS were much higher than other cell based electrical biosensors which are compared in this paper.

Surface binding and uptake of cadmium (Cd2+) by LLC-PK1 cells on permeable membrane supports

International Nuclear Information System (INIS)

Prozialeck, W.C.; Lamar, P.C.

1993-01-01

Recent studies have shown that Cd 2+ has relatively specific damaging effects on cell-cell junctions in the renal epithelial cell line, LLC-PK 1 . The objective of the present studies was to examine the surface binding and uptake of Cd 2+ by LLC-PK 1 cells in relation to the disruption of cell-cell junctions. LLC-PK 1 cells on Falcon Cell Culture Inserts were exposed to CdCl 2 containing trace amounts of 109 Cd 2+ from either the apical or the basolateral compartments, and the accumulation of 109 Cd 2+ was monitored for up to 8 h. The integrity of cell-cell junctions was assessed by monitoring the transepithelial electrical resistance. The results showed that the cells accumulated 3-4 times more Cd 2+ from the basolateral compartment than from the apical compartment. The accumulation of Cd 2+ from the basolateral compartment occurred in two phases: a rapid, exponential phase that occurred in 1-2 h and coincided with a decrease in transepithelial resistance, and a slower, linear phase that continued for 6-8 h. The Cd 2+ that accumulated during the rapid phase was easily removed by washing the cells in EGTA, indicating that most of it was bound to sites on the cell surface. By contrast, most of the Cd 2+ that accumulated during the slower phase could not be removed by EGTA, indicating that it had been taken up by the cells. Additional studies showed that the rapid phase of Cd 2+ accumulation was enhanced when Ca 2+ was present at low concentrations (0.1 mM), and was greatly reduced when Ca 2+ was present at high concentrations (10 mM). These results suggest that ld 2+ damages the junctions between LLC-PK 1 cells by interacting with Ca 2+ -sensitive sites on the basolateral cell surface. (orig.)

Glycoprotein on cell surfaces

International Nuclear Information System (INIS)

Muramatsu, T.

1975-01-01

There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

Rapid fabrication of microfluidic polymer electrolyte membrane fuel cell in PDMS by surface patterning of perfluorinated ion-exchange resin

Energy Technology Data Exchange (ETDEWEB)

Song, Yong-Ak; Han, Jongyoon [Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States); Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States); Batista, Candy [Roxbury Community College, 1234 Columbus Ave., Roxbury Crossing, MA 02120 (United States); Sarpeshkar, Rahul [Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139 (United States)

2008-09-01

In this paper we demonstrate a simple and rapid fabrication method for a microfluidic polymer electrolyte membrane (PEM) fuel cell using polydimethylsiloxane (PDMS), which has become the de facto standard material in BioMEMS. Instead of integrating a Nafion sheet film between two layers of a PDMS device in a traditional ''sandwich format,'' we pattern a perfluorinated ion-exchange resin such as a Nafion resin on a glass substrate using a reversibly bonded PDMS microchannel to generate an ion-selective membrane between the fuel-cell electrodes. After this patterning step, the assembly of the microfluidic fuel cell is accomplished by simple oxygen plasma bonding between the PDMS chip and the glass substrate. In an example implementation, the planar PEM microfluidic fuel cell generates an open circuit voltage of 600-800 mV and delivers a maximum current output of nearly 4 {mu}A. To enhance the power output of the fuel cell we utilize self-assembled colloidal arrays as a support matrix for the Nafion resin. Such arrays allow us to increase the thickness of the ion-selective membrane to 20 {mu}m and increase the current output by 166%. Our novel fabrication method enables rapid prototyping of microfluidic fuel cells to study various ion-exchange resins for the polymer electrolyte membrane. Our work will facilitate the development of miniature, implantable, on-chip power sources for biomedical applications. (author)

Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

Science.gov (United States)

Eguiara, Arrate; Holgado, Olaia; Beloqui, Izaskun; Abalde, Leire; Sanchez, Yolanda; Callol, Carles; Martin, Angel G

2011-11-01

The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.

Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering.

Science.gov (United States)

O'Brien, Paul J; Elahipanah, Sina; Rogozhnikov, Dmitry; Yousaf, Muhammad N

2017-05-24

The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types

Modeling the Excess Cell Surface Stored in a Complex Morphology of Bleb-Like Protrusions.

Directory of Open Access Journals (Sweden)

Maryna Kapustina

2016-03-01

Full Text Available Cells transition from spread to rounded morphologies in diverse physiological contexts including mitosis and mesenchymal-to-amoeboid transitions. When these drastic shape changes occur rapidly, cell volume and surface area are approximately conserved. Consequently, the rounded cells are suddenly presented with a several-fold excess of cell surface whose area far exceeds that of a smooth sphere enclosing the cell volume. This excess is stored in a population of bleb-like protrusions (BLiPs, whose size distribution is shown by electron micrographs to be skewed. We introduce three complementary models of rounded cell morphologies with a prescribed excess surface area. A 2D Hamiltonian model provides a mechanistic description of how discrete attachment points between the cell surface and cortex together with surface bending energy can generate a morphology that satisfies a prescribed excess area and BLiP number density. A 3D random seed-and-growth model simulates efficient packing of BLiPs over a primary rounded shape, demonstrating a pathway for skewed BLiP size distributions that recapitulate 3D morphologies. Finally, a phase field model (2D and 3D posits energy-based constitutive laws for the cell membrane, nematic F-actin cortex, interior cytosol, and external aqueous medium. The cell surface is equipped with a spontaneous curvature function, a proxy for the cell surface-cortex couple, that is a priori unknown, which the model "learns" from the thin section transmission electron micrograph image (2D or the "seed and growth" model image (3D. Converged phase field simulations predict self-consistent amplitudes and spatial localization of pressure and stress throughout the cell for any posited stationary morphology target and cell compartment constitutive properties. The models form a general framework for future studies of cell morphological dynamics in a variety of biological contexts.

Tumor cell surface proteins

International Nuclear Information System (INIS)

Kennel, S.J.; Braslawsky, G.R.; Flynn, K.; Foote, L.J.; Friedman, E.; Hotchkiss, J.A.; Huang, A.H.L.; Lankford, P.K.

1982-01-01

Cell surface proteins mediate interaction between cells and their environment. Unique tumor cell surface proteins are being identified and quantified in several tumor systems to address the following questions: (i) how do tumor-specific proteins arise during cell transformation; (ii) can these proteins be used as markers of tumor cell distribution in vivo; (iii) can cytotoxic drugs be targeted specifically to tumor cells using antibody; and (iv) can solid state radioimmunoassay of these proteins provide a means to quantify transformation frequencies. A tumor surface protein of 180,000 M/sub r/ (TSP-180) has been identified on cells of several lung carcinomas of BALB/c mice. TSP-180 was not detected on normal lung tissue, embryonic tissue, or other epithelial or sarcoma tumors, but it was found on lung carcinomas of other strains of mice. Considerable amino acid sequence homology exists among TSP-180's from several cell sources, indicating that TSP-180 synthesis is directed by normal cellular genes although it is not expressed in normal cells. The regulation of synthesis of TSP-180 and its relationship to normal cell surface proteins are being studied. Monoclonal antibodies (MoAb) to TSP-180 have been developed. The antibodies have been used in immunoaffinity chromatography to isolate TSP-180 from tumor cell sources. This purified tumor antigen was used to immunize rats. Antibody produced by these animals reacted at different sites (epitopes) on the TSP-180 molecule than did the original MoAb. These sera and MoAb from these animals are being used to identify normal cell components related to the TSP-180 molecule

Cell surface area and membrane folding in glioblastoma cell lines differing in PTEN and p53 status.

Directory of Open Access Journals (Sweden)

Simon Memmel

Full Text Available Glioblastoma multiforme (GBM is characterized by rapid growth, invasion and resistance to chemo-/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM, the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm(2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19 showed the highest C m values of 3.7-4.0 µF/cm(2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the

Cells behaviors and genotoxicity on topological surface

International Nuclear Information System (INIS)

Yang, N.; Yang, M.K.; Bi, S.X.; Chen, L.; Zhu, Z.Y.; Gao, Y.T.; Du, Z.

2013-01-01

To investigate different cells behaviors and genotoxicity, which were driven by specific microenvironments, three patterned surfaces (pillars, wide grooves and narrow grooves) and one smooth surface were prepared by template-based technique. Vinculin is a membrane-cytoskeletal protein in focal adhesion plaques and associates with cell–cell and cell–matrix junctions, which can promote cell adhesion and spreading. The immunofluorescence staining of vinculin revealed that the narrow grooves patterned substrate was favorable for L929 cell adhesion. For cell multiplication, the narrow grooves surface was fitted for the proliferation of L929, L02 and MSC cells, the pillars surface was only in favor of L929 cells to proliferate during 7 days of cell cultivation. Cell genetic toxicity was evaluated by cellular micronuclei test (MNT). The results indicated that topological surfaces were more suitable for L929 cells to proliferate and maintain the stability of genome. On the contrary, the narrow grooves surface induced higher micronuclei ratio of L02 and MSC cells than other surfaces. With the comprehensive results of cell multiplication and MNT, it was concluded that the wide grooves surface was best fitted for L02 cells to proliferate and have less DNA damages, and the smooth surface was optimum for the research of MSC cells in vitro. - Highlights: • Different cells behaviors on microstructure surfaces were discussed in this paper. • The expression of cell protein of Vinculin was studied in this research. • Cellular micronuclei test was applied to evaluate cells' genotoxicity. • Cell genotoxicity was first studied in the research field of topological surfaces

Rapid kinetics of lysis in human natural cell-mediated cytotoxicity: some implications

International Nuclear Information System (INIS)

Bloom, E.T.; Babbitt, J.T.

1983-01-01

The entire lytic process of natural cell-mediated cytotoxicity against sensitive target cells can occur rapidly, within minutes. This was demonstrated by 51 chromium release and in single-cell assays. At the cellular level, most of the target cell lysis occurred within 15-30 min after binding to effector cells. The enriched natural killer cell subpopulation of lymphocytes obtained by Percoll density gradient centrifugation (containing greater than 70% large granular lymphocytes (LGL)) was the most rapidly lytic population by 51 chromium release. However, in the single-cell assay, the rate of lysis of bound target cells was quite similar for the LGL-enriched effector subpopulation and the higher density subpopulation of effector cells recognized previously. Both the light and dense effector cells contained similar numbers of target binding cells. Therefore, that the light subpopulation effected lysis more rapidly and to a greater extent than the dense subpopulation suggested that the low-density effector cells probably recycled more rapidly than those of higher density. This was corroborated by the finding that when conjugates were formed at 29 degrees C for the single-cell assay, a significant number of dead unconjugated targets could be observed only on the slides made with the LGL-enriched effector cells but not on those made with dense effector cell. Lysis continued to increase in the chromium-release assay probably because of recycling, recruitment, and/or heterogeneity of the effector cells, and/or because of heterogeneity or delayed death of the target cells

An improved out-cell to in-cell rapid transfer system at the HFEF/South

International Nuclear Information System (INIS)

Bacca, J.P.; Sherman, E.K.

1991-01-01

This paper reports on Argonne National Laboratory's Fuel Cycle Facility (FCF) (formerly named Hot Fuel Examination Facility-South) (HFEF/South) which is currently being refurbished and upgraded in preparation for demonstrating remote, fast reactor metal-fuel reprocessing and refabrication, as part of the Integral Fast Reactor (IFR) Program. Among the FCF hot-cell system upgrades being provided is a newly fabricated, direct, out-of-cell to in-cell, small-item transfer system for the FCF argon cell. This system will enable the rapid transfer of selected small items from the hot cell exterior into the argon cell (argon-gas atmosphere) of the facility, without necessitating the use of formerly employed, very time-consuming, and quite laborious procedures. The new system will be especially valuable for the rapid insertion of IFR fuel processing makeup materials and small tools into the argon cell, and for use in argon cell and overall FCF radioactive contamination-control activities

An improved out-cell to in-cell rapid transfer system at the HFEF-south

International Nuclear Information System (INIS)

Bacca, J.P.; Sherman, E.K.

1990-01-01

The Argonne National Laboratory (ANL) Hot Fuel Examination Facility-South (HFEF-S), located at the ANL-West site of the Idaho National Engineering Laboratory, is currently undergoing extensive refurbishment and modifications in preparation for its use, beginning in 1991, in demonstrating remote recycling of fast reactor, metal-alloy fuel as part of the US Department of Energy liquid-metal reactor, Integral Fast Reactor (IFR) program. Included in these improvements to HFEF-S is a new, small-item, rapid transfer system (RTS). When installed, this system will enable the rapid transfer of small items from the hot-cell exterior into the argon cell (argon-gas atmosphere) of the facility without necessitating the use of time-consuming and laborious procedures. The new RTS will also provide another important function associated with HFEF-S hot-cell operation in the IFR Fuel Recycle Program; namely, the rapid insertion of clean, radioactive contamination-measuring smear paper specimens into the hot cells for area surveys, and the expedited removal of these contaminated (including alpha as well as beta/gamma contamination) smears from the argon cell for transfer to an adjacent health physics field laboratory in the facility for nuclear contamination/radiation counting

Rapid extraction and assay of uranium from environmental surface samples

Energy Technology Data Exchange (ETDEWEB)

Barrett, Christopher A.; Chouyyok, Wilaiwan; Speakman, Robert J.; Olsen, Khris B.; Addleman, Raymond Shane

2017-10-01

Extraction methods enabling faster removal and concentration of uranium compounds for improved trace and low-level assay are demonstrated for standard surface sampling material in support of nuclear safeguards efforts, health monitoring, and other nuclear analysis applications. A key problem with the existing surface sampling swipes is the requirement for complete digestion of sample and sampling matrix. This is a time-consuming and labour-intensive process that limits laboratory throughput, elevates costs, and increases background levels. Various extraction methods are explored for their potential to quickly and efficiently remove different chemical forms of uranium from standard surface sampling material. A combination of carbonate and peroxide solutions is shown to give the most rapid and complete form of uranyl compound extraction and dissolution. This rapid extraction process is demonstrated to be compatible with standard inductive coupled plasma mass spectrometry methods for uranium isotopic assay as well as screening techniques such as x-ray fluorescence. The general approach described has application beyond uranium to other analytes of nuclear forensic interest (e.g., rare earth elements and plutonium) as well as heavy metals for environmental and industrial hygiene monitoring.

Ultrathin Oxide Passivation Layer by Rapid Thermal Oxidation for the Silicon Heterojunction Solar Cell Applications

Directory of Open Access Journals (Sweden)

Youngseok Lee

2012-01-01

Full Text Available It is difficult to deposit extremely thin a-Si:H layer in heterojunction with intrinsic thin layer (HIT solar cell due to thermal damage and tough process control. This study aims to understand oxide passivation mechanism of silicon surface using rapid thermal oxidation (RTO process by examining surface effective lifetime and surface recombination velocity. The presence of thin insulating a-Si:H layer is the key to get high Voc by lowering the leakage current (I0 which improves the efficiency of HIT solar cell. The ultrathin thermal passivation silicon oxide (SiO2 layer was deposited by RTO system in the temperature range 500–950°C for 2 to 6 minutes. The thickness of the silicon oxide layer was affected by RTO annealing temperature and treatment time. The best value of surface recombination velocity was recorded for the sample treated at a temperature of 850°C for 6 minutes at O2 flow rate of 3 Lpm. A surface recombination velocity below 25 cm/s was obtained for the silicon oxide layer of 4 nm thickness. This ultrathin SiO2 layer was employed for the fabrication of HIT solar cell structure instead of a-Si:H, (i layer and the passivation and tunneling effects of the silicon oxide layer were exploited. The photocurrent was decreased with the increase of illumination intensity and SiO2 thickness.

Surface quality in rapid prototype MMD process

Directory of Open Access Journals (Sweden)

Lisandro Vargas Henríquez

2004-09-01

Full Text Available This article summarises a Manufacturing Materials and Processes MSc thesis written for the Mechanical and Electrical Engineering Department. The paper shows the interaction of process, gap (deposition distance and extursion terminal velocity modelled process parameters for CEIF's (Centro de Equipos Interfacultades rapid prototype molten material deposit (MMD Titan SH-1 machine by analysing prototupes improved surface quality and resistence to tension and characterising material. The project applies experimental design criteria for orientating the selection of experimental process parameters. Acrylonitrile-buttadin-styrene (ABS had alredy been mechanically and physicochemically characterised (i.e the material used in the MMD process.

Rapid fabrication of detachable three-dimensional tissues by layering of cell sheets with heating centrifuge.

Science.gov (United States)

Haraguchi, Yuji; Kagawa, Yuki; Hasegawa, Akiyuki; Kubo, Hirotsugu; Shimizu, Tatsuya

2018-01-18

Confluent cultured cells on a temperature-responsive culture dish can be harvested as an intact cell sheet by decreasing temperature below 32°C. A three-dimensional (3-D) tissue can be fabricated by the layering of cell sheets. A resulting 3-D multilayered cell sheet-tissue on a temperature-responsive culture dish can be also harvested without any damage by only temperature decreasing. For shortening the fabrication time of the 3-D multilayered constructs, we attempted to layer cell sheets on a temperature-responsive culture dish with centrifugation. However, when a cell sheet was attached to the culture surface with a conventional centrifuge at 22-23°C, the cell sheet hardly adhere to the surface due to its noncell adhesiveness. Therefore, in this study, we have developed a heating centrifuge. In centrifugation (55g) at 36-37°C, the cell sheet adhered tightly within 5 min to the dish without significant cell damage. Additionally, centrifugation accelerated the cell sheet-layering process. The heating centrifugation shortened the fabrication time by one-fifth compared to a multilayer tissue fabrication without centrifugation. Furthermore, the multilayered constructs were finally detached from the dishes by decreasing temperature. This rapid tissue-fabrication method will be used as a valuable tool in the field of tissue engineering and regenerative therapy. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Surface plasmon resonance sensing: from purified biomolecules to intact cells.

Science.gov (United States)

Su, Yu-Wen; Wang, Wei

2018-04-12

Surface plasmon resonance (SPR) has become a well-recognized label-free technique for measuring the binding kinetics between biomolecules since the invention of the first SPR-based immunosensor in 1980s. The most popular and traditional format for SPR analysis is to monitor the real-time optical signals when a solution containing ligand molecules is flowing over a sensor substrate functionalized with purified receptor molecules. In recent years, rapid development of several kinds of SPR imaging techniques have allowed for mapping the dynamic distribution of local mass density within single living cells with high spatial and temporal resolutions and reliable sensitivity. Such capability immediately enabled one to investigate the interaction between important biomolecules and intact cells in a label-free, quantitative, and single cell manner, leading to an exciting new trend of cell-based SPR bioanalysis. In this Trend Article, we first describe the principle and technical features of two types of SPR imaging techniques based on prism and objective, respectively. Then we survey the intact cell-based applications in both fundamental cell biology and drug discovery. We conclude the article with comments and perspectives on the future developments. Graphical abstract Recent developments in surface plasmon resonance (SPR) imaging techniques allow for label-free mapping the mass-distribution within single living cells, leading to great expansions in biomolecular interactions studies from homogeneous substrates functionalized with purified biomolecules to heterogeneous substrates containing individual living cells.

Rapid surface sampling and archival record system

Energy Technology Data Exchange (ETDEWEB)

Barren, E.; Penney, C.M.; Sheldon, R.B. [GE Corporate Research and Development Center, Schenectady, NY (United States)] [and others

1995-10-01

A number of contamination sites exist in this country where the area and volume of material to be remediated is very large, approaching or exceeding 10{sup 6} m{sup 2} and 10{sup 6} m{sup 3}. Typically, only a small fraction of this material is actually contaminated. In such cases there is a strong economic motivation to test the material with a sufficient density of measurements to identify which portions are uncontaminated, so extensively they be left in place or be disposed of as uncontaminated waste. Unfortunately, since contamination often varies rapidly from position to position, this procedure can involve upwards of one million measurements per site. The situation is complicated further in many cases by the difficulties of sampling porous surfaces, such as concrete. This report describes a method for sampling concretes in which an immediate distinction can be made between contaminated and uncontaminated surfaces. Sample acquisition and analysis will be automated.

Rapid modification of urban land surface temperature during rainfall

Science.gov (United States)

Omidvar, H.; Bou-Zeid, E.; Song, J.; Yang, J.; Arwatz, G.; Wang, Z.; Hultmark, M.; Kaloush, K.

2017-12-01

We study the runoff dynamics and heat transfer over urban pavements during rainfall. A kinematic wave approach is combined with heat storage and transfer schemes to develop a model for impervious (with runoff) and pervious (without runoff) pavements. The resulting framework is a numerical prognostic model that can simulate the temperature fields in the subsurface and runoff layers to capture the rapid cooling of the surface, as well as the thermal pollution advected in the runoff. Extensive field measurements were then conducted over experimental pavements in Arizona to probe the physics and better represent the relevant processes in the model, and then to validate the model. The experimental data and the model results were in very good agreements, and their joint analysis elucidated the physics of the rapid heat transfer from the subsurface to the runoff layer. Finally, we apply the developed model to investigate how the various hydrological and thermal properties of the pavements, as well as ambient environmental conditions, modulate the surface and runoff thermal dynamics, what is the relative importance of each of them, and how we can apply the model mitigate the adverse impacts of urbanization.

Quantitative analysis of rat Ig (sub)classes binding to cell surface antigens

International Nuclear Information System (INIS)

Nilsson, R.; Brodin, T.; Sjoegren, H.-O.

1982-01-01

An indirect 125 I-labeled protein A assay for detection of cell surface-bound rat immunoglobulins is presented. The assay is quantitative and rapid and detects as little as 1 ng of cell surface-bound Ig. It discriminates between antibodies belonging to different IgG subclasses, IgM and IgA. The authors describe the production and specificity control of the reagents used and show that the test can be used for quantitative analysis. A large number of sera from untreated rats are tested to evaluate the frequency of falsely positive responses and variation due to age, sex and strain of rat. With this test it is relatively easy to quantitate the binding of classes and subclasses of rat immunoglobulins in a small volume (6 μl) of untreated serum. (Auth.)

Rapid agonist-induced loss of sup 125 I-. beta. -endorphin opioid receptor sites in NG108-15, but not SK-N-SH neuroblastoma cells

Energy Technology Data Exchange (ETDEWEB)

Cone, R.I.; Lameh, J.; Sadee, W. (Univ. of California, San Francisco (United States))

1991-01-01

The authors have measured {mu} and {delta} opioid receptor sites on intact SK-N-SH and NG108-15 neuroblastoma cells, respectively, in culture. Use of {sup 125}I-{beta}-endorphin ({beta}E) as a tracer, together with {beta}E(6-31) to block high-affinity non-opioid binding in both cell lines, permitted the measurement of cell surface {mu} and {delta} opioid receptor sites. Labeling was at {delta} sites in NG108-15 cells and predominantly at {mu} sites in SK-N-SH cells. Pretreatment with the {mu} and {delta} agonist, DADLE, caused a rapid loss of cell surface {delta} receptor sites in NG108-15 cells, but failed to reduce significantly {mu} receptor density in SK-N-SH cells.

Rapid detection of acetamiprid in foods using surface-enhanced Raman spectroscopy (SERS).

Science.gov (United States)

Wijaya, Wisiani; Pang, Shintaro; Labuza, Theodore P; He, Lili

2014-04-01

Acetamiprid is a neonicotinoid pesticide that is commonly used in modern farming. Acetamiprid residue in food commodities can be a potential harm to human and has been implicated in the honey bee hive die off crisis. In this study, we developed rapid, simple, and sensitive methods to detect acetamiprid in apple juice and on apple surfaces using surface-enhanced Raman spectroscopy (SERS). No pretreatment of apple juice sample was performed. A simple surface swab method was used to recover acetamiprid from the apple surface. Samples were incubated with silver dendrites for several minutes and SERS spectra were taken directly from the silver surface. Detection of a set of 5 apple juice samples can be done within 10 min. The swab-SERS method took 15 min for a set of 5 samples. Resulting spectral data were analyzed using principal component analysis. The highest acetamiprid peak at 634 cm(-1) was used to detect and quantify the amount of acetamiprid spiked in 1:1 water-methanol solvent, apple juice, and on apple surface. The SERS method was able to successfully detect acetamiprid at 0.5 μg/mL (0.5 ppm) in solvent, 3 μg/mL (3 ppm) in apple juice, and 0.125 μg/cm(2) on apple surfaces. The SERS methods provide simple, rapid, and sensitive ways to detect acetamiprid in beverages and on the surfaces of thick skinned fruits and vegetables. © 2014 Institute of Food Technologists®

IL-6 trans-Signaling-Dependent Rapid Development of Cytotoxic CD8+ T Cell Function

Directory of Open Access Journals (Sweden)

Jan P. Böttcher

2014-09-01

Full Text Available Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8+ T cells that use granzyme B (GzmB for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6 trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8+ T cells. Such LSEC-stimulated GzmB-expressing CD8+ T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8+ T cells that may be beneficial for vaccination strategies.

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

Science.gov (United States)

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

2012-01-01

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

Cell behaviour on chemically microstructured surfaces

International Nuclear Information System (INIS)

Magnani, Agnese; Priamo, Alfredo; Pasqui, Daniela; Barbucci, Rolando

2003-01-01

Micropatterned surfaces with different chemical topographies were synthesised in order to investigate the influence of surface chemistry and topography on cell behaviour. The microstructured materials were synthesised by photoimmobilising natural Hyaluronan (Hyal) and its sulphated derivative (HyalS), both adequately functionalised with a photorective moiety, on glass substrates. Four different grating patterns (10, 25, 50 and 100 μm) were used to pattern the hyaluronan. The micropatterned samples were analysed by Secondary Ions Mass Spectrometry, Scanning Electron Microscopy (SEM) and Atomic Force Microscopy to investigate the chemistry and the topography of the surfaces. The spectroscopic and microscopic analysis of the microstructured surfaces revealed that the photoimmobilisation process was successful, demonstrating that the photomask patterns were well reproduced on the sample surface. The influence of chemical topographies on the cell behaviour was then analysed. Human and 3T3 fibroblasts, bovine aortic and human (HGTFN line) endothelial cells were used and their behaviour on the micropatterned surfaces was analysed in terms of adhesion, proliferation, locomotion and orientation. Both chemical and topographical controls were found to be important for cell guidance. By decreasing the stripe dimensions, a more fusiform shape of cell was observed. At the same time, the cell locomotion and orientation parallel to the structure increased. However, differences in cell behaviour were detected according to both cell type and micropattern dimensions

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour

Energy Technology Data Exchange (ETDEWEB)

Irving, Michael; Murphy, Mark F; Lilley, Francis; French, Paul W; Burton, David R [General Engineering Research Institute, Liverpool John Moores University, Liverpool, L3 3AF (United Kingdom); Dixon, Simon [Biomer Technology LTD, 10 Seymour Court, Tudor Road, Manor Park, Runcorn, Cheshire, WA7 1SY (United Kingdom); Sharp, Martin C [General Engineering Research Institute, Liverpool John Moores University, Liverpool, L3 3AF (United Kingdom)

2017-02-01

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72 h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods. - Highlights: • Abrasive polishing can generate nano-scratches on stainless steel to cast polymer films for cell

The use of abrasive polishing and laser processing for developing polyurethane surfaces for controlling fibroblast cell behaviour

International Nuclear Information System (INIS)

Irving, Michael; Murphy, Mark F; Lilley, Francis; French, Paul W; Burton, David R; Dixon, Simon; Sharp, Martin C

2017-01-01

Studies have shown that surfaces having micro and nano-scale features can be used to control cell behaviours including; cell proliferation, migration and adhesion. The aim of this work was to compare the use of laser processing and abrasive polishing to develop micro/nano-patterned polyurethane substrates for controlling fibroblast cell adhesion, migration and proliferation. Laser processing in a directional manner resulted in polyurethane surfaces having a ploughed field effect with micron-scale features. In contrast, abrasive polishing in a directional and random manner resulted in polyurethane surfaces having sub-micron scale features orientated in a linear or random manner. Results show that when compared with flat (non-patterned) polymer, both the laser processed and abrasive polished surface having randomly organised features, promoted significantly greater cell adhesion, while also enhancing cell proliferation after 72 h. In contrast, the abrasive polished surface having linear features did not enhance cell adhesion or proliferation when compared to the flat surface. For cell migration, the cells growing on the laser processed and abrasively polished random surface showed decreased levels of migration when compared to the flat surface. This study shows that both abrasive polishing and laser processing can be used to produce surfaces having features on the nano-scale and micron-scale, respectively. Surfaces produced using both techniques can be used to promote fibroblast cell adhesion and proliferation. Thus both methods offer a viable alternative to using lithographic techniques for developing patterned surfaces. In particular, abrasive polishing is an attractive method due to it being a simple, rapid and inexpensive method that can be used to produce surfaces having features on a comparable scale to more expensive, multi-step methods. - Highlights: • Abrasive polishing can generate nano-scratches on stainless steel to cast polymer films for cell

Neurotrophin responsiveness of sympathetic neurons is regulated by rapid mobilization of the p75 receptor to the cell surface through TrkA activation of Arf6.

Science.gov (United States)

Edward Hickman, F; Stanley, Emily M; Carter, Bruce D

2018-05-22

availability, may provide insight into how and why neurodegenerative processes manifest and reveal new therapeutic targets. Results from this study indicate a novel mechanism by which p75NTR can be rapidly shuttled to the cell surface from existing intracellular pools and explores a unique pathway by which NGF regulates the sympathetic innervation of target tissues, which has profound consequences for the function of these organs. Copyright © 2018 the authors.

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

Science.gov (United States)

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.

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

Science.gov (United States)

Chen, Jianbo

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

Cell behavior on microparticles with different surface morphology

International Nuclear Information System (INIS)

Huang Sha; Fu Xiaobing

2010-01-01

Microparticles can serve as substrates for cell amplification and deliver the cell aggregation to the site of the defect for tissue regeneration. To develop favorable microparticles for cell delivery application, we fabricated and evaluated three types of microparticles that differ in surface properties. The microparticles with varied surface morphology (smooth, pitted and multicavity) were created from chemically crosslinked gelatin particles that underwent various drying treatments. Three types of microparticles were characterized and assessed in terms of the cell behavior of human keratinocytes and fibroblasts seeded on them. The cells could attach, spread and proliferate on all types of microparticles but spread and populated more slowly on the microparticles with smooth surfaces than on those with pitted or multicavity surfaces. Microparticles with a multicavity surface demonstrated the highest cell attachment and growth rate. Furthermore, cells tested on microparticles with a multicavity surface exhibited better morphology and induced the earlier formation of extracellular-based cell-microparticle aggregation than those on microparticles with other surface morphology (smooth and pitted). Thus, microparticles with a multicavity surface show promise for attachment and proliferation of cells in tissue engineering.

Diclofenac in Arabidopsis cells: Rapid formation of conjugates.

Science.gov (United States)

Fu, Qiuguo; Ye, Qingfu; Zhang, Jianbo; Richards, Jaben; Borchardt, Dan; Gan, Jay

2017-03-01

Pharmaceutical and personal care products (PPCPs) are continuously introduced into the soil-plant system, through practices such as agronomic use of reclaimed water and biosolids containing these trace contaminants. Plants may accumulate PPCPs from soil, serving as a conduit for human exposure. Metabolism likely controls the final accumulation of PPCPs in plants, but is in general poorly understood for emerging contaminants. In this study, we used diclofenac as a model compound, and employed 14 C tracing, and time-of-flight (TOF) and triple quadruple (QqQ) mass spectrometers to unravel its metabolism pathways in Arabidopsis thaliana cells. We further validated the primary metabolites in Arabidopsis seedlings. Diclofenac was quickly taken up into A. thaliana cells. Phase I metabolism involved hydroxylation and successive oxidation and cyclization reactions. However, Phase I metabolites did not accumulate appreciably; they were instead rapidly conjugated with sulfate, glucose, and glutamic acid through Phase II metabolism. In particular, diclofenac parent was directly conjugated with glutamic acid, with acyl-glutamatyl-diclofenac accounting for >70% of the extractable metabolites after 120-h incubation. In addition, at the end of incubation, >40% of the spiked diclofenac was in the non-extractable form, suggesting extensive sequestration into cell matter. The rapid formation of non-extractable residue and dominance of diclofenac-glutamate conjugate uncover previously unknown metabolism pathways for diclofenac. In particular, the rapid conjugation of parent highlights the need to consider conjugates of emerging contaminants in higher plants, and their biological activity and human health implications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bone marrow mesenchymal stem cells differentiation and proliferation on the surface of coral implant

International Nuclear Information System (INIS)

Al-Salihi, K.A.; Samsudin, A.R.

2004-01-01

This study was designed to evaluate the ability of natural coral implant to provide an environment for marrow cells to differentiate into osteoblasts and function suitable for mineralized tissue formation. DNA content, alkaline phosptatase (ALP) activity, calcium (Ca) content and mineralized nodules, were measured at day 3, day 7 and day 14, in rat bone marrow stromal cells cultured with coral discs glass discs, while cells alone and coral disc alone cultured as control. DNA content, ALP activity, Ca content measurements showed no difference between coral, glass and cells groups at 3 day which were higher than control (coral disc alone), but there were higher asurement at day 7 and 14 in the cell cultured on coral than on glass discs, control cells and control coral discs. Mineralized nodules formation (both in area and number) was more predominant on the coral surface than in control groups. These results showed that natural coral implant provided excellent and favorable situation for marrow cell to differentiate to osteoblasts, lead to large amount of mineralized tissue formation on coral surface. This in vitro result could explain the rapid bone bonding of coral in vivo. (Author)

Innate lymphoid cells: models of plasticity for immune homeostasis and rapid responsiveness in protection.

Science.gov (United States)

Almeida, F F; Belz, G T

2016-09-01

Innate lymphoid cells (ILCs) have stormed onto the immune landscape as "newly discovered" cell types. These tissue-resident sentinels are enriched at mucosal surfaces and engage in complex cross talk with elements of the adaptive immune system and microenvironment to orchestrate immune homeostasis. Many parallels exist between innate cells and T cells leading to the initial partitioning of ILCs into rather rigid subsets that reflect their "adaptive-like" effector cytokines profiles. ILCs themselves, however, have unique attributes that are only just beginning to be elucidated. These features result in complementarity with, rather than complete duplication of, functions of the adaptive immune system. Key transcription factors determine the pathway of differentiation of progenitors towards an ILC1, ILC2, or ILC3 subset. Once formed, flexibility in the responses of these subsets to stimuli unexpectedly allows transdifferentation between the different subsets and the acquisition of altered phenotypes and function. This provides a mechanism for rapid innate immune responsiveness. Here, we discuss the models of differentiation for maintenance and activation of tissue-resident ILCs in maintaining immune homeostasis and protection.

Surface-modified CMOS IC electrochemical sensor array targeting single chromaffin cells for highly parallel amperometry measurements.

Science.gov (United States)

Huang, Meng; Delacruz, Joannalyn B; Ruelas, John C; Rathore, Shailendra S; Lindau, Manfred

2018-01-01

Amperometry is a powerful method to record quantal release events from chromaffin cells and is widely used to assess how specific drugs modify quantal size, kinetics of release, and early fusion pore properties. Surface-modified CMOS-based electrochemical sensor arrays allow simultaneous recordings from multiple cells. A reliable, low-cost technique is presented here for efficient targeting of single cells specifically to the electrode sites. An SU-8 microwell structure is patterned on the chip surface to provide insulation for the circuitry as well as cell trapping at the electrode sites. A shifted electrode design is also incorporated to increase the flexibility of the dimension and shape of the microwells. The sensitivity of the electrodes is validated by a dopamine injection experiment. Microwells with dimensions slightly larger than the cells to be trapped ensure excellent single-cell targeting efficiency, increasing the reliability and efficiency for on-chip single-cell amperometry measurements. The surface-modified device was validated with parallel recordings of live chromaffin cells trapped in the microwells. Rapid amperometric spikes with no diffusional broadening were observed, indicating that the trapped and recorded cells were in very close contact with the electrodes. The live cell recording confirms in a single experiment that spike parameters vary significantly from cell to cell but the large number of cells recorded simultaneously provides the statistical significance.

Biomolecular strategies for cell surface engineering

Science.gov (United States)

Wilson, John Tanner

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

A nucleation theory of cell surface capping

International Nuclear Information System (INIS)

Coutsias, E.A.; Wester, M.J.; Perelson, A.S.

1997-01-01

We propose a new theory of cell surface capping based on the principles of nucleation. When antibody interacts with cell surface molecules, the molecules initially form small aggregates called patches that later coalesce into a large aggregate called a cap. While a cap can form by patches being pulled together by action of the cell''s cytoskeleton, in the case of some molecules, disruption of the cytoskeleton does not prevent cap formation. Diffusion of large aggregates on a cell surface is slow, and thus we propose that a cap can form solely through the diffusion of small aggregates containing just one or a few cell surface molecules. Here we consider the extreme case in which single molecules are mobile, but aggregates of all larger sizes are immobile. We show that a set of patches in equilibrium with a open-quotes seaclose quotes of free cell surface molecules can undergo a nucleation-type phase transition in which the largest patch will bind free cell surface molecules, deplete the concentration of such molecules in the open-quotes seaclose quotes and thus cause the other patches to shrink in size. We therefore show that a cap can form without patches having to move, collide with each other, and aggregate

Cell Adhesion on Surface-Functionalized Magnesium.

Science.gov (United States)

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

2016-05-18

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

Rapid lentiviral transduction preserves the engraftment potential of Fanca(-/-) hematopoietic stem cells.

Science.gov (United States)

Müller, Lars U W; Milsom, Michael D; Kim, Mi-Ok; Schambach, Axel; Schuesler, Todd; Williams, David A

2008-06-01

Fanconi anemia (FA) is a rare recessive syndrome, characterized by congenital anomalies, bone marrow failure, and predisposition to cancer. Two earlier clinical trials utilizing gamma-retroviral vectors for the transduction of autologous FA hematopoietic stem cells (HSCs) required extensive in vitro manipulation and failed to achieve detectable long-term engraftment of transduced HSCs. As a strategy for minimizing ex vivo manipulation, we investigated the use of a "rapid" lentiviral transduction protocol in a murine Fanca(-/-) model. Importantly, while this and most murine models of FA fail to completely mimic the human hematopoietic phenotype, we observed a high incidence of HSC transplant engraftment failure and low donor chimerism after conventional transduction (CT) of Fanca(-/-) donor cells. In contrast, rapid transduction (RT) of Fanca(-/-) HSCs preserved engraftment to the level achieved in wild-type cells, resulting in long-term multilineage engraftment of gene-modified cells. We also demonstrate the correction of the characteristic hypersensitivity of FA cells against the cross-linking agent mitomycin C (MMC), and provide evidence for the advantage of using pharmacoselection as a means of further increasing gene-modified cells after RT. Collectively, these data support the use of rapid lentiviral transduction for gene therapy in FA.

Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance.

Science.gov (United States)

Lange, K; Gartzke, J

2001-08-01

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.

Development of cell-based quantitative evaluation method for cell cycle-arrest type cancer drugs for apoptosis by high precision surface plasmon resonance sensor

Science.gov (United States)

Ona, Toshihiro; Nishijima, Hiroshi; Kosaihira, Atsushi; Shibata, Junko

2008-04-01

In vitro rapid and quantitative cell-based assay is demanded to verify the efficacy prediction of cancer drugs since a cancer patient may have unconventional aspects of tumor development. Here, we show the rapid and non-label quantitative verifying method and instrumentation of apoptosis for cell cycle-arrest type cancer drugs (Roscovitine and D-allose) by reaction analysis of living liver cancer cells cultured on a sensor chip with a newly developed high precision (50 ndeg s -1 average fluctuation) surface plasmon resonance (SPR) sensor. The time-course cell reaction as the SPR angle change rate for 10 min from 30 min cell culture with a drug was significantly related to cell viability. By the simultaneous detection of differential SPR angle change and fluorescence by specific probes using the new instrument, the SPR angle was related to the nano-order potential decrease in inner mitochondrial membrane potential. The results obtained are universally valid for the cell cycle-arrest type cancer drugs, which mediate apoptosis through different cell-signaling pathways, by a liver cancer cell line of Hep G2 (P<0.001). This system towards the application to evaluate personal therapeutic potentials of drugs using cancer cells from patients in clinical use.

Rapid thyroid dysfunction screening based on serum surface-enhanced Raman scattering and multivariate statistical analysis

Science.gov (United States)

Tian, Dayong; Lü, Guodong; Zhai, Zhengang; Du, Guoli; Mo, Jiaqing; Lü, Xiaoyi

2018-01-01

In this paper, serum surface-enhanced Raman scattering and multivariate statistical analysis are used to investigate a rapid screening technique for thyroid function diseases. At present, the detection of thyroid function has become increasingly important, and it is urgently necessary to develop a rapid and portable method for the detection of thyroid function. Our experimental results show that, by using the Silmeco-based enhanced Raman signal, the signal strength greatly increases and the characteristic peak appears obviously. It is also observed that the Raman spectra of normal and anomalous thyroid function human serum are significantly different. Principal component analysis (PCA) combined with linear discriminant analysis (LDA) was used to diagnose thyroid dysfunction, and the diagnostic accuracy was 87.4%. The use of serum surface-enhanced Raman scattering technology combined with PCA-LDA shows good diagnostic performance for the rapid detection of thyroid function. By means of Raman technology, it is expected that a portable device for the rapid detection of thyroid function will be developed.

Basic Surface Properties of Mononuclear Cells from Didelphis marsupialis

Directory of Open Access Journals (Sweden)

Nacife Valéria Pereira

1998-01-01

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

Tracking Cell Surface GABAB Receptors Using an α-Bungarotoxin Tag*

Science.gov (United States)

Wilkins, Megan E.; Li, Xinyan; Smart, Trevor G.

2008-01-01

GABAB receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABAB receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABAB receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, α-bungarotoxin. By using the α-bungarotoxin binding site-tagged GABAB R1a subunit (R1aBBS), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, α-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABAB receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors. PMID:18812318

Rapid transcriptional pulsing dynamics of high expressing retroviral transgenes in embryonic stem cells.

Directory of Open Access Journals (Sweden)

Mandy Y M Lo

Full Text Available Single cell imaging studies suggest that transcription is not continuous and occurs as discrete pulses of gene activity. To study mechanisms by which retroviral transgenes can transcribe to high levels, we used the MS2 system to visualize transcriptional dynamics of high expressing proviral integration sites in embryonic stem (ES cells. We established two ES cell lines each bearing a single copy, self-inactivating retroviral vector with a strong ubiquitous human EF1α gene promoter directing expression of mRFP fused to an MS2-stem-loop array. Transfection of MS2-EGFP generated EGFP focal dots bound to the mRFP-MS2 stem loop mRNA. These transcription foci colocalized with the transgene integration site detected by immunoFISH. Live tracking of single cells for 20 minutes detected EGFP focal dots that displayed frequent and rapid fluctuations in transcription over periods as short as 25 seconds. Similarly rapid fluctuations were detected from focal doublet signals that colocalized with replicated proviral integration sites by immunoFISH, consistent with transcriptional pulses from sister chromatids. We concluded that retroviral transgenes experience rapid transcriptional pulses in clonal ES cell lines that exhibit high level expression. These events are directed by a constitutive housekeeping gene promoter and may provide precedence for rapid transcriptional pulsing at endogenous genes in mammalian stem cells.

Lactoperoxidase catalyzed radioiodination of cell surface immunoglobulin: incorporated radioactivity may not reflect relative cell surface Ig density

International Nuclear Information System (INIS)

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

1979-01-01

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

Multijunction Solar Cell Technology for Mars Surface Applications

Science.gov (United States)

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

2006-01-01

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

Chemical characterization of solid polymer electrolyte membrane surfaces in LiFePO4 half-cells

Science.gov (United States)

Kyu, Thein; He, Ruixuan; Peng, Fang; Dunn, William E.; Kyu's Group Team, Dr.

High temperature (60 °C) capacity retention of succinonitrile plasticized solid polymer electrolyte membrane (PEM) in a LiFePO4 half-cell was investigated with or without lithium bis(oxalato)borate (LiBOB) modification. Various symmetric cells and half-cells were studied under different thermal and electrochemical conditions. At room temperature cycling, the unmodified PEM in the half-cell appeared stable up to 50 cycles tested. Upon cycling at 60 °C, the capacity decays rapidly and concurrently the cell resistance increased. The chemical compositions of the solid PEM surfaces on both cathode and anode sides were analyzed. New IR bands (including those belonged to amide) were discerned on the unmodified PEM surface of the Li electrode side at 60 °C suggestive of side reaction, but no new bands develop during room temperature cycling. To our astonishment, the side reaction was effectively suppressed upon LiBOB addition (0.4 wt%) into the PEM, contributing to increased high temperature capacity retention at 60°C. Plausible mechanisms of capacity fading and improved cycling performance due to LiBOB modification are discussed.

Monitoring cell culture media degradation using surface enhanced Raman scattering (SERS) spectroscopy.

Science.gov (United States)

Calvet, Amandine; Ryder, Alan G

2014-08-20

The quality of the cell culture media used in biopharmaceutical manufacturing is a crucial factor affecting bioprocess performance and the quality of the final product. Due to their complex composition these media are inherently unstable, and significant compositional variations can occur particularly when in the prepared liquid state. For example photo-degradation of cell culture media can have adverse effects on cell viability and thus process performance. There is therefore, from quality control, quality assurance and process management view points, an urgent demand for the development of rapid and inexpensive tools for the stability monitoring of these complex mixtures. Spectroscopic methods, based on fluorescence or Raman measurements, have now become viable alternatives to more time-consuming and expensive (on a unit analysis cost) chromatographic and/or mass spectrometry based methods for routine analysis of media. Here we demonstrate the application of surface enhanced Raman scattering (SERS) spectroscopy for the simple, fast, analysis of cell culture media degradation. Once stringent reproducibility controls are implemented, chemometric data analysis methods can then be used to rapidly monitor the compositional changes in chemically defined media. SERS shows clearly that even when media are stored at low temperature (2-8°C) and in the dark, significant chemical changes occur, particularly with regard to cysteine/cystine concentration. Copyright © 2014 Elsevier B.V. All rights reserved.

Mpl traffics to the cell surface through conventional and unconventional routes.

Science.gov (United States)

Cleyrat, Cédric; Darehshouri, Anza; Steinkamp, Mara P; Vilaine, Mathias; Boassa, Daniela; Ellisman, Mark H; Hermouet, Sylvie; Wilson, Bridget S

2014-09-01

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hyperkalemia caused by rapid red cell transfusion and the potassium absorption filter

Directory of Open Access Journals (Sweden)

Yasuhiko Imashuku

2017-01-01

Full Text Available We report a case of transient hyperkalemia during hysterectomy after cesarean section, due to preoperatively undiagnosed placenta accreta that caused unforeseen massive hemorrhage and required rapid red cell transfusion. Hyperkalemia-induced by rapid red cell transfusion is a well-known severe complication of transfusion; however, in patients with sudden massive hemorrhage, rapid red cell transfusion is necessary to save their life. In such cases, it is extremely important to monitor serum potassium levels. For an emergency situation, a system should be developed to ensure sufficient preparation for immediate transfusion and laboratory tests. Furthermore, sufficient stock of preparations to treat hyperkalemia, such as calcium preparations, diuretics, glucose, and insulin is required. Moreover, a transfusion filter that absorbs potassium has been developed and is now available for clinical use in Japan. The filter is easy to use and beneficial, and should be prepared when it is available.

Hyperkalemia caused by rapid red cell transfusion and the potassium absorption filter

Science.gov (United States)

Imashuku, Yasuhiko; Kitagawa, Hirotoshi; Mizuno, Takayoshi; Fukushima, Yutaka

2017-01-01

We report a case of transient hyperkalemia during hysterectomy after cesarean section, due to preoperatively undiagnosed placenta accreta that caused unforeseen massive hemorrhage and required rapid red cell transfusion. Hyperkalemia-induced by rapid red cell transfusion is a well-known severe complication of transfusion; however, in patients with sudden massive hemorrhage, rapid red cell transfusion is necessary to save their life. In such cases, it is extremely important to monitor serum potassium levels. For an emergency situation, a system should be developed to ensure sufficient preparation for immediate transfusion and laboratory tests. Furthermore, sufficient stock of preparations to treat hyperkalemia, such as calcium preparations, diuretics, glucose, and insulin is required. Moreover, a transfusion filter that absorbs potassium has been developed and is now available for clinical use in Japan. The filter is easy to use and beneficial, and should be prepared when it is available. PMID:28217070

Rapid fabrication of large-area, corrosion-resistant superhydrophobic Mg alloy surfaces.

Science.gov (United States)

Xu, Wenji; Song, Jinlong; Sun, Jing; Lu, Yao; Yu, Ziyuan

2011-11-01

A superhydrophobic magnesium (Mg) alloy surface was successfully fabricated via a facile electrochemical machining process, and subsequently covered with a fluoroalkylsilane (FAS) film. The surface morphologies and chemical compositions were investigated using a scanning electron microscope (SEM) equipped with an energy-dispersive spectroscopy (EDS) and a Fourier-transform infrared spectrophotometer (FTIR). The results show hierarchal rough structures and an FAS film with a low surface energy on the Mg alloy surfaces, which confers good superhydrophobicity with a water contact angle of 165.2° and a water tilting angle of approximately 2°. The processing conditions, such as the processing time and removal rate per unit area at a constant removal mass per unit area, were investigated to determine their effects on the superhydrophobicity. Interestingly, when the removal mass per unit area is constant at approximately 11.10 mg/cm(2), the superhydrophobicity does not change with the removal rate per unit area. Therefore, a superhydrophobic Mg alloy surface can be rapidly fabricated based on this property. A large-area superhydrophobic Mg alloy surface was also fabricated for the first time using a small-area moving cathode. The corrosion resistance and durability of the superhydrophobic surfaces were also examined.

High-level ethanol production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase

Energy Technology Data Exchange (ETDEWEB)

Kondo, A.; Shigechi, H.; Abe, M.; Uyama, K. [Dept. of Chemical Science and Engineering, Kobe Univ., Nadaku, Kobe (Japan); Matsumoto, T.; Fukuda, H. [Div. of Molecular Science, Kobe Univ., Nadaku, Kobe (Japan); Takahashi, S.; Ueda, M.; Tanaka, A. [Dept. of Synthetic Chemistry and Biological Chemistry, Kyoto Univ., Sakyoku, Kyoto (Japan); Kishimoto, M. [Dept. of Biotechnology, Osaka Univ., Osaka (Japan)

2002-07-01

A Strain of host yeast YF207, which is a tryptophan auxotroph and shows strong flocculation ability, was obtained from Saccharomyces diastaticus ATCC60712 and S. cerevisiae W303-1B by tetrad analysis. The plasmid pGA11, which is a multicopy plasmid for cell-surface expression of the Rhyzopus oryzae glucoamylase/{alpha}-agglutinin fusion protein, was then introduced into this flocculent yeast strain (YF207/pGA11). Yeast YF207/pGA11 grew rapidly under aerobic condition (dissolved oxygen 2.0 ppm), using soluble starch. The harvested cells were used for batch fermentation of soluble starch to ethanol under anaerobic condition and showed high ethanol production rates (0.71 g h{sup -1} I{sup -1}) without a time lag, because glucoamylase was immobilized on the yeast cell surface. During repeated utilization of cells for fermentation, YF207/pGA11 maintained high ethanol production rates over 300 h. Moreover, in fed-batch fermentation with YF207/pGA11 for approximately 120 h, the ethanol concentration reached up to 50 g I{sup -1}. In conclusion, flocculent yeast cells displaying cell-surface glucoamylase are considered to be very effective for the direct fermentation of soluble starch to ethanol. (orig.)

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Directory of Open Access Journals (Sweden)

Craig A Gedye

Full Text Available Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell

Cell surface profiling using high-throughput flow cytometry: a platform for biomarker discovery and analysis of cellular heterogeneity.

Science.gov (United States)

Gedye, Craig A; Hussain, Ali; Paterson, Joshua; Smrke, Alannah; Saini, Harleen; Sirskyj, Danylo; Pereira, Keira; Lobo, Nazleen; Stewart, Jocelyn; Go, Christopher; Ho, Jenny; Medrano, Mauricio; Hyatt, Elzbieta; Yuan, Julie; Lauriault, Stevan; Meyer, Mona; Kondratyev, Maria; van den Beucken, Twan; Jewett, Michael; Dirks, Peter; Guidos, Cynthia J; Danska, Jayne; Wang, Jean; Wouters, Bradly; Neel, Benjamin; Rottapel, Robert; Ailles, Laurie E

2014-01-01

Cell surface proteins have a wide range of biological functions, and are often used as lineage-specific markers. Antibodies that recognize cell surface antigens are widely used as research tools, diagnostic markers, and even therapeutic agents. The ability to obtain broad cell surface protein profiles would thus be of great value in a wide range of fields. There are however currently few available methods for high-throughput analysis of large numbers of cell surface proteins. We describe here a high-throughput flow cytometry (HT-FC) platform for rapid analysis of 363 cell surface antigens. Here we demonstrate that HT-FC provides reproducible results, and use the platform to identify cell surface antigens that are influenced by common cell preparation methods. We show that multiple populations within complex samples such as primary tumors can be simultaneously analyzed by co-staining of cells with lineage-specific antibodies, allowing unprecedented depth of analysis of heterogeneous cell populations. Furthermore, standard informatics methods can be used to visualize, cluster and downsample HT-FC data to reveal novel signatures and biomarkers. We show that the cell surface profile provides sufficient molecular information to classify samples from different cancers and tissue types into biologically relevant clusters using unsupervised hierarchical clustering. Finally, we describe the identification of a candidate lineage marker and its subsequent validation. In summary, HT-FC combines the advantages of a high-throughput screen with a detection method that is sensitive, quantitative, highly reproducible, and allows in-depth analysis of heterogeneous samples. The use of commercially available antibodies means that high quality reagents are immediately available for follow-up studies. HT-FC has a wide range of applications, including biomarker discovery, molecular classification of cancers, or identification of novel lineage specific or stem cell markers.

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

Directory of Open Access Journals (Sweden)

Christian Niehage

Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

Analysis of Pseudomonas aeruginosa cell envelope proteome by capture of surface-exposed proteins on activated magnetic nanoparticles.

Directory of Open Access Journals (Sweden)

Davide Vecchietti

Full Text Available We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria.

Analysis of Pseudomonas aeruginosa Cell Envelope Proteome by Capture of Surface-Exposed Proteins on Activated Magnetic Nanoparticles

Science.gov (United States)

Vecchietti, Davide; Di Silvestre, Dario; Miriani, Matteo; Bonomi, Francesco; Marengo, Mauro; Bragonzi, Alessandra; Cova, Lara; Franceschi, Eleonora; Mauri, Pierluigi; Bertoni, Giovanni

2012-01-01

We report on specific magneto-capturing followed by Multidimensional Protein Identification Technology (MudPIT) for the analysis of surface-exposed proteins of intact cells of the bacterial opportunistic pathogen Pseudomonas aeruginosa. The magneto-separation of cell envelope fragments from the soluble cytoplasmic fraction allowed the MudPIT identification of the captured and neighboring proteins. Remarkably, we identified 63 proteins captured directly by nanoparticles and 67 proteins embedded in the cell envelope fragments. For a high number of proteins, our analysis strongly indicates either surface exposure or localization in an envelope district. The localization of most identified proteins was only predicted or totally unknown. This novel approach greatly improves the sensitivity and specificity of the previous methods, such as surface shaving with proteases that was also tested on P. aeruginosa. The magneto-capture procedure is simple, safe, and rapid, and appears to be well-suited for envelope studies in highly pathogenic bacteria. PMID:23226459

"Rapid Revisit" Measurements of Sea Surface Winds Using CYGNSS

Science.gov (United States)

Park, J.; Johnson, J. T.

2017-12-01

The Cyclone Global Navigation Satellite System (CYGNSS) is a space-borne GNSS-R (GNSS-Reflectometry) mission that launched December 15, 2016 for ocean surface wind speed measurements. CYGNSS includes 8 small satellites in the same LEO orbit, so that the mission provides wind speed products having unprecedented coverage both in time and space to study multi-temporal behaviors of oceanic winds. The nature of CYGNSS coverage results in some locations on Earth experiencing multiple wind speed measurements within a short period of time (a "clump" of observations in time resulting in a "rapid revisit" series of measurements). Such observations could seemingly provide indications of regions experiencing rapid changes in wind speeds, and therefore be of scientific utility. Temporally "clumped" properties of CYGNSS measurements are investigated using early CYGNSS L1/L2 measurements, and the results show that clump durations and spacing vary with latitude. For example, the duration of a clump can extend as long as a few hours at higher latitudes, with gaps between clumps ranging from 6 to as high as 12 hours depending on latitude. Examples are provided to indicate the potential of changes within a clump to produce a "rapid revisit" product for detecting convective activity. Also, we investigate detector design for identifying convective activities. Results from analyses using recent CYGNSS L2 winds will be provided in the presentation.

Rapid surface sampling and archival record system (RSSAR)

International Nuclear Information System (INIS)

Barren, E.; Bracco, A.; Dorn, S.B.

1997-01-01

Purpose is to develop a rapid surface (concrete, steel) contamination measurement system that will provide a ''quick-look'' indication of contamination areas, an archival record, and an automated analysis. A bulk sampling oven is also being developed. The sampling device consists of a sampling head, a quick look detector, and an archiving system (sorbent tube). The head thermally desorbs semi-volatiles, such as PCBs, oils, etc., from concrete and steel surfaces; the volatilized materials are passed through a quick-look detector. Sensitivity of the detector can be attenuated for various contaminant levels. Volatilized materials are trapped in a tube filled with adsorbent. The tubes are housed in a magazine which also archives information about sampling conditions. Analysis of the tubes can be done at a later date. The concrete sampling head is fitted with a tungsten-halogen lamp; in laboratory experiments it has extracted model contaminants by heating the top 4mm of the surface to 250 C within 100-200 s. The steel sampling head has been tested on different types of steels and has extracted model contaminants within 30 s. A mathematical model of heat and mass transport in concrete has been developed. Rate of contaminant removal is at maximum when the moisture content is about 100 kg/m 3 . The system will be useful during decontamination and decommissioning operations

Surface engineering of cardiovascular stent with endothelial cell selectivity for in vivo re-endothelialisation.

Science.gov (United States)

Wei, Yu; Ji, Ying; Xiao, Lin-Lin; Lin, Quan-kui; Xu, Jian-ping; Ren, Ke-feng; Ji, Jian

2013-04-01

The in vivo endothelialisation of materials provides a promising strategy for the rapid re-endothelialisation of a cardiovascular implantation. Although many studies have focused on improving the rapid endothelialisation through the immobilisation of bioactive molecules, it should be noted that the endothelial cells (ECs) will compete with other cell types in vivo. Thus, the efforts to partially enhance the EC growth without considering the cell competition might be misleading and meaningless in vivo. In this study, we demonstrated that the competitive growth of human umbilical vein endothelial cells (HUVECs) over human aortic smooth muscle cells (HASMCs) could be increased through the synergic action of the nonspecific resistance to phosphorylcholine and the specific recognition of the REDV peptide. Further in vivo data indicate that the competitive ability of ECs over SMCs, instead of the number of ECs, is a significantly more important criterion for the development of a pure endothelial layer in vivo and thus the attainment of a better anti-restenosis effect. Consequently, the surface tailoring of a stent to obtain high endothelial cell selectivity is likely an effective design criterion for in situ endothelialisation and a possible future solution for the problem of in-stent restenosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Chimeric antigen receptor T cells form nonclassical and potent immune synapses driving rapid cytotoxicity.

Science.gov (United States)

Davenport, A J; Cross, R S; Watson, K A; Liao, Y; Shi, W; Prince, H M; Beavis, P A; Trapani, J A; Kershaw, M H; Ritchie, D S; Darcy, P K; Neeson, P J; Jenkins, M R

2018-02-27

Chimeric antigen receptor T (CAR-T) cells are effective serial killers with a faster off-rate from dying tumor cells than CAR-T cells binding target cells through their T cell receptor (TCR). Here we explored the functional consequences of CAR-mediated signaling using a dual-specific CAR-T cell, where the same cell was triggered via TCR (tcrCTL) or CAR (carCTL). The carCTL immune synapse lacked distinct LFA-1 adhesion rings and was less reliant on LFA to form stable conjugates with target cells. carCTL receptors associated with the synapse were found to be disrupted and formed a convoluted multifocal pattern of Lck microclusters. Both proximal and distal receptor signaling pathways were induced more rapidly and subsequently decreased more rapidly in carCTL than in tcrCTL. The functional consequence of this rapid signaling in carCTL cells included faster lytic granule recruitment to the immune synapse, correlating with faster detachment of the CTL from the target cell. This study provides a mechanism for how CAR-T cells can debulk large tumor burden quickly and may contribute to further refinement of CAR design for enhancing the quality of signaling and programming of the T cell. Copyright © 2018 the Author(s). Published by PNAS.

Studies on Rapidly Frozen Suspensions of Yeast Cells by Differential Thermal Analysis and Conductometry

Science.gov (United States)

Mazur, Peter

1963-01-01

Few, if any, yeast cells survived rapid cooling to -196°C and subsequent slow warming. After rapid freezing, the suspensions absorbed latent heat of fusion between -15° and 0°C during warming, and the relation between the amount of heat absorbed and the concentration of cells was the same as that in equivalent KCl solutions, indicating that frozen suspensions behave thermally like frozen solutions. The amount of heat absorbed was such that more than 80 per cent of the intracellular solution had to be frozen. The conductometric behavior of frozen suspensions showed that cell solutes were still inside the cells and surrounded by an intact cell membrane at the time heat was being absorbed. Two models are consistent with these findings. The first assumes that intracellular freezing has taken place; the second that all freezable water has left the cells and frozen externally. The latter model is ruled out because rapidly cooled cells do not shrink by an amount equal to the volume of water that would have to be withdrawn to prevent internal freezing. PMID:13934216

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device

Energy Technology Data Exchange (ETDEWEB)

Germain, Todd; Ansari, Megan; Pappas, Dimitri, E-mail: d.pappas@ttu.edu

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. - Highlights: • Microfluidic system switches rapidly between normoxia and hypoxia (5 min). • Observation of rapid adaptation of PC3 cells to hypoxia and normoxia (30 min). • Drug susceptibility in tumor cells restored after chip switched to normoxia for 30 min.

Cell surface of sea urchin micromeres and primary mesenchyme

International Nuclear Information System (INIS)

DeSimone, D.W.

1985-01-01

The cell surface and extracellular matrix (ECM) of the sea urchin embryo were studied during the early morphogenetic events involved in the differentiation of the micromere cell lineage. Sixteen-cell and early cleavage stage blastomeres were isolated and the protein composition of their cell surfaces examined by 125 I-labelling followed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Micromere-specific cell surface proteins are reported for Arbacia punctulata, Strongylocentrotus droebachiensis, and Strongylocentrotus purpuratus. Cell surface glycoproteins were characterized on the basis of lectin binding specificity with a novel lectin affinity transfer technique. Using this procedure, cell-type specific surface proteins, which are also lectin-binding specific, can be detected. In addition, fluorescein conjugated lectins were microinjected into the blastocoels of living S. drobachiensis and Lytechinus pictus embryos and the patterns of lectin bindings observed by fluorescence microscopy. The evidence presented in this thesis suggests that the differentiation of the primary mesenchyme cells is correlated with changes in the molecular composition of the cell-surface and the ECM

Surface plasmon resonance assisted rapid laser joining of glass

Energy Technology Data Exchange (ETDEWEB)

Zolotovskaya, Svetlana A.; Tang, Guang; Abdolvand, Amin, E-mail: a.abdolvand@dundee.ac.uk [School of Engineering, Physics and Mathematics, University of Dundee, Dundee DD1 4HN (United Kingdom); Wang, Zengbo [School of Electronic Engineering, Bangor University, Bangor LL57 1UT (United Kingdom)

2014-08-25

Rapid and strong joining of clear glass to glass containing randomly distributed embedded spherical silver nanoparticles upon nanosecond pulsed laser irradiation (∼40 ns and repetition rate of 100 kHz) at 532 nm is demonstrated. The embedded silver nanoparticles were ∼30–40 nm in diameter, contained in a thin surface layer of ∼10 μm. A joint strength of 12.5 MPa was achieved for a laser fluence of only ∼0.13 J/cm{sup 2} and scanning speed of 10 mm/s. The bonding mechanism is discussed in terms of absorption of the laser energy by nanoparticles and the transfer of the accumulated localised heat to the surrounding glass leading to the local melting and formation of a strong bond. The presented technique is scalable and overcomes a number of serious challenges for a widespread adoption of laser-assisted rapid joining of glass substrates, enabling applications in the manufacture of microelectronic devices, sensors, micro-fluidic, and medical devices.

Osteoblast cell response to surface-modified carbon nanotubes

International Nuclear Information System (INIS)

Zhang Faming; Weidmann, Arne; Nebe, J. Barbara; Burkel, Eberhard

2012-01-01

In order to investigate the interaction of cells with modified multi-walled carbon nanotubes (MWCNTs) for their potential biomedical applications, the MWCNTs were chemically modified with carboxylic acid groups (–COOH), polyvinyl alcohol (PVA) polymer and biomimetic apatite on their surfaces. Additionally, human osteoblast MG-63 cells were cultured in the presence of the surface-modified MWCNTs. The metabolic activities of osteoblastic cells, cell proliferation properties, as well as cell morphology were studied. The surface modification of MWCNTs with biomimetic apatite exhibited a significant increase in the cell viability of osteoblasts, up to 67.23%. In the proliferation phases, there were many more cells in the biomimetic apatite-modified MWCNT samples than in the MWCNTs–COOH. There were no obvious changes in cell morphology in osteoblastic MG-63 cells cultured in the presence of these chemically-modified MWCNTs. The surface modification of MWCNTs with apatite achieves an effective enhancement of their biocompatibility.

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

Science.gov (United States)

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

2007-05-01

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

Activation of intracellular angiotensin AT2 receptors induces rapid cell death in human uterine leiomyosarcoma cells

DEFF Research Database (Denmark)

Zhao, Yi; Lützen, Ulf; Fritsch, Jürgen

2015-01-01

The presence of AT2 receptors in mitochondria and their role in NO generation and cell aging were recently demonstrated in various human and mouse non-tumour cells. We investigated the intracellular distribution of AT2 receptors including their presence in mitochondria and the role in the induction...... agonist, Compound 21 (C21) penetrates the cell membrane of quiescent SK-UT-1 cells, activates intracellular AT2 receptors and induces rapid cell death; approximately 70% of cells died within 24 h. The cells, which escaped from the cell death, displayed activation of the mitochondrial apoptotic pathway, i...

Formation of plasmonic silver nanoparticles using rapid thermal annealing at low temperature and study in reflectance reduction of Si surface

Science.gov (United States)

Barman, Bidyut; Dhasmana, Hrishikesh; Verma, Abhishek; Kumar, Amit; Pratap Chaudhary, Shiv; Jain, V. K.

2017-09-01

This work presents studies of plasmonic silver nanoparticles (AgNPs) formation at low temperatures (200 °C-300 °C) onto Si surface by sputtering followed with rapid thermal processing (RTP) for different time durations(5-30 min). The study reveals that 20 min RTP at all temperatures show minimum average size of AgNPs (60.42 nm) with corresponding reduction in reflectance of Si surface from 40.12% to mere 1.15% only in wavelength region 300-800 nm for RTP at 200 °C. A detailed supporting growth mechanism is also discussed. This low temperature technique can be helpful in achieving efficiency improvement in solar cells via reflectance reduction with additional features such as reproducibility, minimal time and very good adhesion without damaging underlying layers device parameters.

Tracking cell surface GABAB receptors using an alpha-bungarotoxin tag.

Science.gov (United States)

Wilkins, Megan E; Li, Xinyan; Smart, Trevor G

2008-12-12

GABA(B) receptors mediate slow synaptic inhibition in the central nervous system and are important for synaptic plasticity as well as being implicated in disease. Located at pre- and postsynaptic sites, GABA(B) receptors will influence cell excitability, but their effectiveness in doing so will be dependent, in part, on their trafficking to, and stability on, the cell surface membrane. To examine the dynamic behavior of GABA(B) receptors in GIRK cells and neurons, we have devised a method that is based on tagging the receptor with the binding site components for the neurotoxin, alpha-bungarotoxin. By using the alpha-bungarotoxin binding site-tagged GABA(B) R1a subunit (R1a(BBS)), co-expressed with the R2 subunit, we can track receptor mobility using the small reporter, alpha-bungarotoxin-conjugated rhodamine. In this way, the rates of internalization and membrane insertion for these receptors could be measured with fixed and live cells. The results indicate that GABA(B) receptors rapidly turnover in the cell membrane, with the rate of internalization affected by the state of receptor activation. The bungarotoxin-based method of receptor-tagging seems ideally suited to follow the dynamic regulation of other G-protein-coupled receptors.

Pheochromocytoma (PC12 Cell Response on Mechanobactericidal Titanium Surfaces

Directory of Open Access Journals (Sweden)

Jason V. Wandiyanto

2018-04-01

Full Text Available Titanium is a biocompatible material that is frequently used for making implantable medical devices. Nanoengineering of the surface is the common method for increasing material biocompatibility, and while the nanostructured materials are well-known to represent attractive substrata for eukaryotic cells, very little information has been documented about the interaction between mammalian cells and bactericidal nanostructured surfaces. In this study, we investigated the effect of bactericidal titanium nanostructures on PC12 cell attachment and differentiation—a cell line which has become a widely used in vitro model to study neuronal differentiation. The effects of the nanostructures on the cells were then compared to effects observed when the cells were placed in contact with non-structured titanium. It was found that bactericidal nanostructured surfaces enhanced the attachment of neuron-like cells. In addition, the PC12 cells were able to differentiate on nanostructured surfaces, while the cells on non-structured surfaces were not able to do so. These promising results demonstrate the potential application of bactericidal nanostructured surfaces in biomedical applications such as cochlear and neuronal implants.

In vivo stem cell tracking with imageable nanoparticles that bind bioorthogonal chemical receptors on the stem cell surface.

Science.gov (United States)

Lee, Sangmin; Yoon, Hwa In; Na, Jin Hee; Jeon, Sangmin; Lim, Seungho; Koo, Heebeom; Han, Sang-Soo; Kang, Sun-Woong; Park, Soon-Jung; Moon, Sung-Hwan; Park, Jae Hyung; Cho, Yong Woo; Kim, Byung-Soo; Kim, Sang Kyoon; Lee, Taekwan; Kim, Dongkyu; Lee, Seulki; Pomper, Martin G; Kwon, Ick Chan; Kim, Kwangmeyung

2017-09-01

It is urgently necessary to develop reliable non-invasive stem cell imaging technology for tracking the in vivo fate of transplanted stem cells in living subjects. Herein, we developed a simple and well controlled stem cell imaging method through a combination of metabolic glycoengineering and bioorthogonal copper-free click chemistry. Firstly, the exogenous chemical receptors containing azide (-N 3 ) groups were generated on the surfaces of stem cells through metabolic glycoengineering using metabolic precursor, tetra-acetylated N-azidoacetyl-d-mannosamine(Ac 4 ManNAz). Next, bicyclo[6.1.0]nonyne-modified glycol chitosan nanoparticles (BCN-CNPs) were prepared as imageable nanoparticles to deliver different imaging agents. Cy5.5, iron oxide nanoparticles and gold nanoparticles were conjugated or encapsulated to BCN-CNPs for optical, MR and CT imaging, respectively. These imageable nanoparticles bound chemical receptors on the Ac 4 ManNAz-treated stem cell surface specifically via bioorthogonal copper-free click chemistry. Then they were rapidly taken up by the cell membrane turn-over mechanism resulting in higher endocytic capacity compared non-specific uptake of nanoparticles. During in vivo animal test, BCN-CNP-Cy5.5-labeled stem cells could be continuously tracked by non-invasive optical imaging over 15 days. Furthermore, BCN-CNP-IRON- and BCN-CNP-GOLD-labeled stem cells could be efficiently visualized using in vivo MR and CT imaging demonstrating utility of our stem cell labeling method using chemical receptors. These results conclude that our method based on metabolic glycoengineering and bioorthogonal copper-free click chemistry can stably label stem cells with diverse imageable nanoparticles representing great potential as new stem cell imaging technology. Copyright © 2017 Elsevier Ltd. All rights reserved.

Delivery of folates to the cytoplasm of MA104 cells is mediated by a surface membrane receptor that recycles

International Nuclear Information System (INIS)

Kamen, B.A.; Wang, M.T.; Streckfuss, A.J.; Peryea, X.; Anderson, R.G.

1988-01-01

MA104 cells, as well as several other rapidly dividing tissue culture cells, have a folate-binding protein associated with their cell surface. The protein has the properties of a membrane receptor: (a) 5-methyl[ 3 H]tetrahydrofolic acid binds with high affinity (Kd approximately equal to 3 nM); (b) the protein is an integral membrane protein; (c) it appears to deliver physiological concentrations of 5-methyl[ 3 H]tetrahydrofolic acid to the inside of the cell; (d) binding activity is regulated by the concentration of folate within the cell. To better understand the mechanism of action of this receptor, we have studied the pathway of folate internalization. We present evidence that during internalization: (a) folate binds to the membrane receptor; (b) the ligand-receptor complex moves into the cell; (c) the ligand is released from the receptor in an acidic intracellular compartment and moves into the cytoplasm; and (d) the unoccupied receptor returns to the cell surface

Rapid Detection and Identification of miRNAs by Surface-Enhanced Raman Spectroscopy Using Hollow Au Nanoflowers Substrates

Directory of Open Access Journals (Sweden)

Xiaowei Cao

2017-01-01

Full Text Available MicroRNAs (miRNAs are recognized as regulators of gene expression during the biological processes of cells as well as biomarkers of many diseases. Development of rapid and sensitive miRNA profiling methods is crucial for evaluating the pattern of miRNA expression related to normal and diseased states. This work presents a novel hollow Au nanoflowers (HAuNFs substrate for rapid detection and identification of miRNAs by surface-enhanced Raman scattering (SERS spectroscopy. We synthesized the HAuNFs by a seed-mediated growth approach. Then, HAuNFs substrates were fabricated by depositing HAuNFs onto the surfaces of (3-aminopropyltriethoxysilane- (APTES- functionalized ITO glass. The result demonstrated that HAuNFs substrates had very good reproducibility, homogeneous SERS activity, and high SERS effect. The substrates enabled us to successfully obtain the SERS spectra of miR-10a-5p, miR-125a-5p, and miR-196a-5p. The difference spectra among the three kinds of miRNAs were studied to better interpret the spectral differences and identify miRNA expression patterns with high accuracy. The principal component analysis (PCA of the SERS spectra was used to distinguish among the three kinds of miRNAs. Considering its time efficiency, being label-free, and its sensitivity, the SERS based on HAuNFs substrates is very promising for miRNA research and plays an important role in early disease detection and prevention.

Silver nanoparticle-enriched diamond-like carbon implant modification as a mammalian cell compatible surface with antimicrobial properties

Science.gov (United States)

Gorzelanny, Christian; Kmeth, Ralf; Obermeier, Andreas; Bauer, Alexander T.; Halter, Natalia; Kümpel, Katharina; Schneider, Matthias F.; Wixforth, Achim; Gollwitzer, Hans; Burgkart, Rainer; Stritzker, Bernd; Schneider, Stefan W.

2016-01-01

The implant-bone interface is the scene of competition between microorganisms and distinct types of tissue cells. In the past, various strategies have been followed to support bony integration and to prevent bacterial implant-associated infections. In the present study we investigated the biological properties of diamond-like carbon (DLC) surfaces containing silver nanoparticles. DLC is a promising material for the modification of medical implants providing high mechanical and chemical stability and a high degree of biocompatibility. DLC surface modifications with varying silver concentrations were generated on medical-grade titanium discs, using plasma immersion ion implantation-induced densification of silver nanoparticle-containing polyvinylpyrrolidone polymer solutions. Immersion of implants in aqueous liquids resulted in a rapid silver release reducing the growth of surface-bound and planktonic Staphylococcus aureus and Staphylococcus epidermidis. Due to the fast and transient release of silver ions from the modified implants, the surfaces became biocompatible, ensuring growth of mammalian cells. Human endothelial cells retained their cellular differentiation as indicated by the intracellular formation of Weibel-Palade bodies and a high responsiveness towards histamine. Our findings indicate that the integration of silver nanoparticles into DLC prevents bacterial colonization due to a fast initial release of silver ions, facilitating the growth of silver susceptible mammalian cells subsequently. PMID:26955791

Controlled rotation and translation of spherical particles or living cells by surface acoustic waves.

Science.gov (United States)

Bernard, Ianis; Doinikov, Alexander A; Marmottant, Philippe; Rabaud, David; Poulain, Cédric; Thibault, Pierre

2017-07-11

We show experimental evidence of the acoustically-assisted micromanipulation of small objects like solid particles or blood cells, combining rotation and translation, using high frequency surface acoustic waves. This was obtained from the leakage in a microfluidic channel of two standing waves arranged perpendicularly in a LiNbO 3 piezoelectric substrate working at 36.3 MHz. By controlling the phase lag between the emitters, we could, in addition to translation, generate a swirling motion of the emitting surface which, in turn, led to the rapid rotation of spherical polystyrene Janus beads suspended in the channel and of human red and white blood cells up to several rounds per second. We show that these revolution velocities are compatible with a torque caused by the acoustic streaming that develops at the particles surface, like that first described by [F. Busse et al., J. Acoust. Soc. Am., 1981, 69(6), 1634-1638]. This device, based on standard interdigitated transducers (IDTs) adjusted to emit at equal frequencies, opens a way to a large range of applications since it allows the simultaneous control of the translation and rotation of hard objects, as well as the investigation of the response of cells to shear stress.

Fluorescence Exclusion: A Simple Method to Assess Projected Surface, Volume and Morphology of Red Blood Cells Stored in Blood Bank

Directory of Open Access Journals (Sweden)

Camille Roussel

2018-05-01

Full Text Available Red blood cells (RBC ability to circulate is closely related to their surface area-to-volume ratio. A decrease in this ratio induces a decrease in RBC deformability that can lead to their retention and elimination in the spleen. We recently showed that a subpopulation of “small RBC” with reduced projected surface area accumulated upon storage in blood bank concentrates, but data on the volume of these altered RBC are lacking. So far, single cell measurement of RBC volume has remained a challenging task achieved by a few sophisticated methods some being subject to potential artifacts. We aimed to develop a reproducible and ergonomic method to assess simultaneously RBC volume and morphology at the single cell level. We adapted the fluorescence exclusion measurement of volume in nucleated cells to the measurement of RBC volume. This method requires no pre-treatment of the cell and can be performed in physiological or experimental buffer. In addition to RBC volume assessment, brightfield images enabling a precise definition of the morphology and the measurement of projected surface area can be generated simultaneously. We first verified that fluorescence exclusion is precise, reproducible and can quantify volume modifications following morphological changes induced by heating or incubation in non-physiological medium. We then used the method to characterize RBC stored for 42 days in SAG-M in blood bank conditions. Simultaneous determination of the volume, projected surface area and morphology allowed to evaluate the surface area-to-volume ratio of individual RBC upon storage. We observed a similar surface area-to-volume ratio in discocytes (D and echinocytes I (EI, which decreased in EII (7% and EIII (24%, sphero-echinocytes (SE; 41% and spherocytes (S; 47%. If RBC dimensions determine indeed the ability of RBC to cross the spleen, these modifications are expected to induce the rapid splenic entrapment of the most morphologically altered RBC

A Simple Hydrophilic Treatment of SU-8 Surfaces for Cell Culturing and Cell Patterning

DEFF Research Database (Denmark)

Wang, Zhenyu; Stangegaard, Michael; Dufva, Hans Martin

2005-01-01

SU-8, an epoxy-based photoresist, widely used in constitution different mTAS systems, is incompatible with mammalian cell adhesion and culture in its native form. Here, we demonstrate a simple, cheap and robust two-step method to render a SU-8 surface hydrophilic and compatible with cell culture........ The contact angle of SU-8 surface was significantly reduced from 90° to 25° after the surface modification. The treated SU-8 surfaces provided a cell culture environment that was comparable with cell culture flask surface in terms of generation time and morphology....

UNMANNED AIRCRAFT SYSTEMS FOR RAPID NEAR SURFACE GEOPHYSICAL MEASUREMENTS

Directory of Open Access Journals (Sweden)

J. B. Stoll

2013-08-01

Full Text Available This paper looks at some of the unmanned aircraft systems (UAS options and deals with a magnetometer sensor system which might be of interest in conducting rapid near surface geophysical measurements. Few of the traditional airborne geophysical sensors are now capable of being miniaturized to sizes and payload within mini UAS limits (e.g. airborne magnetics, gamma ray spectrometer. Here the deployment of a fluxgate magnetometer mounted on an UAS is presented demonstrating its capability of detecting metallic materials that are buried in the soil. The effectiveness in finding ferrous objects (e.g. UXO, landslides is demonstrated in two case studies.

Semi-automated, occupationally safe immunofluorescence microtip sensor for rapid detection of Mycobacterium cells in sputum.

Directory of Open Access Journals (Sweden)

Shinnosuke Inoue

Full Text Available An occupationally safe (biosafe sputum liquefaction protocol was developed for use with a semi-automated antibody-based microtip immunofluorescence sensor. The protocol effectively liquefied sputum and inactivated microorganisms including Mycobacterium tuberculosis, while preserving the antibody-binding activity of Mycobacterium cell surface antigens. Sputum was treated with a synergistic chemical-thermal protocol that included moderate concentrations of NaOH and detergent at 60°C for 5 to 10 min. Samples spiked with M. tuberculosis complex cells showed approximately 10(6-fold inactivation of the pathogen after treatment. Antibody binding was retained post-treatment, as determined by analysis with a microtip immunosensor. The sensor correctly distinguished between Mycobacterium species and other cell types naturally present in biosafe-treated sputum, with a detection limit of 100 CFU/mL for M. tuberculosis, in a 30-minute sample-to-result process. The microtip device was also semi-automated and shown to be compatible with low-cost, LED-powered fluorescence microscopy. The device and biosafe sputum liquefaction method opens the door to rapid detection of tuberculosis in settings with limited laboratory infrastructure.

MEMS-based dynamic cell-to-cell culture platforms using electrochemical surface modifications

International Nuclear Information System (INIS)

Chang, Jiyoung; Lin, Liwei; Yoon, Sang-Hee; Mofrad, Mohammad R K

2011-01-01

MEMS-based biological platforms with the capability of both spatial placements and time releases of living cells for cell-to-cell culture experiments have been designed and demonstrated utilizing electrochemical surface modification effects. The spatial placement is accomplished by electrochemical surface modification of substrate surfaces to be either adhesive or non-adhesive for living cells. The time control is achieved by the electrical activation of the selective indium tin oxide co-culture electrode to allow the migration of living cells onto the electrode to start the cell-to-cell culture studies. Prototype devices have a three-electrode design with an electrode size of 50 × 50 µm 2 and the separation gaps of 2 µm between them. An electrical voltage of −1.5 V has been used to activate the electrodes independently and sequentially to demonstrate the dynamic cell-to-cell culture experiments of NIH 3T3 fibroblast and Madin Darby canine kidney cells. As such, this MEMS platform could be a basic yet versatile tool to characterize transient cell-to-cell interactions

Memory phenotype CD4 T cells undergoing rapid, nonburst-like, cytokine-driven proliferation can be distinguished from antigen-experienced memory cells.

Directory of Open Access Journals (Sweden)

Souheil-Antoine Younes

2011-10-01

Full Text Available Memory phenotype (CD44(bright, CD25(negative CD4 spleen and lymph node T cells (MP cells proliferate rapidly in normal or germ-free donors, with BrdU uptake rates of 6% to 10% per day and Ki-67 positivity of 18% to 35%. The rapid proliferation of MP cells stands in contrast to the much slower proliferation of lymphocytic choriomeningitis virus (LCMV-specific memory cells that divide at rates ranging from <1% to 2% per day over the period from 15 to 60 days after LCMV infection. Anti-MHC class II antibodies fail to inhibit the in situ proliferation of MP cells, implying a non-T-cell receptor (TCR-driven proliferation. Such proliferation is partially inhibited by anti-IL-7Rα antibody. The sequence diversity of TCRβ CDR3 gene segments is comparable among the proliferating and quiescent MP cells from conventional and germ-free mice, implying that the majority of proliferating MP cells have not recently derived from a small cohort of cells that expand through multiple continuous rounds of cell division. We propose that MP cells constitute a diverse cell population, containing a subpopulation of slowly dividing authentic antigen-primed memory cells and a majority population of rapidly proliferating cells that did not arise from naïve cells through conventional antigen-driven clonal expansion.

Rapid biochemical functionalization of technical surfaces by means of a photobleaching-based maskless projection lithography process

Science.gov (United States)

Waldbaur, Ansgar; Waterkotte, Björn; Leuthold, Juerg; Schmitz, Katja; Rapp, Bastian E.

2013-03-01

MEMS/MOEMS based systems are increasingly applied in the biological and biomedical context, e.g. in form of biosensors or substrates for monitoring biological responses such as cell migration. For such applications, technical surfaces have to be provided with suitable biochemical functionalization. Typical functionalization procedures include wet-chemical techniques based on self-assembled monolayers of thiols on gold or silanes on glass. These processes create binary patterns and are often of limited use if spatially constrained non-binary patterns like surface bound biochemical gradients have to be provided. In order to create gradients or patterns, methods such as direct spotting or dip pen nanolithography can be used. Here, gradients can be emulated by varying the spot density or the concentration of the solutions employed. However, these methods are serial in nature and are thus of limited use if large surface areas have to be patterned. We present a technique to generate gradients of biochemical function by a photobleaching-based process allowing fast large-scale patterning. The process is based on photobleaching resulting in light-induced coupling of a fluorescently tagged biomolecule to a technical surface by concerted bleaching of the fluorophore. We custom designed a maskless projection lithography system based on a digital mirror device that allows the rapid creation of 8-bit grayscale protein patterns on any technical surface from digital data (e.g. bitmap files). We demonstrate how this process can be used to obtain patterns of several cm2 lateral size at micrometer resolution within minutes.

Hepatitis B virus surface antigen and anti-hepatitis C virus rapid tests underestimate hepatitis prevalence among HIV-infected patients.

Science.gov (United States)

Hønge, Bl; Jespersen, S; Medina, C; Té, Ds; da Silva, Zj; Ostergaard, L; Laursen, Al; Wejse, C; Krarup, H; Erikstrup, C

2014-10-01

In the case of coinfection with HIV and hepatitis B virus (HBV) and/or hepatitis C virus (HCV), hepatic disease progression is often accelerated, with higher rates of liver cirrhosis and liver-related mortality. We aimed to evaluate the performance of the rapid tests used routinely to detect HBV surface antigen (HBsAg) and anti-HCV among HIV-infected patients in Guinea-Bissau. Blood samples from HIV-infected patients in Guinea-Bissau were stored after testing for HBsAg and anti-HCV with rapid tests. Samples were subsequently re-tested for HBsAg and anti-HCV in Denmark. Two rapid tests were used in Guinea-Bissau: HBsAg Strip Ref 2034 (VEDA.LAB, Alençon, France; sensitivity 62.3%; specificity 99.2%) and HEPA-SCAN (Bhat Bio-Tech, Bangalore, India; sensitivity 57.1%; specificity 99.7%). In the two tests the ability to obtain the correct outcome depended on the antigen and antibody concentrations, respectively. Sex, age, CD4 cell count and antiretroviral therapy status did not differ between false negative and true positive samples in either of the tests. The study is limited by a low number of anti-HCV positive samples. New diagnostic rapid tests should always be evaluated in the setting in which they will be used before implementation. © 2014 British HIV Association.

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

Science.gov (United States)

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

2017-03-01

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

Study on residual stress across the pipes' thickness using outer surface rapid heating. Development of pipe outer surface irradiated laser stress improvement process (L-SIP)

International Nuclear Information System (INIS)

Ohta, Takahiro; Terasaki, Toshio

2009-01-01

The new process called L-SIP (outer surface irradiated Laser Stress Improvement Process) is developed to improve the tensile residual stress of the inner surface near the butt welded joints of pipes in the compression stress. The temperature gradient occurs in the thickness of pipes in heating the outer surface rapidly by laser beam. By the thermal expansion difference between the inner surface and the outer surface, the compression plastic strain generates near the outer surface and the tensile plastic strain generates near the inner surface of pipes. The compression stress occurs near the inner surface of pipes by the plastic deformation. In this paper, the theoretical equation which calculates residual stress distribution from the inherent strain distribution in the thickness of pipes is derived. And, the relation between the distribution of temperature and the residual stress in the thickness is examined for various pipes size. (1) By rapidly heating from the outer surface, the residual stress near the inner surface of the pipe is improved to the compression stress. (2) Pipes size hardly affects the distribution of the residual stress in the stainless steel pipes for piping (JISG3459). (3) The temperature rising area from the outside is smaller, the area of the compression residual stress near the inner surface becomes wider. (author)

Rapid Column-Free Enrichment of Mononuclear Cells from Solid Tissues

Science.gov (United States)

Scoville, Steven D.; Keller, Karen A.; Cheng, Stephanie; Zhang, Michael; Zhang, Xiaoli; Caligiuri, Michael A.; Freud, Aharon G.

2015-01-01

We have developed a rapid negative selection method to enrich rare mononuclear cells from human tissues. Unwanted and antibody-tethered cells are selectively depleted during a Ficoll separation step, and there is no need for magnetic-based reagents and equipment. The new method is fast, customizable, inexpensive, remarkably efficient, and easy to perform, and per sample the overall cost is less than one-tenth the cost associated with a magnetic column-based method. PMID:26223896

Surface-modified gold nanorods for specific cell targeting

Science.gov (United States)

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

2012-05-01

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

ERK/CANP rapid signaling mediates 17β-estradiol-induced proliferation of human breast cancer cell line MCF-7 cells.

Science.gov (United States)

Wang, Guo-Sheng; Huang, Yan-Gang; Li, Huan; Bi, Shi-Jie; Zhao, Jin-Long

2014-01-01

17β-estradiol (E2) exerts its functions through both genomic and non-genomic signaling pathways. Because E2 is important in breast cancer development, we investigated whether its actions in promoting breast cancer cell proliferation occur through the non-genomic signaling pathway via extracellular signal-regulated kinase 1/2 (ERK1/2)/calcium-activated neutral protease (CANP). MCF-7 breast cancer cells were treated with ERKl/2 inhibitor (PD98059) or CANP inhibitor (calpeptin) before exposure to 1×10(-8) M E2. MTT colorimetry and flow cytometry were used to analyze effects on cell proliferation and cell cycle progression, respectively. Expression of phosphorylated-ERK (p-ERK), total ERK, and Capn4 proteins were assessed by Western blotting. Cell proliferation increased in cells treated with E2 for 24 h (P<0.05), and the proportion of cells in G0/G1 was decreased, accompanied by accelerated G1/S. Calpeptin pre-treatment significantly inhibited the E2-induced proliferation of MCF-7 cells (P<0.05), while also ameliorating the effects of E2 on cell cycle progression. Further, expression of p-ERK was rapidly up-regulated (after 10 min) by E2 (P<0.05), an effect that persisted 16 h after E2 exposure but which was significantly inhibited by PD98059 (P<0.05). Finally, expression of Capn4 protein was rapidly up-regulated in E2-exposed cells (P<0.05), but this change was significantly inhibited by PD98059 or calpeptin (P<0.05) pre-treatment. Thus, the rapid, non-genomic ERK/CANP signaling pathway mediates E2-induced proliferation of human breast cancer cells.

Cost-effective and rapid blood analysis on a cell-phone.

Science.gov (United States)

Zhu, Hongying; Sencan, Ikbal; Wong, Justin; Dimitrov, Stoyan; Tseng, Derek; Nagashima, Keita; Ozcan, Aydogan

2013-04-07

We demonstrate a compact and cost-effective imaging cytometry platform installed on a cell-phone for the measurement of the density of red and white blood cells as well as hemoglobin concentration in human blood samples. Fluorescent and bright-field images of blood samples are captured using separate optical attachments to the cell-phone and are rapidly processed through a custom-developed smart application running on the phone for counting of blood cells and determining hemoglobin density. We evaluated the performance of this cell-phone based blood analysis platform using anonymous human blood samples and achieved comparable results to a standard bench-top hematology analyser. Test results can either be stored on the cell-phone memory or be transmitted to a central server, providing remote diagnosis opportunities even in field settings.

The location of splenic NKT cells favours their rapid activation by blood-borne antigen

Science.gov (United States)

Barral, Patricia; Sánchez-Niño, María Dolores; van Rooijen, Nico; Cerundolo, Vincenzo; Batista, Facundo D

2012-01-01

Natural killer T (NKT) cells play an important role in mounting protective responses to blood-borne infections. However, though the spleen is the largest blood filter in the body, the distribution and dynamics of NKT cells within this organ are not well characterized. Here we show that the majority of NKT cells patrol around the marginal zone (MZ) and red pulp (RP) of the spleen. In response to lipid antigen, these NKT cells become arrested and rapidly produce cytokines, while the small proportion of NKT cells located in the white pulp (WP) exhibit limited activation. Importantly, disruption of the splenic MZ by chemical or genetic approaches results in a severe reduction in NKT cell activation indicating the need of cooperation between both MZ macrophages and dendritic cells for efficient NKT cell responses. Thus, the location of splenic NKT cells in the MZ and RP facilitates their access to blood-borne antigen and enables the rapid initiation of protective immune responses. PMID:22505026

The location of splenic NKT cells favours their rapid activation by blood-borne antigen.

Science.gov (United States)

Barral, Patricia; Sánchez-Niño, María Dolores; van Rooijen, Nico; Cerundolo, Vincenzo; Batista, Facundo D

2012-05-16

Natural killer T (NKT) cells play an important role in mounting protective responses to blood-borne infections. However, though the spleen is the largest blood filter in the body, the distribution and dynamics of NKT cells within this organ are not well characterized. Here we show that the majority of NKT cells patrol around the marginal zone (MZ) and red pulp (RP) of the spleen. In response to lipid antigen, these NKT cells become arrested and rapidly produce cytokines, while the small proportion of NKT cells located in the white pulp (WP) exhibit limited activation. Importantly, disruption of the splenic MZ by chemical or genetic approaches results in a severe reduction in NKT cell activation indicating the need of cooperation between both MZ macrophages and dendritic cells for efficient NKT cell responses. Thus, the location of splenic NKT cells in the MZ and RP facilitates their access to blood-borne antigen and enables the rapid initiation of protective immune responses.

Interactions between endothelial progenitor cells (EPC) and titanium implant surfaces.

Science.gov (United States)

Ziebart, Thomas; Schnell, Anne; Walter, Christian; Kämmerer, Peer W; Pabst, Andreas; Lehmann, Karl M; Ziebart, Johanna; Klein, Marc O; Al-Nawas, Bilal

2013-01-01

Endothelial cells play an important role in peri-implant angiogenesis during early bone formation. Therefore, interactions between endothelial progenitor cells (EPCs) and titanium dental implant surfaces are of crucial interest. The aim of our in vitro study was to investigate the reactions of EPCs in contact with different commercially available implant surfaces. EPCs from buffy coats were isolated by Ficoll density gradient separation. After cell differentiation, EPC were cultured for a period of 7 days on different titanium surfaces. The test surfaces varied in roughness and hydrophilicity: acid-etched (A), sand-blasted-blasted and acid-etched (SLA), hydrophilic A (modA), and hydrophilic SLA (modSLA). Plastic and fibronectin-coated plastic surfaces served as controls. Cell numbers and morphology were analyzed by confocal laser scanning microscopy. Secretion of vascular endothelial growth factor (VEGF)-A was measured by enzyme-linked immunosorbent assay and expressions of iNOS and eNOS were investigated by real-time polymerase chain reaction. Cell numbers were higher in the control groups compared to the cells of titanium surfaces. Initially, hydrophilic titanium surfaces (modA and modSLA) showed lower cell numbers than hydrophobic surfaces (A and SLA). After 7 days smoother surfaces (A and modA) showed increased cell numbers compared to rougher surfaces (SLA and modSLA). Cell morphology of A, modA, and control surfaces was characterized by a multitude of pseudopodia and planar cell soma architecture. SLA and modSLA promoted small and plump cell soma with little quantity of pseudopodia. The lowest VEGF level was measured on A, the highest on modSLA. The highest eNOS and iNOS expressions were found on modA surfaces. The results of this study demonstrate that biological behaviors of EPCs can be influenced by different surfaces. The modSLA surface promotes an undifferentiated phenotype of EPCs that has the ability to secrete growth factors in great quantities. In

Quantitative analysis of topoisomerase IIα to rapidly evaluate cell proliferation in brain tumors

International Nuclear Information System (INIS)

Oda, Masashi; Arakawa, Yoshiki; Kano, Hideyuki; Kawabata, Yasuhiro; Katsuki, Takahisa; Shirahata, Mitsuaki; Ono, Makoto; Yamana, Norikazu; Hashimoto, Nobuo; Takahashi, Jun A.

2005-01-01

Immunohistochemical cell proliferation analyses have come into wide use for evaluation of tumor malignancy. Topoisomerase IIα (topo IIα), an essential nuclear enzyme, has been known to have cell cycle coupled expression. We here show the usefulness of quantitative analysis of topo IIα mRNA to rapidly evaluate cell proliferation in brain tumors. A protocol to quantify topo IIα mRNA was developed with a real-time RT-PCR. It took only 3 h to quantify from a specimen. A total of 28 brain tumors were analyzed, and the level of topo IIα mRNA was significantly correlated with its immuno-staining index (p < 0.0001, r = 0.9077). Furthermore, it sharply detected that topo IIα mRNA decreased in growth-inhibited glioma cell. These results support that topo IIα mRNA may be a good and rapid indicator to evaluate cell proliferate potential in brain tumors

Functional dynamics of cell surface membrane proteins.

Science.gov (United States)

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

2014-04-01

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

A single-cell technique for the measurement of membrane potential, membrane conductance, and the efflux of rapidly penetrating solutes in Amphiuma erythrocytes.

Science.gov (United States)

Stoner, L C; Kregenow, F M

1980-10-01

We describe a single-cell technique for measuring membrane potential, membrane resistance, and the efflux of rapidly penetrating solutes such as Cl and H2O. Erythrocytes from Amphiuma means were aspirated into a Sylgard (Dow Corning Corp.)-coated capillary. The aspirated cell separated a solution within the capillary from a solution in the bath. Each of these two solutions was contiguous with approximately 5% of the total membrane surface. Microelectrodes placed concentrically within the capillary permit the measurement of intracellular voltage, specific membrane resistance, and the electrical seal between the two solutions. The intracellular voltage averaged -17.7 mV (pH 7.6) and changed as either intra- or extracellular chloride was varied. The average specific membrane resistance measured by passing current across the exposed membrane surface was 110 ohm-cm2. 36Cl and tritiated H2O fluxes (0.84 +/- 0.05 x 10(-6) M . cm-2 . min-1 and 6.4 +/- 1.5 x 10(-3) M . cm-2 . min-1, respectively) were determined by noting the rate at which isotope leaves the cell and crosses the membrane exposed to the bath. Our measured values for the flux of 36Cl and tritiated H2O approximate reported values for free-floating cells. 36Cl efflux, in addition, is inhibited by 4-acetamido-4'-isothiocyano-stilbene 2,2'-disulfonic acid (SITS) and furosemide, known inhibitors of the anion exchange mechanism responsible for the rapid anion fluxes of red blood cells. One can also demonstrate directly that > 89% of 36Cl efflux is "electrically silent" by analyzing the flux in the presence of an imposed transcellular voltage.

Control of cell behavior on PTFE surface using ion beam irradiation

International Nuclear Information System (INIS)

Kitamura, Akane; Kobayashi, Tomohiro; Meguro, Takashi; Suzuki, Akihiro; Terai, Takayuki

2009-01-01

A polytetrafluoroethylene (PTFE) surface is smooth and biologically inert, so that cells cannot attach to it. Ion beam irradiation of the PTFE surface forms micropores and a melted layer, and the surface is finally covered with a large number of small protrusions. Recently, we found that cells could adhere to this irradiated PTFE surface and spread over the surface. Because of their peculiar attachment behavior, these surfaces can be used as biological tools. However, the factors regulating cell adhesion are still unclear, although some new functional groups formed by irradiation seem to contribute to this adhesion. To control cell behavior on PTFE surfaces, we must determine the effects of the outermost irradiated surface on cell adhesion. In this study, we removed the thin melted surface layer by postirradiation annealing and investigated cell behavior on the surface. On the surface irradiated with 3 x 10 16 ions/cm 2 , cells spread only on the remaining parts of the melted layer. From these results, it is clear that the melted layer had a capacity for cell attachment. When the surface covered with protrusions was irradiated with a fluence of 1 x 10 17 ions/cm 2 , the distribution of cells changed after the annealing process from 'sheet shaped' into multicellular aggregates with diameters of around 50 μm. These results indicate that we can control cell behavior on PTFE surfaces covered with protrusions using irradiation and subsequent annealing. Multicellular spheroids can be fabricated for tissue engineering using this surface.

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network.

Science.gov (United States)

Raub, T J; Koroly, M J; Roberts, R M

1990-04-01

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the

Skin surface hydration decreases rapidly during long distance flights.

Science.gov (United States)

Guéhenneux, Sabine; Gardinier, Sophie; Morizot, Frederique; Le Fur, Isabelle; Tschachler, Erwin

2012-05-01

Dehydration of the stratum corneum leads to sensations and symptoms of 'dry skin' such as skin tightness and itchiness. As these complaints are frequently experienced by airline travellers, the aim of this study was to investigate the changes in skin surface hydration during long distance flights. The study was performed on four healthy Caucasian, and on four Japanese women aged 29-39 years, travelling on long distance flights. They had stopped using skin care products at least 12 h before, and did not apply them during the flights. The air temperature and relative humidity inside the cabin, as well as skin capacitance of the face and forearm of participants, were registered at several time points before and during the flights. Relative humidity of the aircraft cabin dropped to levels below 10% within 2 h after take-off and stayed at this value throughout the flight. Skin capacitance decreased rapidly on both the face and forearms with most pronounced changes on the cheeks where it decreased by up to 37%. Our results demonstrate that during long distance flights, the aircraft cabin environment leads to a rapid decrease in stratum corneum hydration, an alteration, which probably accounts for the discomfort experienced by long distance aircraft travellers. © 2011 John Wiley & Sons A/S.

Self assembled temperature responsive surfaces for generation of cell patches for bone tissue engineering

International Nuclear Information System (INIS)

Valmikinathan, Chandra M; ChangWei; Xu Jiahua; Yu Xiaojun

2012-01-01

One of the major challenges in the fabrication of tissue engineered scaffolds is the ability of the scaffold to biologically mimic autograft-like tissues. One of the alternate approaches to achieve this is by the application of cell seeded scaffolds with optimal porosity and mechanical properties. However, the current approaches for seeding cells on scaffolds are not optimal in terms of seeding efficiencies, cell penetration into the scaffold and more importantly uniform distribution of cells on the scaffold. Also, recent developments in scaffold geometries to enhance surface areas, pore sizes and porosities tend to further complicate the scenario. Cell sheet-based approaches for cell seeding have demonstrated a successful approach to generate scaffold-free tissue engineering approaches. However, the method of generating the temperature responsive surface is quite challenging and requires carcinogenic reagents and gamma rays. Therefore, here, we have developed temperature responsive substrates by layer-by-layer self assembly of smart polymers. Multilayer thin films prepared from tannic acid and poly N-isopropylacrylamide were fabricated based on their electrostatic and hydrogen bonding interactions. Cell attachment and proliferation studies on these thin films showed uniform cell attachment on the substrate, matching tissue culture plates. Also, the cells could be harvested as cell patches and sheets from the scaffolds, by reducing the temperature for a short period of time, and seeded onto porous scaffolds for tissue engineering applications. An enhanced cell seeding efficiency on scaffolds was observed using the cell patch-based technique as compared to seeding cells in suspension. Owing to the already pre-existent cell–cell and cell–extracellular matrix interactions, the cell patch showed the ability to reattach rapidly onto scaffolds and showed enhanced ability to proliferate and differentiate into a bone-like matrix. (paper)

Effect of rapid thermal treatment on optical properties of porous silicon surface doped lithium

Energy Technology Data Exchange (ETDEWEB)

Haddadi, Ikbel, E-mail: haded.ikbel@yahoo.fr; Slema, Sonia Ben; Amor, Sana Ben; Bousbih, Rabaa; Bardaoui, Afrah; Dimassi, Wissem; Ezzaouia, Hatem

2015-04-15

In this paper, we have studied the effect of rapid thermal annealing on the optical properties of porous silicon layers doped with lithium (Li/PS). Surface modification of As-deposited Li/PS samples through thermal annealing were investigated by varying the temperature from 100 °C to 800 °C in an infrared (IR) heated belt furnace. A decrease in the reflectivity to about 6% for Li/PS annealed at 200 °C was obtained. From Photoluminescence (PL) spectra, a blue-shift of the gap was observed when the temperature is increased to 800 °C; we correlate these results to the change in chemical composition of the layers in order to find the optimized conditions for a potential application in silicon solar cells. - Highlights: • We have varied the annealing temperature of PS doped with Li. • PL intensity shows significant variation as function of temperature. • We observe reduce of Si–O–Li bands with increasing temperature. • Concurrent with the loss of Li we observe a decrease of the PL.

Surface deformation during an action potential in pearled cells

Science.gov (United States)

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

2017-11-01

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

Rapid activation of Rac GTPase in living cells by force is independent of Src.

Directory of Open Access Journals (Sweden)

Yeh-Chuin Poh

2009-11-01

Full Text Available It is well known that mechanical forces are crucial in regulating functions of every tissue and organ in a human body. However, it remains unclear how mechanical forces are transduced into biochemical activities and biological responses at the cellular and molecular level. Using the magnetic twisting cytometry technique, we applied local mechanical stresses to living human airway smooth muscle cells with a magnetic bead bound to the cell surface via transmembrane adhesion molecule integrins. The temporal and spatial activation of Rac, a small guanosine triphosphatase, was quantified using a fluorescent resonance energy transfer (FRET method that measures changes in Rac activity in response to mechanical stresses by quantifying intensity ratios of ECFP (enhanced cyan fluorescent protein as a donor and YPet (a variant yellow fluorescent protein as an acceptor of the Rac biosensor. The applied stress induced rapid activation (less than 300 ms of Rac at the cell periphery. In contrast, platelet derived growth factor (PDGF induced Rac activation at a much later time (>30 sec. There was no stress-induced Rac activation when a mutant form of the Rac biosensor (RacN17 was transfected or when the magnetic bead was coated with transferrin or with poly-L-lysine. It is known that PDGF-induced Rac activation depends on Src activity. Surprisingly, pre-treatment of the cells with specific Src inhibitor PP1 or knocking-out Src gene had no effects on stress-induced Rac activation. In addition, eliminating lipid rafts through extraction of cholesterol from the plasma membrane did not prevent stress-induced Rac activation, suggesting a raft-independent mechanism in governing the Rac activation upon mechanical stimulation. Further evidence indicates that Rac activation by stress depends on the magnitudes of the applied stress and cytoskeletal integrity. Our results suggest that Rac activation by mechanical forces is rapid, direct and does not depend on Src

Surface-Enhanced Raman Scattering Nanoparticles as Optical Labels for Imaging Cell Surface Proteins

Science.gov (United States)

MacLaughlin, Christina M.

Assaying the expression of cell surface proteins has widespread application for characterizing cell type, developmental stage, and monitoring disease transformation. Immunophenotyping is conducted by treating cells with labelled targeting moieties that have high affinity for relevant surface protein(s). The sensitivity and specificity of immunophenotyping is defined by the choice of contrast agent and therefore, the number of resolvable signals that can be used to simultaneously label cells. Narrow band width surface-enhanced Raman scattering (SERS) nanoparticles are proposed as optical labels for multiplexed immunophenotying. Two types of surface coatings were investigated to passivate the gold nanoparticles, incorporate SERS functionality, and to facilitate attachment of targeting antibodies. Thiolated poly(ethylene glycol) forms dative bonds with the gold surface and is compatible with multiple physisorbed Raman-active reporter molecules. Ternary lipid bilayers are used to encapsulate the gold nanoparticles particles, and incorporate three different classes of Raman reporters. TEM, UV-Visible absorbance spectroscopy, DLS, and electrophoretic light scattering were used characterize the particle coating. Colourimetric protein assay, and secondary antibody labelling were used to quantify the antibody conjugation. Three different in vitromodels were used to investigate the binding efficacy and specificity of SERS labels for their biomarker targets. Primary human CLL cells, LY10 B lymphoma, and A549 adenocarcinoma lines were targeted. Dark field imaging was used to visualize the colocalization of SERS labels with cells, and evidence of receptor clustering was obtained based on colour shifts of the particles' Rayleigh scattering. Widefield, and spatially-resolved Raman spectra were used to detect labels singly, and in combination from labelled cells. Fluorescence flow cytometry was used to test the particles' binding specificity, and SERS from labelled cells was also

Rapid surface enhanced Raman scattering detection method for chloramphenicol residues

Science.gov (United States)

Ji, Wei; Yao, Weirong

2015-06-01

Chloramphenicol (CAP) is a widely used amide alcohol antibiotics, which has been banned from using in food producing animals in many countries. In this study, surface enhanced Raman scattering (SERS) coupled with gold colloidal nanoparticles was used for the rapid analysis of CAP. Density functional theory (DFT) calculations were conducted with Gaussian 03 at the B3LYP level using the 3-21G(d) and 6-31G(d) basis sets to analyze the assignment of vibrations. Affirmatively, the theoretical Raman spectrum of CAP was in complete agreement with the experimental spectrum. They both exhibited three strong peaks characteristic of CAP at 1104 cm-1, 1344 cm-1, 1596 cm-1, which were used for rapid qualitative analysis of CAP residues in food samples. The use of SERS as a method for the measurements of CAP was explored by comparing use of different solvents, gold colloidal nanoparticles concentration and absorption time. The method of the detection limit was determined as 0.1 μg/mL using optimum conditions. The Raman peak at 1344 cm-1 was used as the index for quantitative analysis of CAP in food samples, with a linear correlation of R2 = 0.9802. Quantitative analysis of CAP residues in foods revealed that the SERS technique with gold colloidal nanoparticles was sensitive and of a good stability and linear correlation, and suited for rapid analysis of CAP residue in a variety of food samples.

Observation of reversible, rapid changes in drug susceptibility of hypoxic tumor cells in a microfluidic device.

Science.gov (United States)

Germain, Todd; Ansari, Megan; Pappas, Dimitri

2016-09-14

Hypoxia is a major stimulus for increased drug resistance and for survival of tumor cells. Work from our group and others has shown that hypoxia increases resistance to anti-cancer compounds, radiation, and other damage-pathway cytotoxic agents. In this work we utilize a microfluidic culture system capable of rapid switching of local oxygen concentrations to determine changes in drug resistance in prostate cancer cells. We observed rapid adaptation to hypoxia, with drug resistance to 2 μM staurosporine established within 30 min of hypoxia. Annexin-V/Sytox Green apoptosis assays over 9 h showed 78.0% viability, compared to 84.5% viability in control cells (normoxic cells with no staurosporine). Normoxic cells exposed to the same staurosporine concentration had a viability of 48.6% after 9 h. Hypoxia adaptation was rapid and reversible, with Hypoxic cells treated with 20% oxygen for 30 min responding to staurosporine with 51.6% viability after drug treatment for 9 h. Induction of apoptosis through the receptor-mediated pathway, which bypasses anti-apoptosis mechanisms induced by hypoxia, resulted in 39.4 ± 7% cell viability. The rapid reversibility indicates co-treatment of oxygen with anti-cancer compounds may be a potential therapeutic target. Copyright © 2016 Elsevier B.V. All rights reserved.

Chitosan as coagulant on cyanobacteria in lake restoration management may cause rapid cell lysis.

Science.gov (United States)

Mucci, Maíra; Noyma, Natalia Pessoa; de Magalhães, Leonardo; Miranda, Marcela; van Oosterhout, Frank; Guedes, Iamê Alves; Huszar, Vera L M; Marinho, Marcelo Manzi; Lürling, Miquel

2017-07-01

Combining coagulant and ballast to remove cyanobacteria from the water column is a promising restoration technique to mitigate cyanobacterial nuisance in surface waters. The organic, biodegradable polymer chitosan has been promoted as a coagulant and is viewed as non-toxic. In this study, we show that chitosan may rapidly compromise membrane integrity and kill certain cyanobacteria leading to release of cell contents in the water. A strain of Cylindrospermopsis raciborskii and one strain of Planktothrix agardhii were most sensitive. A 1.3 h exposure to a low dose of 0.5 mg l -1 chitosan already almost completely killed these cultures resulting in release of cell contents. After 24 h, reductions in PSII efficiencies of all cyanobacteria tested were observed. EC50 values varied from around 0.5 mg l -1 chitosan for the two sensitive strains, via about 5 mg l -1 chitosan for an Aphanizomenon flos-aquae strain, a toxic P. agardhii strain and two Anabaena cylindrica cultures, to more than 8 mg l -1 chitosan for a Microcystis aeruginosa strain and another A. flos-aquae strain. Differences in sensitivity to chitosan might be related to polymeric substances that surround cyanobacteria. Rapid lysis of toxic strains is likely and when chitosan flocking and sinking of cyanobacteria is considered in lake restoration, flocculation efficacy studies should be complemented with investigation on the effects of chitosan on the cyanobacteria assemblage being targeted. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Rapid microbial respiration of oil from the Deepwater Horizon spill in offshore surface waters of the Gulf of Mexico

International Nuclear Information System (INIS)

Edwards, Bethanie R; Reddy, Christopher M; Carmichael, Catherine A; Longnecker, Krista; Van Mooy, Benjamin A S; Camilli, Richard

2011-01-01

The Deepwater Horizon oil spill was one of the largest oil spills in history, and the fate of this oil within the Gulf of Mexico ecosystem remains to be fully understood. The goal of this study-conducted in mid-June of 2010, approximately two months after the oil spill began-was to understand the key role that microbes would play in the degradation of the oil in the offshore oligotrophic surface waters near the Deepwater Horizon site. As the utilization of organic carbon by bacteria in the surface waters of the Gulf had been previously shown to be phosphorus limited, we hypothesized that bacteria would be unable to rapidly utilize the oil released from the Macondo well. Although phosphate was scarce throughout the sampling region and microbes exhibited enzymatic signs of phosphate stress within the oil slick, microbial respiration within the slick was enhanced by approximately a factor of five. An incubation experiment to determine hydrocarbon degradation rates confirmed that a large fraction of this enhanced respiration was supported by hydrocarbon degradation. Extrapolating our observations to the entire area of the slick suggests that microbes had the potential to degrade a large fraction of the oil as it arrived at the surface from the well. These observations decidedly refuted our hypothesis. However, a concomitant increase in microbial abundance or biomass was not observed in the slick, suggesting that microbial growth was nutrient limited; incubations amended with nutrients showed rapid increases in cell number and biomass, which supported this conclusion. Our study shows that the dynamic microbial community of the Gulf of Mexico supported remarkable rates of oil respiration, despite a dearth of dissolved nutrients.

Rapid construction of mechanically- confined multi- cellular structures using dendrimeric intercellular linker.

Science.gov (United States)

Mo, Xuejun; Li, Qiushi; Yi Lui, Lena Wai; Zheng, Baixue; Kang, Chiang Huen; Nugraha, Bramasta; Yue, Zhilian; Jia, Rui Rui; Fu, Hong Xia; Choudhury, Deepak; Arooz, Talha; Yan, Jie; Lim, Chwee Teck; Shen, Shali; Hong Tan, Choon; Yu, Hanry

2010-10-01

Tissue constructs that mimic the in vivo cell-cell and cell-matrix interactions are especially useful for applications involving the cell- dense and matrix- poor internal organs. Rapid and precise arrangement of cells into functional tissue constructs remains a challenge in tissue engineering. We demonstrate rapid assembly of C3A cells into multi- cell structures using a dendrimeric intercellular linker. The linker is composed of oleyl- polyethylene glycol (PEG) derivatives conjugated to a 16 arms- polypropylenimine hexadecaamine (DAB) dendrimer. The positively charged multivalent dendrimer concentrates the linker onto the negatively charged cell surface to facilitate efficient insertion of the hydrophobic oleyl groups into the cellular membrane. Bringing linker- treated cells into close proximity to each other via mechanical means such as centrifugation and micromanipulation enables their rapid assembly into multi- cellular structures within minutes. The cells exhibit high levels of viability, proliferation, three- dimensional (3D) cell morphology and other functions in the constructs. We constructed defined multi- cellular structures such as rings, sheets or branching rods that can serve as potential tissue building blocks to be further assembled into complex 3D tissue constructs for biomedical applications. 2010 Elsevier Ltd. All rights reserved.

Rapid and label-free separation of Burkitt's lymphoma cells from red blood cells by optically-induced electrokinetics.

Directory of Open Access Journals (Sweden)

Wenfeng Liang

Full Text Available Early stage detection of lymphoma cells is invaluable for providing reliable prognosis to patients. However, the purity of lymphoma cells in extracted samples from human patients' marrow is typically low. To address this issue, we report here our work on using optically-induced dielectrophoresis (ODEP force to rapidly purify Raji cells' (a type of Burkitt's lymphoma cell sample from red blood cells (RBCs with a label-free process. This method utilizes dynamically moving virtual electrodes to induce negative ODEP force of varying magnitudes on the Raji cells and RBCs in an optically-induced electrokinetics (OEK chip. Polarization models for the two types of cells that reflect their discriminate electrical properties were established. Then, the cells' differential velocities caused by a specific ODEP force field were obtained by a finite element simulation model, thereby established the theoretical basis that the two types of cells could be separated using an ODEP force field. To ensure that the ODEP force dominated the separation process, a comparison of the ODEP force with other significant electrokinetics forces was conducted using numerical results. Furthermore, the performance of the ODEP-based approach for separating Raji cells from RBCs was experimentally investigated. The results showed that these two types of cells, with different concentration ratios, could be separated rapidly using externally-applied electrical field at a driven frequency of 50 kHz at 20 Vpp. In addition, we have found that in order to facilitate ODEP-based cell separation, Raji cells' adhesion to the OEK chip's substrate should be minimized. This paper also presents our experimental results of finding the appropriate bovine serum albumin concentration in an isotonic solution to reduce cell adhesion, while maintaining suitable medium conductivity for electrokinetics-based cell separation. In short, we have demonstrated that OEK technology could be a promising tool for

Enhanced compatibility of chemically modified titanium surface with periodontal ligament cells

International Nuclear Information System (INIS)

Kado, T.; Hidaka, T.; Aita, H.; Endo, K.; Furuichi, Y.

2012-01-01

Highlights: ► Cell-adhesive molecules were covalently immobilized on a Ti surface. ► Immobilized cell-adhesive molecules maintained native function on the Ti surface. ► Immobilized collagen enhanced adhesion of periodontal ligament cells to the Ti. - Abstract: A simple chemical modification method was developed to immobilize cell-adhesive molecules on a titanium surface to improve its compatibility with human periodontal ligament cells (HPDLCs).The polished titanium disk was immersed in 1% (v/v) p-vinylbenzoic acid solution for 2 h to introduce carboxyl groups onto the surface. After rinsing with distilled deionized water, the titanium disk was dipped into 1.47% 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution containing 0.1 mg/ml Gly-Arg-Gly-Asp-Ser (GRGDS), human plasma fibronectin (pFN), or type I collagen from calf skin (Col) to covalently immobilize the cell-adhesive molecules on the titanium surface via formation of peptide bonds. X-ray photoelectron spectroscopy analyses revealed that cell-adhesive molecules were successfully immobilized on the titanium surfaces. The Col-immobilized titanium surface revealed higher values regarding nano rough characteristics than the as-polished titanium surface under scanning probe microscopy. The number of HPDLCs attached to both the pFN- and Col-immobilized titanium surfaces was twice that attached to the as-polished titanium surfaces. The cells were larger with the cellular processes that stretched to a greater extent on the pFN- and Col-immobilized titanium surfaces than on the as-polished titanium surface (p < 0.05). HPDLCs on the Col-immobilized titanium surfaces showed more extensive expression of vinculin at the tips of cell projections and more contiguously along the cell outline than on the as-polished, GRGDS-immobilized and pFN-immobilized titanium surfaces. It was concluded that cell-adhesive molecules successfully immobilized on the titanium surface and improved the compatibility of the surface

Rapid expansion of T cells: Effects of culture and cryopreservation and importance of short-term cell recovery.

Science.gov (United States)

Sadeghi, Arian; Ullenhag, Gustav; Wagenius, Gunnar; Tötterman, Thomas H; Eriksson, Fredrik

2013-06-01

Successful cell therapy relies on the identification and mass expansion of functional cells for infusion. Cryopreservation of cells is an inevitable step in most cell therapies which also entails consequences for the frozen cells. This study assessed the impact of cryopreservation and the widely used protocol for rapid expansion of T lymphocytes. The effects on cell viability, immunocompetence and the impact on apoptotic and immunosuppressive marker expression were analyzed using validated assays. Cryopreservation of lymphocytes during the rapid expansion protocol did not affect cell viability. Lymphocytes that underwent mass expansion or culture in high dose IL-2 were unable to respond to PHA stimulation by intracellular ATP production immediately after thawing (ATP = 16 ± 11 ng/ml). However, their reactivity to PHA was regained within 48 hours of recovery (ATP = 356 ± 61 ng/ml). Analysis of mRNA levels revealed downregulation of TGF-β and IL-10 at all time points. Culture in high dose IL-2 led to upregulation of p73 and BCL-2 mRNA levels while FoxP3 expression was elevated after culture in IL-2 and artificial TCR stimuli. FoxP3 levels decreased after short-term recovery without IL-2 or stimulation. Antigen specificity, as determined by IFNγ secretion, was unaffected by cryopreservation but was completely lost after addition of high dose IL-2 and artificial TCR stimuli. In conclusion, allowing short-time recovery of mass expanded and cryopreserved cells before reinfusion could enhance the outcome of adoptive cell therapy as the cells regain immune competence and specificity.

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

Science.gov (United States)

Yi, Shaohua; Long, Fei; Cheng, Juanbo; Huang, Daixin

2017-12-19

Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

A rapid and sensitive method for measuring N-acetylglucosaminidase activity in cultured cells.

Directory of Open Access Journals (Sweden)

Victor Mauri

Full Text Available A rapid and sensitive method to quantitatively assess N-acetylglucosaminidase (NAG activity in cultured cells is highly desirable for both basic research and clinical studies. NAG activity is deficient in cells from patients with Mucopolysaccharidosis type IIIB (MPS IIIB due to mutations in NAGLU, the gene that encodes NAG. Currently available techniques for measuring NAG activity in patient-derived cell lines include chromogenic and fluorogenic assays and provide a biochemical method for the diagnosis of MPS IIIB. However, standard protocols require large amounts of cells, cell disruption by sonication or freeze-thawing, and normalization to the cellular protein content, resulting in an error-prone procedure that is material- and time-consuming and that produces highly variable results. Here we report a new procedure for measuring NAG activity in cultured cells. This procedure is based on the use of the fluorogenic NAG substrate, 4-Methylumbelliferyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside (MUG, in a one-step cell assay that does not require cell disruption or post-assay normalization and that employs a low number of cells in 96-well plate format. We show that the NAG one-step cell assay greatly discriminates between wild-type and MPS IIIB patient-derived fibroblasts, thus providing a rapid method for the detection of deficiencies in NAG activity. We also show that the assay is sensitive to changes in NAG activity due to increases in NAGLU expression achieved by either overexpressing the transcription factor EB (TFEB, a master regulator of lysosomal function, or by inducing TFEB activation chemically. Because of its small format, rapidity, sensitivity and reproducibility, the NAG one-step cell assay is suitable for multiple procedures, including the high-throughput screening of chemical libraries to identify modulators of NAG expression, folding and activity, and the investigation of candidate molecules and constructs for applications in

Cell surface alteration in Epstein-Barr virus-transformed cells from patients with extreme insulin resistance

International Nuclear Information System (INIS)

Gorden, D.L.; Robert, A.; Moncada, V.Y.; Taylor, S.I.; Muehlhauser, J.C.; Carpentier, J.L.

1990-01-01

An abnormality was detected in the morphology of the cell surface of Epstein-Barr virus-transformed lymphocytes of patients with genetic forms of insulin resistance. In cells from two patients with leprechaunism and two patients with type A extreme insulin resistance, scanning electron microscopy demonstrated a decrease in the percentage of the cell surface occupied by microvilli in cells from the patients with leprechaunism and type A insulin resistance compared with control cells. When cells from a healthy control subject and one of the patients with leprechaunism (Lep/Ark-1) were incubated with 125 I-labeled insulin, there was a decrease in the percentage of 125 I-insulin associated with microvilli on the cell surface. Thus, the decreased localization of insulin receptors with the microvillous region of the cell surface was in proportion to the decrease in microvilli

Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

Science.gov (United States)

Zheng, Guoying; Guan, Binbin; Hu, Penghui; Qi, Xingying; Wang, Pingting; Kong, Yu; Liu, Zihao; Gao, Ping; Li, Rui; Zhang, Xu; Wu, Xudong; Sui, Lei

2018-04-27

To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs. © 2018 John Wiley

Cell surface engineering of industrial microorganisms for biorefining applications.

Science.gov (United States)

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.

Surface acoustic wave actuated cell sorting (SAWACS).

Science.gov (United States)

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

2010-03-21

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

Inspecting rapidly moving surfaces for small defects using CNN cameras

Science.gov (United States)

Blug, Andreas; Carl, Daniel; Höfler, Heinrich

2013-04-01

A continuous increase in production speed and manufacturing precision raises a demand for the automated detection of small image features on rapidly moving surfaces. An example are wire drawing processes where kilometers of cylindrical metal surfaces moving with 10 m/s have to be inspected for defects such as scratches, dents, grooves, or chatter marks with a lateral size of 100 μm in real time. Up to now, complex eddy current systems are used for quality control instead of line cameras, because the ratio between lateral feature size and surface speed is limited by the data transport between camera and computer. This bottleneck is avoided by "cellular neural network" (CNN) cameras which enable image processing directly on the camera chip. This article reports results achieved with a demonstrator based on this novel analogue camera - computer system. The results show that computational speed and accuracy of the analogue computer system are sufficient to detect and discriminate the different types of defects. Area images with 176 x 144 pixels are acquired and evaluated in real time with frame rates of 4 to 10 kHz - depending on the number of defects to be detected. These frame rates correspond to equivalent line rates on line cameras between 360 and 880 kHz, a number far beyond the available features. Using the relation between lateral feature size and surface speed as a figure of merit, the CNN based system outperforms conventional image processing systems by an order of magnitude.

Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

International Nuclear Information System (INIS)

Rieber, E.P.; Linke, R.P.; Riethmueller, G.; Heyden, H.W. von; Waller, H.D.

1976-01-01

Using 125 I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab') 2 -fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of μ-chains was detected. γ-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria. (orig.) [de

Fc-receptors and surface immunoglobulins in cells of the hairy cell leukemia

Energy Technology Data Exchange (ETDEWEB)

Rieber, E P; Linke, R P; Riethmueller, G [Tuebingen Univ. (Germany, F.R.). Abt. fuer Experimentelle Chirurgie und Immunologie; Heyden, H.W. von; Waller, H D [Tuebingen Univ. (Germany, F.R.). Abt. Innere Medizin 2

1976-01-01

Using /sup 125/I-labelled aggregated IgG in a quantitative assay a strong expression of Fc-receptors was found on the leukemic cells of a patient with hairy cell leukemia. The Fc-receptor activity on these cells was much higher than that on monocytes and B-lymphocytes from normal blood. Surface immunoglobulins were detected by radioautography using radioactively labelled (Fab')/sub 2/-fragments of monospecific antibodies directed against immunoglobulin heavy chains. Prior to radioautography the cells were stained for the tartrate resistant acid phosphatase. It is found that all cells containing this enzyme bore delta-chains on their surface. On more than 90% of these cells a simultaneous expression of ..mu..-chains was detected. ..gamma..-chains could only be demonstrated on cells which were negative for the tartrate resistant acid phosphatase; part of these cells, however, were hairy cells by morphological criteria.

Rapid desensitization of adrenaline- and neuropeptide Y-stimulated Ca2+ mobilization in HEL-cells

NARCIS (Netherlands)

Michel, M. C.

1994-01-01

1. Desensitization of Gs-coupled receptors, the beta 2-adrenoceptor for example, involves rapid and slower components but little is known regarding the existence of rapid desensitization of Gi-coupled receptors and its possible mechanisms. In HEL-cells stimulation of alpha 2A-adrenoceptors by

Cell Surface Enzymatic Engineering-Based Approaches to Improve Cellular Therapies

KAUST Repository

AbuElela, Ayman; Sakashita, Kosuke; Merzaban, Jasmeen

2014-01-01

The cell surface represents the interface between the cell and its environment. As such, the cell surface controls cell–cell interactions and functions such as adhesion and migration, and will transfer external cues to regulate processes

A comparison of cryopreservation methods: Slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells

Directory of Open Access Journals (Sweden)

Sandra Ferry

2011-09-01

Full Text Available Abstract Background The finding of human umbilical cord blood as one of the most likely sources of hematopoietic stem cells offers a less invasive alternative for the need of hematopoietic stem cell transplantation. Due to the once-in-a-life time chance of collecting it, an optimum cryopreservation method that can preserve the life and function of the cells contained is critically needed. Methods Until now, slow-cooling has been the routine method of cryopreservation; however, rapid-cooling offers a simple, efficient, and harmless method for preserving the life and function of the desired cells. Therefore, this study was conducted to compare the effectiveness of slow- and rapid-cooling to preserve umbilical cord blood of mononucleated cells suspected of containing hematopoietic stem cells. The parameters used in this study were differences in cell viability, malondialdehyde content, and apoptosis level. The identification of hematopoietic stem cells themselves was carried out by enumerating CD34+ in a flow cytometer. Results Our results showed that mononucleated cell viability after rapid-cooling (91.9% was significantly higher than that after slow-cooling (75.5%, with a p value = 0.003. Interestingly, the malondialdehyde level in the mononucleated cell population after rapid-cooling (56.45 μM was also significantly higher than that after slow-cooling (33.25 μM, with a p value p value = 0.138. However, CD34+ enumeration was much higher in the population that underwent slow-cooling (23.32 cell/μl than in the one that underwent rapid-cooling (2.47 cell/μl, with a p value = 0.001. Conclusions Rapid-cooling is a potential cryopreservation method to be used to preserve the umbilical cord blood of mononucleated cells, although further optimization of the number of CD34+ cells after rapid-cooling is critically needed.

A system for applying rapid warming or cooling stimuli to cells during patch clamp recording or ion imaging.

Science.gov (United States)

Reid, G; Amuzescu, B; Zech, E; Flonta, M L

2001-10-15

We describe a system for superfusing small groups of cells at a precisely controlled and rapidly adjustable local temperature. Before being applied to the cell or cells under study, solutions are heated or cooled in a chamber of small volume ( approximately 150 microl) and large surface area, sandwiched between four small Peltier elements. The current through the Peltier elements is controlled by a microprocessor using a PID (proportional-integral-derivative) feedback algorithm. The chamber can be heated to at least 60 degrees C and cooled to 0 degrees C, changing its temperature at a maximum rate of about 7 degrees C per second; temperature ramps can be followed under feedback control at up to 4 degrees C per second. Temperature commands can be applied from the digital-to-analogue converter of any laboratory interface or generated digitally by the microprocessor. The peak-to-peak noise contributed by the system does not exceed that contributed by a patch pipette, holder and headstage, making it suitable for single channel as well as whole cell recordings.

Studies of cell biomechanics with surface micro-/nano-technology

International Nuclear Information System (INIS)

Wang Dong; Zhang Wei; Jiang Xingyu

2011-01-01

We report the recent progress in our studies of cell biology using micro-/nano-technology. Cells have a size of several to tens of microns, which makes them easily manipulated by micro-/nano-technology. The shape of the cell influences the alignment of the actin cytoskeleton, which bears the main forces of the cell, maintains the shape,and mediates a series of biochemical reactions. We invented a stretching device and studied the real-time actin filament dynamics under stretch. We found that one stretch cycle shortened the actin filaments and promoted their reassemble process. Cell migration is a complex mechanical process. We found that cell geometry determines the cell polarity and migration direction. We fabricated three-dimensional surfaces to mimic the topography in vivo, and further built a cell culture model by integrating the three-dimensional surface, microfluidics, cell patterning,and coculturing of multiple cell types. We also investigated the neuronal guidance by surface patterning. (authors)

Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties

DEFF Research Database (Denmark)

Couchman, J R; Austria, R; Woods, A

1988-01-01

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild-type BHK cells and an adhesion-defective ricin......-resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased...... sulfation, reduced affinity for fibronectin and decreased half-life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin-resistant mutant. Whether this results from...

Surface modification induced phase transformation and structure variation on the rapidly solidified recast layer of titanium

Energy Technology Data Exchange (ETDEWEB)

Tsai, Ming-Hung [Department of Mechanical Engineering and Graduate Institute of Mechanical and Precision Engineering, National Kaoshiung University of Applied Sciences, Kaoshiung 807, Taiwan (China); School of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Haung, Chiung-Fang [School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Division of Family and Operative Dentistry, Department of Dentistry, Taipei Medical University Hospital, Taipei 110, Taiwan (China); Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei 110, Taiwan (China); Shyu, Shih-Shiun [Department of Dentistry, Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, New Taipei City 231, Taiwan (China); Chou, Yen-Ru [Research Center for Biomedical Devices and Prototyping Production, Taipei Medical University, Taipei 110, Taiwan (China); Graduate Institute of Biomedical Materials and Tissue Engineering, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Research Center for Biomedical Implants and Microsurgery Devices, Taipei Medical University, Taipei 110, Taiwan (China); Lin, Ming-Hong [Department of Mechanical Engineering and Graduate Institute of Mechanical and Precision Engineering, National Kaoshiung University of Applied Sciences, Kaoshiung 807, Taiwan (China); Peng, Pei-Wen, E-mail: apon@tmu.edu.tw [School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei 110, Taiwan (China); and others

2015-08-15

In this study, neodymium-doped yttrium orthovanadate (Nd:YVO{sub 4}) as a laser source with different scanning speeds was used on biomedical Ti surface. The microstructural and biological properties of laser-modified samples were investigated by means of optical microscope, electron microscope, X-ray diffraction, surface roughness instrument, contact angle and cell cytotoxicity assay. After laser modification, the rough volcano-like recast layer with micro-/nanoporous structure and wave-like recast layer with nanoporous structure were generated on the surfaces of laser-modified samples, respectively. It was also found out that, an α → (α + rutile-TiO{sub 2}) phase transition occurred on the recast layers of laser-modified samples. The Ti surface becomes hydrophilic at a high speed laser scanning. Moreover, the cell cytotoxicity assay demonstrated that laser-modified samples did not influence the cell adhesion and proliferation behaviors of osteoblast (MG-63) cell. The laser with 50 mm/s scanning speed induced formation of rough volcano-like recast layer accompanied with micro-/nanoporous structure, which can promote cell adhesion and proliferation of MG-63 cell on Ti surface. The results indicated that the laser treatment was a potential technology to enhance the biocompatibility for titanium. - Highlights: • Laser induced the formation of recast layer with micro-/nanoporous structure on Ti. • An α → (α + rutile-TiO{sub 2}) phase transition was observed within the recast layer. • The Ti surface becomes hydrophilic at a high speed laser scanning. • Laser-modified samples exhibit good biocompatibility to osteoblast (MG-63) cell.

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

Science.gov (United States)

Polliack, Aaron; Tadmor, Tamar

2011-06-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Liu, Yun; Zhang, Rui; Lian, Zhongshuai; Wang, Shihui; Wright, Aaron T.

2014-08-01

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

Investigation of back surface fields effect on bifacial solar cells

Science.gov (United States)

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

2012-11-01

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

Rapid acquisition and model-based analysis of cell-free transcription–translation reactions from nonmodel bacteria

Science.gov (United States)

Wienecke, Sarah; Ishwarbhai, Alka; Tsipa, Argyro; Aw, Rochelle; Kylilis, Nicolas; Bell, David J.; McClymont, David W.; Jensen, Kirsten; Biedendieck, Rebekka

2018-01-01

Native cell-free transcription–translation systems offer a rapid route to characterize the regulatory elements (promoters, transcription factors) for gene expression from nonmodel microbial hosts, which can be difficult to assess through traditional in vivo approaches. One such host, Bacillus megaterium, is a giant Gram-positive bacterium with potential biotechnology applications, although many of its regulatory elements remain uncharacterized. Here, we have developed a rapid automated platform for measuring and modeling in vitro cell-free reactions and have applied this to B. megaterium to quantify a range of ribosome binding site variants and previously uncharacterized endogenous constitutive and inducible promoters. To provide quantitative models for cell-free systems, we have also applied a Bayesian approach to infer ordinary differential equation model parameters by simultaneously using time-course data from multiple experimental conditions. Using this modeling framework, we were able to infer previously unknown transcription factor binding affinities and quantify the sharing of cell-free transcription–translation resources (energy, ribosomes, RNA polymerases, nucleotides, and amino acids) using a promoter competition experiment. This allows insights into resource limiting-factors in batch cell-free synthesis mode. Our combined automated and modeling platform allows for the rapid acquisition and model-based analysis of cell-free transcription–translation data from uncharacterized microbial cell hosts, as well as resource competition within cell-free systems, which potentially can be applied to a range of cell-free synthetic biology and biotechnology applications. PMID:29666238

Interaction of progenitor bone cells with different surface modifications of titanium implant

Energy Technology Data Exchange (ETDEWEB)

Chen, Wen-Cheng, E-mail: wencchen@fcu.edu.tw [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Chen, Ya-Shun [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Ko, Chia-Ling [Advanced Medical Devices and Composites Laboratory, Department of Fiber and Composite Materials, College of Engineering, Feng Chia University, Taichung 40724, Taiwan (China); Dental Medical Devices and Materials Research Center, College of Dental Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Lin, Yi; Kuo, Tzu-Huang; Kuo, Hsien-Nan [Medical Device Development Division, Metal Industries Research and Development Centre, Kaohsiung 82151, Taiwan (China)

2014-04-01

Changes in the physical and chemical properties of Ti surfaces can be attributed to cell performance, which improves surface biocompatibility. The cell proliferation, mineralization ability, and gene expression of progenitor bone cells (D1 cell) were compared on five different Ti surfaces, namely, mechanical grinding (M), electrochemical modification through potentiostatic anodization (ECH), sandblasting and acid etching (SLA), sandblasting, hydrogen peroxide treatment, and heating (SAOH), and sandblasting, alkali heating, and etching (SMART). SAOH treatment produced the most hydrophilic surface, whereas SLA produced the most hydrophobic surface. Cell activity indicated that SLA and SMART produced significantly rougher surfaces and promoted D1 cell attachment within 1 day of culturing, whereas SAOH treatment produced moderate roughness (Ra = 1.26 μm) and accelerated the D1 cell proliferation up to 7 days after culturing. The ECH surface significantly promoted alkaline phosphatase (ALP) expression and osteocalcin (OCN) secretion in the D1 cells compared with the other surface groups. The ECH and SMART-treated Ti surfaces resulted in maximum ALP and OCN expressions during the D1 cell culture. SLA, SAOH, and SMART substrate surfaces were rougher and exhibited better cell metabolic responses during the early stage of cell attachment, proliferation, and morphologic expressions within 1 day of D1 cell culture. The D1 cells cultured on the ECH and SMART substrates exhibited higher differentiation, and higher ALP and OCN expressions after 10 days of culture. Thus, the ECH and SMART treatments promote better ability of cell mineralization in vitro, which demonstrate their great potential for clinical use. - Highlights: • Progenitor bone cells onto Ti with different modifications are characterized. • Surface roughness and hydrophilicity encourage early stage cell attachment. • Composition and surface treatments are more vital in bone cell mineralization. �

Interaction of progenitor bone cells with different surface modifications of titanium implant

International Nuclear Information System (INIS)

Chen, Wen-Cheng; Chen, Ya-Shun; Ko, Chia-Ling; Lin, Yi; Kuo, Tzu-Huang; Kuo, Hsien-Nan

2014-01-01

Changes in the physical and chemical properties of Ti surfaces can be attributed to cell performance, which improves surface biocompatibility. The cell proliferation, mineralization ability, and gene expression of progenitor bone cells (D1 cell) were compared on five different Ti surfaces, namely, mechanical grinding (M), electrochemical modification through potentiostatic anodization (ECH), sandblasting and acid etching (SLA), sandblasting, hydrogen peroxide treatment, and heating (SAOH), and sandblasting, alkali heating, and etching (SMART). SAOH treatment produced the most hydrophilic surface, whereas SLA produced the most hydrophobic surface. Cell activity indicated that SLA and SMART produced significantly rougher surfaces and promoted D1 cell attachment within 1 day of culturing, whereas SAOH treatment produced moderate roughness (Ra = 1.26 μm) and accelerated the D1 cell proliferation up to 7 days after culturing. The ECH surface significantly promoted alkaline phosphatase (ALP) expression and osteocalcin (OCN) secretion in the D1 cells compared with the other surface groups. The ECH and SMART-treated Ti surfaces resulted in maximum ALP and OCN expressions during the D1 cell culture. SLA, SAOH, and SMART substrate surfaces were rougher and exhibited better cell metabolic responses during the early stage of cell attachment, proliferation, and morphologic expressions within 1 day of D1 cell culture. The D1 cells cultured on the ECH and SMART substrates exhibited higher differentiation, and higher ALP and OCN expressions after 10 days of culture. Thus, the ECH and SMART treatments promote better ability of cell mineralization in vitro, which demonstrate their great potential for clinical use. - Highlights: • Progenitor bone cells onto Ti with different modifications are characterized. • Surface roughness and hydrophilicity encourage early stage cell attachment. • Composition and surface treatments are more vital in bone cell mineralization. �

Electrostatic behavior of the charge-regulated bacterial cell surface.

Science.gov (United States)

Hong, Yongsuk; Brown, Derick G

2008-05-06

The electrostatic behavior of the charge-regulated surfaces of Gram-negative Escherichia coli and Gram-positive Bacillus brevis was studied using numerical modeling in conjunction with potentiometric titration and electrophoretic mobility data as a function of solution pH and electrolyte composition. Assuming a polyelectrolytic polymeric bacterial cell surface, these experimental and numerical analyses were used to determine the effective site numbers of cell surface acid-base functional groups and Ca(2+) sorption coefficients. Using effective site concentrations determined from 1:1 electrolyte (NaCl) experimental data, the charge-regulation model was able to replicate the effects of 2:1 electrolyte (CaCl(2)), both alone and as a mixture with NaCl, on the measured zeta potential using a single Ca(2+) surface binding constant for each of the bacterial species. This knowledge is vital for understanding how cells respond to changes in solution pH and electrolyte composition as well as how they interact with other surfaces. The latter is especially important due to the widespread use of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory in the interpretation of bacterial adhesion. As surface charge and surface potential both vary on a charge-regulated surface, accurate modeling of bacterial interactions with surfaces ultimately requires use of an electrostatic model that accounts for the charge-regulated nature of the cell surface.

Cell patterning without chemical surface modification: Cell cell interactions between printed bovine aortic endothelial cells (BAEC) on a homogeneous cell-adherent hydrogel

Science.gov (United States)

Chen, C. Y.; Barron, J. A.; Ringeisen, B. R.

2006-10-01

Cell printing offers the unique ability to directly deposit one or multiple cell types directly onto a surface without the need to chemically pre-treat the surface with lithographic methods. We utilize biological laser printing (BioLP ™) to form patterns of bovine aortic endothelial cells (BAECs) onto a homogeneous cell adherent hydrogel surface. These normal cells are shown to retain near-100% viability post-printing. In order to determine whether BAECs encountered shear and/or heat stress during printing, immunocytochemical staining experiments were performed to detect potential expression of heat shock proteins (HSP) by the deposited cells. Printed BAECs expressed HSP at levels similar to negative control cells, indicating that the BioLP process does not expose cells to damaging levels of stress. However, HSP expression was slightly higher at the highest laser energy studied, suggesting more stress was present under these extreme conditions. Printed BAECs also showed preferential asymmetric growth and migration towards each other and away from the originally printed pattern, demonstrating a retained ability for the cells to communicate post-printing.

Integration of nanoparticle cell lysis and microchip PCR for one-step rapid detection of bacteria.

Science.gov (United States)

Wan, Weijie; Yeow, John T W

2012-04-01

This paper describes an integrated microchip system as an efficient and cost-effective solution involving Nanotechnology and Lab-on-a-Chip technology for the rapid detection of bacteria. The system is based on using surface-modified gold nanoparticles for efficient cell lysis followed by microchip PCR without having to remove the nanoparticles from the PCR solution. Poly(quaternary ammonium) modified gold nanoparticles are used to provide a novel and efficient cell lysis method without the need to go through time-consuming, expensive and complicated microfabrication processes as most of current cell lysis methods for Lab-on-a-Chip applications do. It also facilitates the integration of cell lysis and PCR by sharing the same reaction chamber as PCR uses. It is integrated with a prototype microchip PCR system consisting of a physical microchip PCR device and an automated temperature control mechanism. The research work explores solutions for the problem of PCR inhibition caused by gold nanoparticles as well as for the problem of non-specific PCR amplification in the integrated microchip system. It also explores the possibility of greatly reducing PCR cycling time to achieve the same result compared to the protocol for a regular PCR machine. The simplicity of the setup makes it easy to be integrated with other Lab-on-a-Chip functional modules to create customized solutions for target applications.

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

Science.gov (United States)

Boheler, Kenneth R; Gundry, Rebekah L

2017-01-01

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

Surface contact stimulates the just-in-time deployment of bacterial adhesins.

Science.gov (United States)

Li, Guanglai; Brown, Pamela J B; Tang, Jay X; Xu, Jing; Quardokus, Ellen M; Fuqua, Clay; Brun, Yves V

2012-01-01

The attachment of bacteria to surfaces provides advantages such as increasing nutrient access and resistance to environmental stress. Attachment begins with a reversible phase, often mediated by surface structures such as flagella and pili, followed by a transition to irreversible attachment, typically mediated by polysaccharides. Here we show that the interplay between pili and flagellum rotation stimulates the rapid transition between reversible and polysaccharide-mediated irreversible attachment. We found that reversible attachment of Caulobacter crescentus cells is mediated by motile cells bearing pili and that their contact with a surface results in the rapid pili-dependent arrest of flagellum rotation and concurrent stimulation of polar holdfast adhesive polysaccharide. Similar stimulation of polar adhesin production by surface contact occurs in Asticcacaulis biprosthecum and Agrobacterium tumefaciens. Therefore, single bacterial cells respond to their initial contact with surfaces by triggering just-in-time adhesin production. This mechanism restricts stable attachment to intimate surface interactions, thereby maximizing surface attachment, discouraging non-productive self-adherence, and preventing curing of the adhesive. © 2011 Blackwell Publishing Ltd.

Human Adipose-Derived Stem Cells on Rapid Prototyped Three-Dimensional Hydroxyapatite/Beta-Tricalcium Phosphate Scaffold.

Science.gov (United States)

Canciani, Elena; Dellavia, Claudia; Ferreira, Lorena Maria; Giannasi, Chiara; Carmagnola, Daniela; Carrassi, Antonio; Brini, Anna Teresa

2016-05-01

In the study, we assess a rapid prototyped scaffold composed of 30/70 hydroxyapatite (HA) and beta-tricalcium-phosphate (β-TCP) loaded with human adipose-derived stem cells (hASCs) to determine cell proliferation, differentiation toward osteogenic lineage, adhesion and penetration on/into the scaffold.In this in vitro study, hASCs isolated from fat tissue discarded after plastic surgery were expanded, characterized, and then loaded onto the scaffold. Cells were tested for: viability assay (Alamar Blue at days 3, 7 and Live/Dead at day 32), differentiation index (alkaline phosphatase activity at day 14), scaffold adhesion (standard error of the mean analysis at days 5 and 18), and penetration (ground sections at day 32).All the hASC populations displayed stemness markers and the ability to differentiate toward adipogenic and osteogenic lineages.Cellular vitality increased between 3 and 7 days, and no inhibitory effect by HA/β-TCP was observed. Under osteogenic stimuli, scaffold increased alkaline phosphatase activity of +243% compared with undifferentiated samples. Human adipose-derived stem cells adhered on HA/β-TCP surface through citoplasmatic extensions that occupied the macropores and built networks among them. Human adipose derived stem cells were observed in the core of HA/β-TCP. The current combination of hASCs and HA/β-TCP scaffold provided encouraging results. If authors' data will be confirmed in preclinical models, the present engineering approach could represent an interesting tool in treating large bone defects.

Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries

Directory of Open Access Journals (Sweden)

Marco Cavallari

2017-07-01

Full Text Available Unbiased and simultaneous identification of a specific antibody and its target antigen has been difficult without prior knowledge of at least one interaction partner. Immunization with complex mixtures of antigens such as whole organisms and tissue extracts including tumoral ones evokes a highly diverse immune response. During such a response, antibodies are generated against a variety of epitopes in the mixture. Here, we propose a surface display design that is suited to simultaneously identify camelid single domain antibodies and their targets. Immune libraries of single-domain antigen recognition fragments from camelid heavy chain-only antibodies (VHH were attached to the peptidoglycan of Gram-positive Staphylococcus aureus employing its endogenous housekeeping sortase enzyme. The sortase transpeptidation reaction covalently attached the VHH to the bacterial peptidoglycan. The reversible nature of the reaction allowed the recovery of the VHH from the bacterial surface and the use of the VHH in downstream applications. These staphylococcal surface display libraries were used to rapidly identify VHH as well as their targets by immunoprecipitation (IP. Our novel bacterial surface display platform was stable under harsh screening conditions, allowed fast target identification, and readily permitted the recovery of the displayed VHH for downstream analysis.

Development of a qPCR method to rapidly assess the function of NKT cells.

Science.gov (United States)

Sohn, Silke; Tiper, Irina; Japp, Emily; Sun, Wenji; Tkaczuk, Katherine; Webb, Tonya J

2014-05-01

NKT cells comprise a rare, but important subset of T cells which account for ~0.2% of the total circulating T cell population. NKT cells are known to have anti-tumor functions and rapidly produce high levels of cytokines following activation. Several clinical trials have sought to exploit the effector functions of NKT cells. While some studies have shown promise, NKT cells are approximately 50% lower in cancer patients compared to healthy donors of the same age and gender, thus limiting their therapeutic efficacy. These studies indicate that baseline levels of activation should be assessed before initiating an NKT cell based immunotherapeutic strategy. The goal of this study was to develop a sensitive method to rapidly assess NKT cell function. We utilized artificial antigen presenting cells in combination with qPCR in order to determine NKT cell function in peripheral blood mononuclear cells from healthy donors and breast cancer patients. We found that NKT cell activation can be detected by qPCR, but not by ELISA, in healthy donors as well as in breast cancer patients following four hour stimulation. This method utilizing CD1d-expressing aAPCs will enhance our knowledge of NKT cell biology and could potentially be used as a novel tool in adoptive immunotherapeutic strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

Assessing the Nano-Dynamics of the Cell Surface

Energy Technology Data Exchange (ETDEWEB)

Bae, Chil Man [Dept. of Physiology and Biophysics, State University of New York, Buffalo (United States); Park, Ik Keun [Mechanical Engineering, Seoul National University of Technology, Seoul (Korea, Republic of); Bulter, Peter J. [Dept. of Bioengineering, The Pennsylvania State University, University Park (United States)

2012-06-15

It is important to know the mechanism of cell membrane fluctuation because it can be readout for the nanomechanical interaction between cytoskeleton and plasma membrane. Traditional techniques, however, have drawbacks such as probe contact with the cell surface, complicate analysis, and limit spatial and temporal resolution. In this study, we developed a new system for non-contact measurement of nano-scale localized-cell surface dynamics using modified-scanning ion-conductance microscopy. With 2 nm resolution, we determined that endothelial cells have local membrane fluctuations of -20 nm, actin depolymerization causes increase in fluctuation amplitude, and ATP depletion abolishes all membrane fluctuations.

Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells.

Directory of Open Access Journals (Sweden)

Yi-Chun Kuo

Full Text Available Surface plasmon resonance (SPR biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs. OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

Stem cell responses to plasma surface modified electrospun polyurethane scaffolds.

Science.gov (United States)

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

2014-07-01

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

Cell Surface Enzymatic Engineering-Based Approaches to Improve Cellular Therapies

KAUST Repository

AbuElela, Ayman

2014-06-06

The cell surface represents the interface between the cell and its environment. As such, the cell surface controls cell–cell interactions and functions such as adhesion and migration, and will transfer external cues to regulate processes such as survival, death, and differentiation. Redefining the cell surface by temporarily (or permanently) modifying the molecular landscape of the plasma membrane affects the way in which the cell interacts with its environment and influences the information that is relayed into the cell along downstream signaling pathways. This chapter outlines the role of key enzymes, the glycosyltransferases, in posttranslationally modifying proteins and lipids to fine-tune cells, ability to migrate. These enzymes are critical in controlling the formation of a platform structure, sialyl Lewis x (sLex), on circulating cells that plays a central role in the recognition and recruitment by selectin counter receptors on endothelial cells that line blood vessels of tissues throughout the body. By developing methods to manipulate the activity of these enzymes and hence the cell surface structures that result, treatments can be envisioned that direct the migration of therapeutic cells to specific locations throughout the body and also to inhibit metastasis of detrimental cells such as circulating tumor cells.

Rapid detection of urinary polyomavirus BK by heterodyne-based surface plasmon resonance biosensor

Science.gov (United States)

Su, Li-Chen; Tian, Ya-Chung; Chang, Ying-Feng; Chou, Chien; Lai, Chao-Sung

2014-01-01

In renal transplant patients, immunosuppressive therapy may result in the reactivation of polyomavirus BK (BKV), leading to polyomavirus-associated nephropathy (PVAN), which inevitably causes allograft failure. Since the treatment outcomes of PVAN remain unsatisfactory, early identification and continuous monitoring of BKV reactivation and reduction of immunosuppressants are essential to prevent PVAN development. The present study demonstrated that the developed dual-channel heterodyne-based surface plasmon resonance (SPR) biosensor is applicable for the rapid detection of urinary BKV. The use of a symmetrical reference channel integrated with the poly(ethylene glycol)-based low-fouling self-assembled monolayer to reduce the environmental variations and the nonspecific noise was proven to enhance the sensitivity in urinary BKV detection. Experimentally, the detection limit of the biosensor for BKV detection was estimated to be around 8500 copies/mL. In addition, urine samples from five renal transplant patients were tested to rapidly distinguish PVAN-positive and PVAN-negative renal transplant patients. By virtue of its simplicity, rapidity, and applicability, the SPR biosensor is a remarkable potential to be used for continuous clinical monitoring of BKV reactivation.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism

KAUST Repository

Maruyama, Daisuke

2015-05-01

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization. Two female gametes (the egg cell and the central cell) in flowering plants coordinately prevent attractions of excess number of pollen tubes via two mechanisms to inactivate persistent synergid cell. © 2015 Elsevier Inc.

Rapid Surface Sampling and Archival Record (RSSAR) System. Topical report, October 1, 1993--December 31, 1994

International Nuclear Information System (INIS)

1997-01-01

This report describes the results of Phase 1 efforts to develop a Rapid Surface Sampling and Archival Record (RSSAR) System for the detection of semivolatile organic contaminants on concrete, transite, and metal surfaces. The characterization of equipment and building surfaces for the presence of contaminants as part of building decontamination and decommissioning activities is an immensely large tacks of concern to both government and industry. Contaminated and clean materials must be clearly identified and segregated so that the clean materials can be recycled or reused, if possible, or disposed of more cheaply as nonhazardous waste. Characterization of building and equipment surfaces will be needed during initial investigations, during cleanup operations, and during the final confirmatory process, increasing the total number of samples well beyond that needed for initial characterization. This multiplicity of information places a premium on the ability to handle and track data as efficiently as possible. Aware of the shortcomings of traditional surface characterization technology, GE, with DOE support has undertaken a 12-month effort to complete Phase 1 of a proposed four-phase program to develop the RSSAR system. The objectives of this work are to provide instrumentation to cost-effectively sample concrete and steel surfaces, provide a quick-look indication for the presence or absence of contaminants, and collect samples for later, more detailed analysis in a readily accessible and addressable form. The Rapid Surface Sampling and Archival Record (RSSAR) System will be a modular instrument made up of several components: (1) sampling heads for concrete surfaces, steel surfaces, and bulk samples; (2) quick-look detectors for photoionization and ultraviolet; (3) multisample trapping module to trap and store vaporized contaminants in a manner suitable for subsequent detailed lab-based analyses

Carrier population control and surface passivation in solar cells

KAUST Repository

Cuevas, Andres

2018-05-02

Controlling the concentration of charge carriers near the surface is essential for solar cells. It permits to form regions with selective conductivity for either electrons or holes and it also helps to reduce the rate at which they recombine. Chemical passivation of the surfaces is equally important, and it can be combined with population control to implement carrier-selective, passivating contacts for solar cells. This paper discusses different approaches to suppress surface recombination and to manipulate the concentration of carriers by means of doping, work function and charge. It also describes some of the many surface-passivating contacts that are being developed for silicon solar cells, restricted to experiments performed by the authors.

Cell surface N-glycans influence the level of functional E-cadherin at the cell–cell border

Directory of Open Access Journals (Sweden)

M. Kristen Hall

2014-01-01

Full Text Available E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell–cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell–cell border, and consequently reduced the strength of cell–cell interactions. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell–cell border and thereby control E-cadherin mediated cell–cell adhesion.

Rapid metabolism of exogenous angiotensin II by catecholaminergic neuronal cells in culture media.

Science.gov (United States)

Basu, Urmi; Seravalli, Javier; Madayiputhiya, Nandakumar; Adamec, Jiri; Case, Adam J; Zimmerman, Matthew C

2015-02-01

Angiotensin II (AngII) acts on central neurons to increase neuronal firing and induce sympathoexcitation, which contribute to the pathogenesis of cardiovascular diseases including hypertension and heart failure. Numerous studies have examined the precise AngII-induced intraneuronal signaling mechanism in an attempt to identify new therapeutic targets for these diseases. Considering the technical challenges in studying specific intraneuronal signaling pathways in vivo, especially in the cardiovascular control brain regions, most studies have relied on neuronal cell culture models. However, there are numerous limitations in using cell culture models to study AngII intraneuronal signaling, including the lack of evidence indicating the stability of AngII in culture media. Herein, we tested the hypothesis that exogenous AngII is rapidly metabolized in neuronal cell culture media. Using liquid chromatography-tandem mass spectrometry, we measured levels of AngII and its metabolites, Ang III, Ang IV, and Ang-1-7, in neuronal cell culture media after administration of exogenous AngII (100 nmol/L) to a neuronal cell culture model (CATH.a neurons). AngII levels rapidly declined in the media, returning to near baseline levels within 3 h of administration. Additionally, levels of Ang III and Ang-1-7 acutely increased, while levels of Ang IV remained unchanged. Replenishing the media with exogenous AngII every 3 h for 24 h resulted in a consistent and significant increase in AngII levels for the duration of the treatment period. These data indicate that AngII is rapidly metabolized in neuronal cell culture media, and replenishing the media at least every 3 h is needed to sustain chronically elevated levels. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Nanolayer surface passivation schemes for silicon solar cells

NARCIS (Netherlands)

Dingemans, G.

2011-01-01

This thesis is concerned with nanolayer surface passivation schemes and corresponding deposition processes, for envisaged applications in crystalline silicon solar cells. Surface passivation, i.e. the reduction of electronic recombination processes at semiconductor surfaces, is essential for

Rapid and sensitive detection of malachite green in aquaculture water by electrochemical preconcentration and surface-enhanced Raman scattering.

Science.gov (United States)

Xu, Kai-Xuan; Guo, Mei-Hong; Huang, Yu-Ping; Li, Xiao-Dong; Sun, Jian-Jun

2018-04-01

A highly sensitive and rapid method of in-situ surface-enhanced Raman spectroscopy (SERS) combining with electrochemical preconcentration (EP) in detecting malachite green (MG) in aquaculture water was established. Ag nanoparticles (AgNPs) were synthesized and spread onto the surface of gold electrodes after centrifuging to produce SERS-active substrates. After optimizing the pH values, preconcentration potentials and times, in-situ EP-SERS detection was carried out. A sensitive and rapid analysis of the low-concentration MG was accomplished within 200s and the limit of detection was 2.4 × 10 -16 M. Copyright © 2017 Elsevier B.V. All rights reserved.

Rapid bacterial antibiotic susceptibility test based on simple surface-enhanced Raman spectroscopic biomarkers

Science.gov (United States)

Liu, Chia-Ying; Han, Yin-Yi; Shih, Po-Han; Lian, Wei-Nan; Wang, Huai-Hsien; Lin, Chi-Hung; Hsueh, Po-Ren; Wang, Juen-Kai; Wang, Yuh-Lin

2016-03-01

Rapid bacterial antibiotic susceptibility test (AST) and minimum inhibitory concentration (MIC) measurement are important to help reduce the widespread misuse of antibiotics and alleviate the growing drug-resistance problem. We discovered that, when a susceptible strain of Staphylococcus aureus or Escherichia coli is exposed to an antibiotic, the intensity of specific biomarkers in its surface-enhanced Raman scattering (SERS) spectra drops evidently in two hours. The discovery has been exploited for rapid AST and MIC determination of methicillin-susceptible S. aureus and wild-type E. coli as well as clinical isolates. The results obtained by this SERS-AST method were consistent with that by the standard incubation-based method, indicating its high potential to supplement or replace existing time-consuming methods and help mitigate the challenge of drug resistance in clinical microbiology.

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

Science.gov (United States)

Cai, Rong; Kawazoe, Naoki; Chen, Guoping

2015-02-01

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

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

International Nuclear Information System (INIS)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

2012-01-01

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K 3 ) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of ATP

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis

Energy Technology Data Exchange (ETDEWEB)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting, E-mail: BTZhu@kumc.edu

2012-07-15

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K{sub 3}) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ∼ 12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. -- Highlights: ► Menadione causes mitochondrial superoxide accumulation and injury. ► Menadione-induced cell death is caspase-independent, due to rapid depletion of

Electrical pulse – mediated enhanced delivery of silver nanoparticles into living suspension cells for surface enhanced Raman spectroscopy

International Nuclear Information System (INIS)

Lin, J; Li, B; Feng, S; Chen, G; Li, Y; Huang, Z; Chen, R; Yu, Y; Huang, H; Lin, S; Li, C; Su, Y; Zeng, H

2012-01-01

Electrical pulse-mediated enhanced silver nanoparticles delivery is a much better method for intracellular surface-enhanced Raman spectroscopy (SERS) measurements of suspension cells. Robust and high-quality SERS spectra of living suspension cells were obtained based on an electroporation-SERS method, which can overcomes the shortcoming of non-uniform distribution of silver nanoparticles localized in the cell cytoplasm after electroporation and reduces the amount variance of silver nanoparticles delivered into different cells. The electroporation parameters include three 150 V (375 V/cm) electric pulses of 1, 5, and 5 ms durations respectively. Our results indicate that considerable amount of silver nanoparticles can be rapidly delivered into the human promyelocytic leukemia HL60 cells, and the satisfied SERS spectra were obtained while the viability of the treated cells was highly maintained (91.7%). The electroporation-SERS method offers great potential approach in delivering silver nanoparticles into living suspension cells, which is useful for widely biomedical applications including the real-time intracellular SERS analysis of living cells

Radioimmunoassay to quantitatively measure cell surface immunoglobulins

International Nuclear Information System (INIS)

Krishman, E.C.; Jewell, W.R.

1975-01-01

A radioimmunoassay techniques developed to quantitatively measure the presence of immunoglobulins on the surface of cells, is described. The amount of immunoglobulins found on different tumor cells varied from 200 to 1140 ng/10 6 cells. Determination of immunoglobulins on the peripheral lymphocytes obtained from different cancer patients varied between 340 to 1040 ng/10 6 cells. Cultured tumor cells, on the other hand, were found to contain negligible quantities of human IgG [pt

Simulating the Surface Relief of Nanoaerosols Obtained via the Rapid Cooling of Droplets

Science.gov (United States)

Tovbin, Yu. K.; Zaitseva, E. S.; Rabinovich, A. B.

2018-03-01

An approach is formulated that theoretically describes the structure of a rough surface of small aerosol particles obtained from a liquid droplet upon its rapid cooling. The problem consists of two stages. In the first stage, a concentration profile of the droplet-vapor transition region is calculated. In the second stage, local fractions of vacant sites and their pairs are found on the basis of this profile, and the rough structure of a frozen droplet surface transitioning to the solid state is calculated. Model parameters are the temperature of the initial droplet and those of the lateral interaction between droplet atoms. Information on vacant sites inside the region of transition allows us to identify adsorption centers and estimate the monolayer capacity, compared to that of the total space of the region of transition. The approach is oriented toward calculating adsorption isotherms on real surfaces.

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

International Nuclear Information System (INIS)

Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu; Rosenmann, Daniel; Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei

2016-01-01

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. - Highlights: • Surface topographical effects the growth patterns and cell function of cancer cells • Nanoscale surface topography on polymer filters for circulating tumor cell culture • Membrane fabricated directly on polymer surfaces utilized for polymer etching • Nanotopography alters cell shape, phenotype and growth patterns of cancer cells • Nanoscale surface topography dictates monolayering or 3D structured cell culture

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

Energy Technology Data Exchange (ETDEWEB)

Makarova, Olga V. [Creatv MicroTech, Inc., 2242 West Harrison St., Chicago 60612, IL (United States); Adams, Daniel L., E-mail: dan@creatvmicrotech.com [Creatv MicroTech, Inc., 1 Deer Park Drive, Monmouth Junction, NJ 08852 (United States); Divan, Ralu; Rosenmann, Daniel [Center for Nanoscale Materials, Argonne National Laboratory, 9700 South Cass Ave., Argonne 60439, IL (United States); Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei [Creatv MicroTech, Inc., 11609 Lake Potomac Drive, Potomac 20854, MD (United States)

2016-09-01

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. - Highlights: • Surface topographical effects the growth patterns and cell function of cancer cells • Nanoscale surface topography on polymer filters for circulating tumor cell culture • Membrane fabricated directly on polymer surfaces utilized for polymer etching • Nanotopography alters cell shape, phenotype and growth patterns of cancer cells • Nanoscale surface topography dictates monolayering or 3D structured cell culture.

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

Science.gov (United States)

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

2016-05-01

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

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

Science.gov (United States)

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

2014-09-01

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

Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

International Nuclear Information System (INIS)

Petit, Chad M.; Melancon, Jeffrey M.; Chouljenko, Vladimir N.; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G.

2005-01-01

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion

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

International Nuclear Information System (INIS)

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

2016-01-01

Graphical abstract: - Highlights: • The nanocrystalline diamond (NCD) surface is functionalized with F or O. • The cell adhesion and growth are evaluated on the functionalized NCD surface. • The cell adhesion and growth depend on the wettability of the surface. • Cell patterning was achieved by using of hydrophilic and hydrophobic surfaces. • Neuroblastoma cells were arrayed on the micro-patterned NCD surface. - Abstract: Nanocrystalline diamond (NCD) film surfaces were modified with fluorine or oxygen by plasma treatment in an O_2 or C_3F_8 gas environment in order to induce wettability. The oxygenated-NCD (O-NCD) film surface was hydrophilic and the fluorinated-NCD (F-NCD) surface was hydrophobic. The efficiency of early cell adhesion, which is dependent on the wettability of the cell culture plate and necessary for the growth and proliferation of cells, was 89.62 ± 3.92% on the O-NCD film and 7.78 ± 0.77% on the F-NCD film surface after 3 h of cell culture. The wettability of the NCD film surface was artificially modified using a metal mask and plasma treatment to fabricate a micro-pattern. Four types of micro-patterns were fabricated (line, circle, mesh, and word) on the NCD film surface. We precisely arrayed the neuroblastoma cells on the micro-patterned NCD film surfaces by controlling the surface wettability and cell seeding density. The neuroblastoma cells adhered and proliferated along the O-NCD film surface.

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

Energy Technology Data Exchange (ETDEWEB)

Park, Young-Sang; Son, Hyeong-Guk; Kim, Dae-Hoon; Oh, Hong-Gi; Lee, Da-Som; Kim, Min-Hye; Lim, Ki-Moo; Song, Kwang-Soup, E-mail: kssong10@kumoh.ac.kr

2016-01-15

Graphical abstract: - Highlights: • The nanocrystalline diamond (NCD) surface is functionalized with F or O. • The cell adhesion and growth are evaluated on the functionalized NCD surface. • The cell adhesion and growth depend on the wettability of the surface. • Cell patterning was achieved by using of hydrophilic and hydrophobic surfaces. • Neuroblastoma cells were arrayed on the micro-patterned NCD surface. - Abstract: Nanocrystalline diamond (NCD) film surfaces were modified with fluorine or oxygen by plasma treatment in an O{sub 2} or C{sub 3}F{sub 8} gas environment in order to induce wettability. The oxygenated-NCD (O-NCD) film surface was hydrophilic and the fluorinated-NCD (F-NCD) surface was hydrophobic. The efficiency of early cell adhesion, which is dependent on the wettability of the cell culture plate and necessary for the growth and proliferation of cells, was 89.62 ± 3.92% on the O-NCD film and 7.78 ± 0.77% on the F-NCD film surface after 3 h of cell culture. The wettability of the NCD film surface was artificially modified using a metal mask and plasma treatment to fabricate a micro-pattern. Four types of micro-patterns were fabricated (line, circle, mesh, and word) on the NCD film surface. We precisely arrayed the neuroblastoma cells on the micro-patterned NCD film surfaces by controlling the surface wettability and cell seeding density. The neuroblastoma cells adhered and proliferated along the O-NCD film surface.

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

Science.gov (United States)

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

2011-04-01

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

Modification of surface/neuron interfaces for neural cell-type specific responses: a review

International Nuclear Information System (INIS)

Chen, Cen; Kong, Xiangdong; Lee, In-Seop

2016-01-01

Surface/neuron interfaces have played an important role in neural repair including neural prostheses and tissue engineered scaffolds. This comprehensive literature review covers recent studies on the modification of surface/neuron interfaces. These interfaces are identified in cases both where the surfaces of substrates or scaffolds were in direct contact with cells and where the surfaces were modified to facilitate cell adhesion and controlling cell-type specific responses. Different sources of cells for neural repair are described, such as pheochromocytoma neuronal-like cell, neural stem cell (NSC), embryonic stem cell (ESC), mesenchymal stem cell (MSC) and induced pluripotent stem cell (iPS). Commonly modified methods are discussed including patterned surfaces at micro- or nano-scale, surface modification with conducting coatings, and functionalized surfaces with immobilized bioactive molecules. These approaches to control cell-type specific responses have enormous potential implications in neural repair. (paper)

The impact of different nanoparticle surface chemistry and size on uptake and toxicity in a murine macrophage cell line

International Nuclear Information System (INIS)

Clift, Martin J.D.; Rothen-Rutishauser, Barbara; Brown, David M.; Duffin, Rodger; Donaldson, Ken; Proudfoot, Lorna; Guy, Keith; Stone, Vicki

2008-01-01

This study investigated the uptake, kinetics and cellular distribution of different surface coated quantum dots (QDs) before relating this to their toxicity. J774.A1 cells were treated with organic, COOH and NH 2 (PEG) surface coated QDs (40 nM). Model 20 nm and 200 nm COOH-modified coated polystyrene beads (PBs) were also examined (50 μg ml -1 ). The potential for uptake of QDs was examined by both fixed and live cell confocal microscopy as well as by flow cytometry over 2 h. Both the COOH 20 nm and 200 nm PBs were clearly and rapidly taken up by the J774.A1 cells, with uptake of 20 nm PBs being relatively quicker and more extensive. Similarly, COOH QDs were clearly taken up by the macrophages. Uptake of NH 2 (PEG) QDs was not detectable by live cell imaging however, was observed following 3D reconstruction of fixed cells, as well as by flow cytometry. Cells treated with organic QDs, monitored by live cell imaging, showed only a small amount of uptake in a relatively small number of cells. This uptake was insufficient to be detected by flow cytometry. Imaging of fixed cells was not possible due to a loss in cell integrity related to cytotoxicity. A significant reduction (p 2 (PEG) QDs, 20 nm and 200 nm PBs at pH 4.0 (indicative of an endosome) after 2 h, suggesting reduced stability. No evidence of exocytosis was found over 2 h. These findings confirm that surface coating has a significant influence on the mode of NP interaction with cells, as well as the subsequent consequences of that interaction

Probes for anionic cell surface detection

Science.gov (United States)

Smith, Bradley D.

2013-03-05

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

Rapid surface accumulation of NMDA receptors increases glutamatergic excitation during status epilepticus.

Science.gov (United States)

Naylor, David E; Liu, Hantao; Niquet, Jerome; Wasterlain, Claude G

2013-06-01

After 1h of lithium-pilocarpine status epilepticus (SE), immunocytochemical labeling of NMDA receptor NR1 subunits reveals relocation of subunits from the interior to the cell surface of dentate gyrus granule cells and CA3 pyramidal cells. Simultaneously, an increase in NMDA-miniature excitatory postsynaptic currents (mEPSC) as well as an increase in NMDA receptor-mediated tonic currents is observed in hippocampal slices after SE. Mean-variance analysis of NMDA-mEPSCs estimates that the number of functional postsynaptic NMDA receptors per synapse increases 38% during SE, and antagonism by ifenprodil suggests that an increase in the surface representation of NR2B-containing NMDA receptors is responsible for the augmentation of both the phasic and tonic excitatory currents with SE. These results provide a potential mechanism for an enhancement of glutamatergic excitation that maintains SE and may contribute to excitotoxic injury during SE. Therapies that directly antagonize NMDA receptors may be a useful therapeutic strategy during refractory SE. Copyright © 2013 Elsevier Inc. All rights reserved.

Cell surface hydrophobicity of dental plaque microorganisms in situ.

OpenAIRE

Rosenberg, M; Judes, H; Weiss, E

1983-01-01

The cell surface hydrophobicity of bacteria obtained directly from human tooth surfaces was assayed by measuring their adherence to liquid hydrocarbons. Fresh samples of supragingival dental plaque were washed and dispersed in buffer. Adherence of the plaque microorganisms to hexadecane, octane, and xylene was tested turbidimetrically and by direct microscopic observation. The results clearly show that the vast majority of bacteria comprising dental plaque exhibit pronounced cell surface hydr...

Large-volume constant-concentration sampling technique coupling with surface-enhanced Raman spectroscopy for rapid on-site gas analysis

Science.gov (United States)

Zhang, Zhuomin; Zhan, Yisen; Huang, Yichun; Li, Gongke

2017-08-01

In this work, a portable large-volume constant-concentration (LVCC) sampling technique coupling with surface-enhanced Raman spectroscopy (SERS) was developed for the rapid on-site gas analysis based on suitable derivatization methods. LVCC sampling technique mainly consisted of a specially designed sampling cell including the rigid sample container and flexible sampling bag, and an absorption-derivatization module with a portable pump and a gas flowmeter. LVCC sampling technique allowed large, alterable and well-controlled sampling volume, which kept the concentration of gas target in headspace phase constant during the entire sampling process and made the sampling result more representative. Moreover, absorption and derivatization of gas target during LVCC sampling process were efficiently merged in one step using bromine-thiourea and OPA-NH4+ strategy for ethylene and SO2 respectively, which made LVCC sampling technique conveniently adapted to consequent SERS analysis. Finally, a new LVCC sampling-SERS method was developed and successfully applied for rapid analysis of trace ethylene and SO2 from fruits. It was satisfied that trace ethylene and SO2 from real fruit samples could be actually and accurately quantified by this method. The minor concentration fluctuations of ethylene and SO2 during the entire LVCC sampling process were proved to be samples were achieved in range of 95.0-101% and 97.0-104% respectively. It is expected that portable LVCC sampling technique would pave the way for rapid on-site analysis of accurate concentrations of trace gas targets from real samples by SERS.

Macromolecular cell surface engineering for accelerated and reversible cellular aggregation.

OpenAIRE

Amaral, A. J.; Pasparakis, G.

2015-01-01

We report the synthesis of two simple copolymers that induce rapid cell aggregation within minutes in a fully reversible manner. The polymers can act as self-supporting "cellular glues" or as "drivers" of 3D cell spheroids/aggregates formation at minute concentrations.

Fabrication of Biomolecule Microarrays Using Rapid Photochemical Surface Patterning in Thiol-Ene-Based Microfluidic Devices.

Science.gov (United States)

Jönsson, Alexander; Lafleur, Josiane P

2018-01-01

In many biochip applications, it is advantageous to be able to immobilize biomolecules at specific locations on the surface of solid supports. In this protocol, we describe a photochemical surface patterning procedure based on thiol-ene/yne photochemistry which allows for the simple and rapid selective patterning of biomolecules on thiol-ene solid supports. We describe the preparation of solid supports which are required for the immobilization, including porous monoliths, as well as two different immobilization schemes based on biotin-streptavidin interactions and covalent linkage via free amino groups respectively.

Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells.

Directory of Open Access Journals (Sweden)

Tadanobu Takahashi

Full Text Available Mumps viruses show diverse cytopathic effects (CPEs of infected cells and viral plaque formation (no CPE or no plaque formation in some cases depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac, was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study, even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.

Autonomous molecular cascades for evaluation of cell surfaces

Science.gov (United States)

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

2013-08-01

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

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

Science.gov (United States)

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.

Surface segregation of chromium in rapidly solidified Al studied by RBS and SPEM

International Nuclear Information System (INIS)

Tashlykova-Bushkevich, I.I.; Amati, M.; Aleman, B.; Gregoratti, L.; Kiskinova, M.; Ryabuhin, O.V.; Shepelevich, V.G.

2013-01-01

The present study demonstrates the advances of using scanning photoelectron microscopy and imaging accomplished by RBS and AFM to investigate the surface segregation of alloying elements in RS aluminum. Depth profiling of elemental composition indicates that RS microstructure evolution is influenced by solute-nanostructured defect interactions in Al-Cr alloys. It was found that Cr 2p and 3p core level photoemission spectra exhibits foil surface impoverishment of chromium. In agreement with dope depth profiling as carried out by RBS, the revealed phenomenon can be attributed to the fact that Cr drastically reduces the concentration of vacancies compared with RS pure Al, and affects H behaviour in RS Al-Cr alloys. Obtained results indicate that the surface microstructure of the Al alloy foils at the sub-micrometer scale, as far as the high density of quenched-in vacancies is concerned, is essential to elucidate how the microstructural morphology resulting from rapid solidification affects hydrogen trapping at lattice defects. (authors)

ER Alpha Rapid Signaling Is Required for Estrogen Induced Proliferation and Migration of Vascular Endothelial Cells.

Directory of Open Access Journals (Sweden)

Qing Lu

Full Text Available Estrogen promotes the proliferation and migration of vascular endothelial cells (ECs, which likely underlies its ability to accelerate re-endothelialization and reduce adverse remodeling after vascular injury. In previous studies, we have shown that the protective effects of E2 (the active endogenous form of estrogen in vascular injury require the estrogen receptor alpha (ERα. ERα transduces the effects of estrogen via a classical DNA binding, "genomic" signaling pathway and via a more recently-described "rapid" signaling pathway that is mediated by a subset of ERα localized to the cell membrane. However, which of these pathways mediates the effects of estrogen on endothelial cells is poorly understood. Here we identify a triple point mutant version of ERα (KRR ERα that is specifically defective in rapid signaling, but is competent to regulate transcription through the "genomic" pathway. We find that in ECs expressing wild type ERα, E2 regulates many genes involved in cell migration and proliferation, promotes EC migration and proliferation, and also blocks the adhesion of monocytes to ECs. ECs expressing KRR mutant ERα, however, lack all of these responses. These observations establish KRR ERα as a novel tool that could greatly facilitate future studies into the vascular and non-vascular functions of ERα rapid signaling. Further, they support that rapid signaling through ERα is essential for many of the transcriptional and physiological responses of ECs to E2, and that ERα rapid signaling in ECs, in vivo, may be critical for the vasculoprotective and anti-inflammatory effects of estrogen.

Survival of plant tissue at super-low temperatures v. An electron microscope study of ice in cortical cells cooled rapidly.

Science.gov (United States)

Sakai, A; Otsuka, K

1967-12-01

Experiments were carried out with cortical cells in twig bark of mulberry trees in winter in order to clarify the mechanism of survival at super-low temperatures with rapid cooling and rewarming. Attention was given to the relation between the existence of intracellular ice crystals and survival.Cortical cells were cooled rapidly by direct immersion into liquid nitrogen or isopentane cooled at various temperatures. After immersion, they were freeze-substituted with absolute ethanol at -78 degrees . They were then embedded, sectioned and examined under the electron microscope for the presence and distribution of cavities left after ice removal.Cells were found to remain alive and contain no ice cavities when immersed rapidly into isopentane baths kept below -60 degrees . Those cells at intermediate temperatures from -20 degrees to -45 degrees , were almost all destroyed. It was also observed that many ice cavities were contained in the cells immersed rapidly into isopentane baths at -30 degrees . The data seem to indicate that no ice crystals were formed when cooled rapidly by direct immersion into isopentane baths below -60 degrees or into liquid nitrogen.The tissue sections immersed in liquid nitrogen were rapidly transferred to isopentane baths at temperatures ranging from -70 degrees to -10 degrees before rapid rewarming. There was little damage when samples were held at temperatures below -50 degrees for 10 minutes or below -60 degrees for 16 hours. No cavities were found in these cells. Above -45 degrees , and especially at -30 degrees , however, all cells were completely destroyed even when exposed only for 1 minute. Many ice cavities were observed throughout these cells. The results obtained may be explained in terms of the growth rate of intracellular ice crystals.

Imaging and reconstruction of cell cortex structures near the cell surface

Science.gov (United States)

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

2017-11-01

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

Methods To Identify Aptamers against Cell Surface Biomarkers

Directory of Open Access Journals (Sweden)

Frédéric Ducongé

2011-09-01

Full Text Available Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment. During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells.

LANTHANUM STAINING OF THE SURFACE COAT OF CELLS

Science.gov (United States)

Shea, Stephen M.

1971-01-01

Among the techniques which have been reported to stain the surface coat of cells, for electron microscopy, is lanthanum staining en bloc. Similarly, the presence of the cationic dye, Alcian blue 8GX, in a primary glutaraldehyde fixative has been reported to improve the preservation of the surface coat of cells of many types; however, the preserved coat is not very electron opaque unless thin sections are counterstained. The present paper shows that for several rat tissues lanthanum staining en bloc is an effective electron stain for the cell surface, giving excellent contrast, if combined sequentially with prefixation in an aldehyde fixative containing Alcian blue. The cationic substance cetylpyridinium chloride was found to have a similar effect to that of Alcian blue in enhancing the lanthanum staining of the surface coat material of the brush border of intestinal epithelial cells. The patterns of lanthanum staining obtained for the tissues studied strikingly resemble those reported in the literature where tissues are stained by several standard methods for demonstrating mucosubstances at the ultrastructural level. This fact and the reproduction of the effect of Alcian blue by cetylpyridinium chloride constitute a persuasive empirical argument that the material visualized is a mucopolysaccharide or mucopolysaccharide-protein complex. PMID:4108476

Osteoclast cell-surface specializations and nuclear kinetics during egg-laying in Japanese quail

International Nuclear Information System (INIS)

Miller, S.C.

1981-01-01

Medullary bone deposits serve as a reservoir of labile calcium for egg-shell calcification in birds. Quantitative transmission-electron-microscope methods and light-microscope autoradiographic cell-population-kinetic analyses were used to determine changes in cell-surface specializations and population dynamics of medullary bone osteoclasts during egg-laying in Japanese quail. Prior to egg-shell formation, from 0 to about 8 hours after the previous oviposition, very few osteoclast profiles had ruffled borders. The appearance of ruffled borders coincided with the beginning of egg-shell calcification, about 9-10 hours after the previous oviposition. During egg-shell calcification, about 10-21 hours after the previous oviposition, most osteoclast profiles had ruffled borders. Ruffled borders disappeared at the completion of egg-shell calcification and commencement of egg-shell pigmentation. Thus, functional activities of medullary bone osteoclasts appear to be closely synchronized with egg-shell calcification during egg-laying. From 1 to 48 hours after a single injection of 3H-thymidine (3H-TdR), very few labeled osteoclast nuclei were seen during egg-laying. Following multiple injections of 3H-TdR, the percentage of labeled nuclei reached a peak at about 170 hours after the first injection. At this peak-labeling time, relatively few of the osteoclast profiles that had labeled nuclei had two or more; although the average number of nuclei per osteoclast profile was about 3.6. These kinetic data suggest that the medullary bone osteoclast population has a prolonged rate of turnover compared to rapid changes in cell activities associated with each 24-hour egg-laying cycle; and collectively they would suggest that rapid changes in osteoclast functions occur independently of changes in cell-population dynamics

Controlling human corneal stromal stem cell contraction to mediate rapid cell and matrix organization of real architecture for 3-dimensional tissue equivalents.

Science.gov (United States)

Mukhey, Dev; Phillips, James B; Daniels, Julie T; Kureshi, Alvena K

2018-02-01

The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype. For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils

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

Science.gov (United States)

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

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

Directory of Open Access Journals (Sweden)

Thiel Cora S

2012-01-01

Full Text Available Abstract In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

Encapsulant Adhesion to Surface Metallization on Photovoltaic Cells

Energy Technology Data Exchange (ETDEWEB)

Tracy, Jared; Bosco, Nick; Dauskardt, Reinhold

2017-11-01

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

Responses of fibroblasts and glial cells to nanostructured platinum surfaces

Energy Technology Data Exchange (ETDEWEB)

Pennisi, C P; Sevcencu, C; Yoshida, K [Center for Sensory-Motor Interaction (SMI), Aalborg University, Aalborg (Denmark); Dolatshahi-Pirouz, A; Foss, M; Larsen, A Nylandsted; Besenbacher, F [Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus (Denmark); Hansen, J Lundsgaard [Department of Physics and Astronomy, Aarhus University, Aarhus (Denmark); Zachar, V, E-mail: cpennisi@hst.aau.d [Laboratory for Stem Cell Research, Aalborg University (Denmark)

2009-09-23

The chronic performance of implantable neural prostheses is affected by the growth of encapsulation tissue onto the stimulation electrodes. Encapsulation is associated with activation of connective tissue cells at the electrode's metallic contacts, usually made of platinum. Since surface nanotopography can modulate the cellular responses to materials, the aim of the present work was to evaluate the 'in vitro' responses of connective tissue cells to platinum strictly by modulating its surface nanoroughness. Using molecular beam epitaxy combined with sputtering, we produced platinum nanostructured substrates consisting of irregularly distributed nanopyramids and investigated their effect on the proliferation, cytoskeletal organization and cellular morphology of primary fibroblasts and transformed glial cells. Cells were cultured on these substrates and their responses to surface roughness were studied. After one day in culture, the fibroblasts were more elongated and their cytoskeleton less mature when cultured on rough substrates. This effect increased as the roughness of the surface increased and was associated with reduced cell proliferation throughout the observation period (4 days). Morphological changes also occurred in glial cells, but they were triggered by a different roughness scale and did not affect cellular proliferation. In conclusion, surface nanotopography modulates the responses of fibroblasts and glial cells to platinum, which may be an important factor in optimizing the tissue response to implanted neural electrodes.

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

Science.gov (United States)

Tanaka, Tsutomu; Kondo, Akihiko

2015-02-01

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

Touching Textured Surfaces: Cells in Somatosensory Cortex Respond Both to Finger Movement and to Surface Features

Science.gov (United States)

Darian-Smith, Ian; Sugitani, Michio; Heywood, John; Karita, Keishiro; Goodwin, Antony

1982-11-01

Single neurons in Brodmann's areas 3b and 1 of the macaque postcentral gyrus discharge when the monkey rubs the contralateral finger pads across a textured surface. Both the finger movement and the spatial pattern of the surface determine this discharge in each cell. The spatial features of the surface are represented unambiguously only in the responses of populations of these neurons, and not in the responses of the constituent cells.

Interfacing biomembrane mimetic polymer surfaces with living cells - Surface modification for reliable bioartificial liver

International Nuclear Information System (INIS)

Iwasaki, Yasuhiko; Takami, Utae; Sawada, Shin-ichi; Akiyoshi, Kazunari

2008-01-01

The surface design used for reducing nonspecific biofouling is one of the most important issues for the fabrication of medical devices. We present here a newly synthesized a carbohydrate-immobilized phosphorylcholine polymer for surface modification of medical devices to control the interface with living cells. A random copolymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), n-butyl methacrylate (BMA), and 2-lactobionamidoethyl methacrylate (LAMA) was synthesized by conventional radical polymerization. The monomer feeding ratio in the copolymer was adjusted to 24/75/1 (MPC/BMA/LAMA). The copolymer (PMBL1.0) could be coated by solvent evaporation from an ethanol solution. Cells of the human hepatocellular liver carcinoma cell line (HepG2) having asialoglycoprotein receptors (ASGPRs) were seeded on PMBL1.0 or poly(BMA) (PBMA)-coated PET plates. On PBMA, many adherent cells were observed and were well spread with monolayer adhesion. HepG2 adhesion was observed on PMBL1.0 because the cell has ASGPRs. Furthermore, some of the cells adhering to PMBL1.0 had a spheroid formation and similarly shaped spheroids were scattered on the surface. According to confocal laser microscopic observation after 96 h cultivation, it was found that albumin production preferentially occurred in the center of the spheroid. The albumin production of the cells that adhered to PBMA was sparse. The amount of albumin production per unit cell that adhered to PMBL1.0 was determined by ELISA and was significantly higher than that which adhered to PBMA. Long-term cultivation of HepG2 was also performed using hollow fiber mini-modules coated with PMBL1.0. The concentration of albumin produced from HepG2 increased continuously for one month. In the mini-module, the function of HepG2 was effectively preserved for that period. On the hollow fiber membrane, spheroid formation of HepG2 cells was also observed. In conclusion, PMBL1.0 can provide a suitable surface for the cultivation of

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

Directory of Open Access Journals (Sweden)

Ying Huang

2013-11-01

Ischemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage. Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and

Mapping Cellular Hierarchy by Single-Cell Analysis of the Cell Surface Repertoire

OpenAIRE

Guo, Guoji; Luc, Sidinh; Marco, Eugenio; Lin, Ta-Wei; Peng, Cong; Kerenyi, Marc A.; Beyaz, Semir; Kim, Woojin; Xu, Jian; Das, Partha Pratim; Neff, Tobias; Zou, Keyong; Yuan, Guo-Cheng; Orkin, Stuart H.

2013-01-01

Stem cell differentiation pathways are most often studied at the population level, whereas critical decisions are executed at the level of single cells. We have established a highly multiplexed, quantitative PCR assay to profile in an unbiased manner a panel of all commonly used cell surface markers (280 genes) from individual cells. With this method we analyzed over 1500 single cells throughout the mouse hematopoietic system, and illustrate its utility for revealing important biological insi...

Early depletion of proliferating B cells of germinal center in rapidly progressive simian immunodeficiency virus infection

International Nuclear Information System (INIS)

Zhang Zhiqiang; Casimiro, Danilo R.; Schleif, William A.; Chen, Minchun; Citron, Michael; Davies, Mary-Ellen; Burns, Janine; Liang, Xiaoping; Fu, Tong-Ming; Handt, Larry; Emini, Emilio A.; Shiver, John W.

2007-01-01

Lack of virus specific antibody response is commonly observed in both HIV-1-infected humans and SIV-infected monkeys with rapid disease progression. However, the mechanisms underlying this important observation still remain unclear. In a titration study of a SIVmac239 viral stock, three out of six animals with viral inoculation rapidly progressed to AIDS within 5 months. Unexpectedly, there was no obvious depletion of CD4 + T cells in both peripheral and lymph node (LN) compartments in these animals. Instead, progressive depletion of proliferating B cells and disruption of the follicular dendritic cell (FDC) network in germinal centers (GC) was evident in the samples collected at as early as 20 days after viral challenge. This coincided with undetectable, or weak and transient, virus-specific antibody responses over the course of infection. In situ hybridization of SIV RNA in the LN samples revealed a high frequency of SIV productively infected cells and large amounts of accumulated viral RNA in the GCs in these animals. Early severe depletion of GC proliferating B cells and disruption of the FDC network may thus result in an inability to mount a virus-specific antibody response in rapid progressors, which has been shown to contribute to accelerated disease progression of SIV infection

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface.

Directory of Open Access Journals (Sweden)

Ivan E Collier

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

Surface modification of closed plastic bags for adherent cell cultivation

Science.gov (United States)

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

2011-07-01

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

Mechano-chemical signaling maintains the rapid movement of Dictyostelium cells

International Nuclear Information System (INIS)

Lombardi, M.L.; Knecht, D.A.; Lee, J.

2008-01-01

The survival of Dictyostelium cells depends on their ability to efficiently chemotax, either towards food or to form multicellular aggregates. Although the involvement of Ca 2+ signaling during chemotaxis is well known, it is not clear how this regulates cell movement. Previously, fish epithelial keratocytes have been shown to display transient increases in intracellular calcium ([Ca 2+ ] i ) that are mediated by stretch-activated calcium channels (SACs), which play a role in retraction of the cell body [J. Lee, A. Ishihara, G. Oxford, B. Johnson, and K. Jacobson, Regulation of cell movement is mediated by stretch-activated calcium channels. Nature, 1999. 400(6742): p. 382-6.]. To investigate the involvement of SACs in Dictyostelium movement we performed high resolution calcium imaging in wild-type (NC4A2) Dictyostelium cells to detect changes in [Ca 2+ ] i . We observed small, brief, Ca 2+ transients in randomly moving wild-type cells that were dependent on both intracellular and extracellular sources of calcium. Treatment of cells with the SAC blocker gadolinium (Gd 3+ ) inhibited transients and decreased cell speed, consistent with the involvement of SACs in regulating Dictyostelium motility. Additional support for SAC activity was given by the increase in frequency of Ca 2+ transients when Dictyostelium cells were moving on a more adhesive substratum or when they were mechanically stretched. We conclude that mechano-chemical signaling via SACs plays a major role in maintaining the rapid movement of Dictyostelium cells

Organic Electrochemical Transistors for the Detection of Cell Surface Glycans.

Science.gov (United States)

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

2018-05-23

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

Rapid Fatal Outcome from Pulmonary Arteries Compression in Transitional Cell Carcinoma

Directory of Open Access Journals (Sweden)

Ioannis A. Voutsadakis

2009-01-01

Full Text Available Transitional cell carcinoma of the urinary bladder is a malignancy that metastasizes frequently to lymph nodes including the mediastinal lymph nodes. This occurrence may produce symptoms due to compression of adjacent structures such as the superior vena cava syndrome or dysphagia from esophageal compression. We report the case of a 59-year-old man with metastatic transitional cell carcinoma for whom mediastinal lymphadenopathy led to pulmonary artery compression and a rapidly fatal outcome. This rare occurrence has to be distinguished from pulmonary embolism, a much more frequent event in cancer patients, in order that proper and prompt treatment be initiated.

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

Science.gov (United States)

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

2016-01-01

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

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

Science.gov (United States)

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

2002-08-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Zangi, Sepideh [Department of Chemical Engineering, Shahrood Branch, Islamic Azad University, P.O. Box 36155-163, Shahrood (Iran, Islamic Republic of); Hejazi, Iman [Department of Polymer Engineering & Color Technology, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Seyfi, Javad, E-mail: Jseyfi@gmail.com [Department of Chemical Engineering, Shahrood Branch, Islamic Azad University, P.O. Box 36155-163, Shahrood (Iran, Islamic Republic of); Hejazi, Ehsan [Department of Clinical Nutrition and Dietetics, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran (Iran, Islamic Republic of); Khonakdar, Hossein Ali [Department of Polymer Engineering, Faculty of Engineering, South Tehran Branch, Islamic Azad University, P.O. Box 19585-466, Tehran (Iran, Islamic Republic of); Davachi, Seyed Mohammad [School of Chemical Engineering, University of Tehran, P.O. Box 11155-4563, Tehran (Iran, Islamic Republic of)

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. - Highlights: • A novel method is presented for fabrication of superhydrophobic surfaces. • The presence of nanoparticles in non-solvent bath notably promoted phase separation. • Topography had a more notable impact on cell adhesion than superhydrophobicity. • Nano-scale topographical features highly impeded cell adhesion on polymer surfaces.

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

International Nuclear Information System (INIS)

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

2016-01-01

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. - Highlights: • A novel method is presented for fabrication of superhydrophobic surfaces. • The presence of nanoparticles in non-solvent bath notably promoted phase separation. • Topography had a more notable impact on cell adhesion than superhydrophobicity. • Nano-scale topographical features highly impeded cell adhesion on polymer surfaces.

Fabrication of cell container arrays with overlaid surface topographies.

Science.gov (United States)

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

2012-02-01

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

Large-volume constant-concentration sampling technique coupling with surface-enhanced Raman spectroscopy for rapid on-site gas analysis.

Science.gov (United States)

Zhang, Zhuomin; Zhan, Yisen; Huang, Yichun; Li, Gongke

2017-08-05

In this work, a portable large-volume constant-concentration (LVCC) sampling technique coupling with surface-enhanced Raman spectroscopy (SERS) was developed for the rapid on-site gas analysis based on suitable derivatization methods. LVCC sampling technique mainly consisted of a specially designed sampling cell including the rigid sample container and flexible sampling bag, and an absorption-derivatization module with a portable pump and a gas flowmeter. LVCC sampling technique allowed large, alterable and well-controlled sampling volume, which kept the concentration of gas target in headspace phase constant during the entire sampling process and made the sampling result more representative. Moreover, absorption and derivatization of gas target during LVCC sampling process were efficiently merged in one step using bromine-thiourea and OPA-NH 4 + strategy for ethylene and SO 2 respectively, which made LVCC sampling technique conveniently adapted to consequent SERS analysis. Finally, a new LVCC sampling-SERS method was developed and successfully applied for rapid analysis of trace ethylene and SO 2 from fruits. It was satisfied that trace ethylene and SO 2 from real fruit samples could be actually and accurately quantified by this method. The minor concentration fluctuations of ethylene and SO 2 during the entire LVCC sampling process were proved to be gas targets from real samples by SERS. Copyright © 2017 Elsevier B.V. All rights reserved.

Cells responding to surface structure of calcium phosphate ceramics for bone regeneration.

Science.gov (United States)

Zhang, Jingwei; Sun, Lanying; Luo, Xiaoman; Barbieri, Davide; de Bruijn, Joost D; van Blitterswijk, Clemens A; Moroni, Lorenzo; Yuan, Huipin

2017-11-01

Surface structure largely affects the inductive bone-forming potential of calcium phosphate (CaP) ceramics in ectopic sites and bone regeneration in critical-sized bone defects. Surface-dependent osteogenic differentiation of bone marrow stromal cells (BMSCs) partially explained the improved bone-forming ability of submicron surface structured CaP ceramics. In this study, we investigated the possible influence of surface structure on different bone-related cells, which may potentially participate in the process of improved bone formation in CaP ceramics. Besides BMSCs, the response of human brain vascular pericytes (HBVP), C2C12 (osteogenic inducible cells), MC3T3-E1 (osteogenic precursors), SV-HFO (pre-osteoblasts), MG63 (osteoblasts) and SAOS-2 (mature osteoblasts) to the surface structure was evaluated in terms of cell proliferation, osteogenic differentiation and gene expression. The cells were cultured on tricalcium phosphate (TCP) ceramics with either micron-scaled surface structure (TCP-B) or submicron-scaled surface structure (TCP-S) for up to 14 days, followed by DNA, alkaline phosphatase (ALP) and quantitative polymerase chain reaction gene assays. HBVP were not sensitive to surface structure with respect to cell proliferation and osteogenic differentiation, but had downregulated angiogenesis-related gene expression (i.e. vascular endothelial growth factor) on TCP-S. Without additional osteogenic inducing factors, submicron-scaled surface structure enhanced ALP activity and osteocalcin gene expression of human (h)BMSCs and C2C12 cells, favoured the proliferation of MC3T3-E1, MG63 and SAOS-2, and increased ALP activity of MC3T3-E1 and SV-HFO. The results herein indicate that cells with osteogenic potency (either osteogenic inducible cells or osteogenic cells) could be sensitive to surface structure and responded to osteoinductive submicron-structured CaP ceramics in cell proliferation, ALP production or osteogenic gene expression, which favour bone

Development of a cell microarray chip for detection of circulating tumor cells

Science.gov (United States)

Yamamura, S.; Yatsushiro, S.; Abe, K.; Baba, Y.; Kataoka, M.

2012-03-01

Detection of circulating tumor cells (CTCs) in the peripheral blood of metastatic cancer patients has clinical significance in earlier diagnosis of metastases. In this study, a novel cell microarray chip for accurate and rapid detection of tumor cells from human leukocytes was developed. The chip with 20,944 microchambers (105 μm diameter and 50 μm depth) was made from polystyrene, and the surface was rendered to hydrophilic by means of reactive-ion etching, which led to the formation of mono-layers of leukocytes on the microchambers. As the model of CTCs detection, we spiked human bronchioalveolar carcinoma (H1650) cells into human T lymphoblastoid leukemia (CEM) cells suspension and detected H1650 cells using the chip. A CEM suspension contained with H1650 cells was dispersed on the chip surface, followed by 10 min standing to allow the cells to settle down into the microchambers. About 30 CEM cells were accommodated in each microchamber, over 600,000 CEM cells in total being on a chip. We could detect 1 H1650 cell per 106 CEM cells on the microarray by staining with fluorescence-conjugated antibody (Anti-Cytokeratin) and cell membrane marker (DiD). Thus, this cell microarray chip has highly potential to be a novel tool of accurate and rapid detection of CTCs.

A novel, rapid and automated conductometric method to evaluate surfactant-cells interactions by means of critical micellar concentration analysis.

Science.gov (United States)

Tiecco, Matteo; Corte, Laura; Roscini, Luca; Colabella, Claudia; Germani, Raimondo; Cardinali, Gianluigi

2014-07-25

Conductometry is widely used to determine critical micellar concentration and micellar aggregates surface properties of amphiphiles. Current conductivity experiments of surfactant solutions are typically carried out by manual pipetting, yielding some tens reading points within a couple of hours. In order to study the properties of surfactant-cells interactions, each amphiphile must be tested in different conditions against several types of cells. This calls for complex experimental designs making the application of current methods seriously time consuming, especially because long experiments risk to determine alterations of cells, independently of the surfactant action. In this paper we present a novel, accurate and rapid automated procedure to obtain conductometric curves with several hundreds reading points within tens of minutes. The method was validated with surfactant solutions alone and in combination with Saccharomyces cerevisiae cells. An easy-to use R script, calculates conductometric parameters and their statistical significance with a graphic interface to visualize data and results. The validations showed that indeed the procedure works in the same manner with surfactant alone or in combination with cells, yielding around 1000 reading points within 20 min and with high accuracy, as determined by the regression analysis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

CELLISA: reporter cell-based immunization and screening of hybridomas specific for cell surface antigens.

Science.gov (United States)

Chen, Peter; Mesci, Aruz; Carlyle, James R

2011-01-01

Monoclonal antibodies (mAbs) specific for cell surface antigens are an invaluable tool to study immune receptor expression and function. Here, we outline a generalized reporter cell-based approach to the generation and high-throughput screening of mAbs specific for cell surface antigens. Termed CELLISA, this technology hinges upon the capture of hybridoma supernatants in mAb arrays that facilitate ligation of an antigen of interest displayed on BWZ reporter cells in the form of a CD3ζ-fusion chimeric antigen receptor (zCAR); in turn, specific mAb-mediated cross-linking of zCAR on BWZ cells results in the production of β-galactosidase enzyme (β-gal), which can be assayed colorimetrically. Importantly, the BWZ reporter cells bearing the zCAR of interest may be used for immunization as well as screening. In addition, serial immunizations employing additional zCAR- or native antigen-bearing cell lines can be used to increase the frequency of the desired antigen-specific hybridomas. Finally, the use of a cohort of epitope-tagged zCAR (e.g., zCAR(FLAG)) variants allows visualization of the cell surface antigen prior to immunization, and coimmunization using these variants can be used to enhance the immunogenicity of the target antigen. Employing the CELLISA strategy, we herein describe the generation of mAb directed against an uncharacterized natural killer cell receptor protein.

Laser-assisted modification of polystyrene surfaces for cell culture applications

International Nuclear Information System (INIS)

Pfleging, Wilhelm; Bruns, Michael; Welle, Alexander; Wilson, Sandra

2007-01-01

Laser-assisted patterning and modification of polystyrene (PS) was investigated with respect to applications in micro-fluidics and cell culture. For this purpose the wettability, the adsorption of proteins and the adhesion of animal cells were investigated as function of laser- and processing parameters. The change of surface chemistry was characterized by X-ray photoelectron spectroscopy. The local formation of chemical structures suitable for improved cell adhesion was realized on PS surfaces by UV laser irradiation. Above and below the laser ablation threshold two different mechanisms affecting cell adhesion were detected. In the first case the debris deposited on and along laser irradiated areas was responsible for improved cell adhesion, while in the second case a photolytic activation of the polymer surface including a subsequent oxidization in oxygen or ambient air is leading to a highly localized alteration of protein adsorption from cell culture media and finally to increased cell adhesion. Laser modifications of PS using suitable exposure doses and an appropriate choice of the processing gas (helium or oxygen) enabled a highly localized control of wetting. The dynamic advancing contact angle could be adjusted between 2 o and 150 o . The hydrophilic and hydrophobic behaviour are caused by chemical and topographical surface changes

Rapid characterization of the biomechanical properties of drug-treated cells in a microfluidic device

International Nuclear Information System (INIS)

Zhang, Xiaofei; Zhang, Yang; Bai, Guohua; Tan, Qiulin; Sun, Dong; Chu, Henry K; Wang, Kaiqun

2015-01-01

Cell mechanics is closely related to many cell functions. Recent studies have suggested that the deformability of cells can be an effective biomarker to indicate the onset and progression of diseases. In this paper, a microfluidic chip is designed for rapid characterization of the mechanics of drug-treated cells through stretching with dielectrophoresis (DEP) force. This chip was fabricated using PDMS and micro-electrodes were integrated and patterned on the ITO layer of the chip. Leukemia NB4 cells were considered and the effect of all-trans retinoic acid (ATRA) drug on NB4 cells were examined via the microfluidic chip. To induce a DEP force onto the cell, a relatively weak ac voltage was utilized to immobilize a cell at one side of the electrodes. The applied voltage was then increased to 3.5 V pp and the cell started to be stretched along the applied electric field lines. The elongation of the cell was observed using an optical microscope and the results showed that both types of cells were deformed by the induced DEP force. The strain of the NB4 cell without the drug treatment was recorded to be about 0.08 (time t = 180 s) and the drug-treated NB4 cell was about 0.21 (time t = 180 s), indicating a decrease in the stiffness after drug treatment. The elastic modulus of the cell was also evaluated and the modulus changed from 140 Pa to 41 Pa after drug treatment. This microfluidic chip can provide a simple and rapid platform for measuring the change in the biomechanical properties of cells and can potentially be used as the tool to determine the biomechanical effects of different drug treatments for drug discovery and development applications. (paper)

Efficient Isolation and Quantitative Proteomic Analysis of Cancer Cell Plasma Membrane Proteins for Identification of Metastasis-Associated Cell Surface Markers

DEFF Research Database (Denmark)

Lund, Rikke; Leth-Larsen, Rikke; Jensen, Ole N

2009-01-01

Cell surface membrane proteins are involved in central processes such as cell signaling, cell-cell interactions, ion and solute transport, and they seem to play a pivotal role in several steps of the metastatic process of cancer cells. The low abundance and hydrophobic nature of cell surface...... membrane proteins complicate their purification and identification by MS. We used two isogenic cell lines with opposite metastatic capabilities in nude mice to optimize cell surface membrane protein purification and to identify potential novel markers of metastatic cancer. The cell surface membrane...... proteins were isolated by centrifugation/ultracentrifugation steps, followed by membrane separation using a Percoll/sucrose density gradient. The gradient fractions containing the cell surface membrane proteins were identified by enzymatic assays. Stable isotope labeling of the proteome of the metastatic...

High resolution imaging of surface patterns of single bacterial cells

International Nuclear Information System (INIS)

Greif, Dominik; Wesner, Daniel; Regtmeier, Jan; Anselmetti, Dario

2010-01-01

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

An Update on Ocular Surface Epithelial Stem Cells: Cornea and Conjunctiva

Directory of Open Access Journals (Sweden)

Tiago Ramos

2015-01-01

Full Text Available The human ocular surface (front surface of the eye is formed by two different types of epithelia: the corneal epithelium centrally and the conjunctival epithelium that surrounds this. These two epithelia are maintained by different stem cell populations (limbal stem cells for the corneal epithelium and the conjunctival epithelial stem cells. In this review, we provide an update on our understanding of these epithelia and their stem cells systems, including embryology, new markers, and controversy around the location of these stem cells. We also provide an update on the translation of this understanding into clinical applications for the treatment of debilitating ocular surface diseases.

A microfluidics-based technique for automated and rapid labeling of cells for flow cytometry

International Nuclear Information System (INIS)

Patibandla, Phani K; Estrada, Rosendo; Kannan, Manasaa; Sethu, Palaniappan

2014-01-01

Flow cytometry is a powerful technique capable of simultaneous multi-parametric analysis of heterogeneous cell populations for research and clinical applications. In recent years, the flow cytometer has been miniaturized and made portable for application in clinical- and resource-limited settings. The sample preparation procedure, i.e. labeling of cells with antibodies conjugated to fluorescent labels, is a time consuming (∼45 min) and labor-intensive procedure. Microfluidics provides enabling technologies to accomplish rapid and automated sample preparation. Using an integrated microfluidic device consisting of a labeling and washing module, we demonstrate a new protocol that can eliminate sample handling and accomplish sample and reagent metering, high-efficiency mixing, labeling and washing in rapid automated fashion. The labeling module consists of a long microfluidic channel with an integrated chaotic mixer. Samples and reagents are precisely metered into this device to accomplish rapid and high-efficiency mixing. The mixed sample and reagents are collected in a holding syringe and held for up to 8 min following which the mixture is introduced into an inertial washing module to obtain ‘analysis-ready’ samples. The washing module consists of a high aspect ratio channel capable of focusing cells to equilibrium positions close to the channel walls. By introducing the cells and labeling reagents in a narrow stream at the center of the channel flanked on both sides by a wash buffer, the elution of cells into the wash buffer away from the free unbound antibodies is accomplished. After initial calibration experiments to determine appropriate ‘holding time’ to allow antibody binding, both modules were used in conjunction to label MOLT-3 cells (T lymphoblast cell line) with three different antibodies simultaneously. Results confirm no significant difference in mean fluorescence intensity values for all three antibodies labels (p < 0.01) between the

Influence of engineered surface on cell directionality and motility

International Nuclear Information System (INIS)

Tang, Qing Yuan; Pang, Stella W; Tong, Wing Yin; Shi, Peng; Lam, Yun Wah; Shi, Jue

2014-01-01

Control of cell migration is important in numerous key biological processes, and is implicated in pathological conditions such as cancer metastasis and inflammatory diseases. Many previous studies indicated that cell migration could be guided by micropatterns fabricated on cell culture surfaces. In this study, we designed a polydimethylsiloxane cell culture substrate with gratings punctuated by corners and ends, and studied its effects on the behavior of MC3T3-E1 osteoblast cells. MC3T3-E1 cells elongated and aligned with the gratings, and the migration paths of the cells appeared to be guided by the grating pattern. Interestingly, more than 88% of the cells cultured on these patterns were observed to reverse their migration directions at least once during the 16 h examination period. Most of the reversal events occurred at the corners and the ends of the pattern, suggesting these localized topographical features induce an abrupt loss in directional persistence. Moreover, the cell speed was observed to increase temporarily right after each directional reversal. Focal adhesion complexes were more well-established in cells on the angular gratings than on flat surfaces, but the formation of filipodia appeared to be imbalanced at the corners and the ends, possibly leading to the loss of directional persistence. This study describes the first engineered cell culture surface that consistently induces changes in the directional persistence of adherent cells. This will provide an experimental model for the study of this phenomenon and a valuable platform to control the cell motility and directionality, which can be used for cell screening and selection. (paper)

Adhesion of yeast cells on surface of polymers produced by radiation polymerization

International Nuclear Information System (INIS)

Lu, Zhaoxin; Takehisa, Masaaki; Xie Zongchuan.

1995-01-01

The adhesion of yeast (Saccharomyces formesences) cells on polymers was studied thermodynamically. The polymers were laminally prepared by means of radiation polymerization. By measuring contact angles, we calculated dispersion component and polar component of surface free energy of the polymers and the cells, and interfacial free energy between the polymer and the cells. Then interfacial free energy change of the cell adhesion to surface of the polymer was evaluated. The adhesion behavior of yeast cells on the polymers was observed by optical microscope. From above results, we conclude that the initial adhesion of the cells is related to the surface free energy of the polymer, but the irreversible adhesion may be close to the polar component in surface free energy. The high polar component is favourable the irreversible adhesion of yeast cells. (author)

Multiplex flow cytometry barcoding and antibody arrays identify surface antigen profiles of primary and metastatic colon cancer cell lines.

Directory of Open Access Journals (Sweden)

Kumar Sukhdeo

Full Text Available Colon cancer is a deadly disease affecting millions of people worldwide. Current treatment challenges include management of disease burden as well as improvements in detection and targeting of tumor cells. To identify disease state-specific surface antigen signatures, we combined fluorescent cell barcoding with high-throughput flow cytometric profiling of primary and metastatic colon cancer lines (SW480, SW620, and HCT116. Our multiplexed technique offers improvements over conventional methods by permitting the simultaneous and rapid screening of cancer cells with reduced effort and cost. The method uses a protein-level analysis with commercially available antibodies on live cells with intact epitopes to detect potential tumor-specific targets that can be further investigated for their clinical utility. Multiplexed antibody arrays can easily be applied to other tumor types or pathologies for discovery-based approaches to target identification.

Radioimmunoassay for antibodies against surface membrane antigens using adhering cells

Energy Technology Data Exchange (ETDEWEB)

Tax, A; Manson, L A [Wistar Inst. of Anatomy and Biology, Philadelphia, Pa. (USA)

1976-07-01

A radioimmunoassay using cells adhering to plastic is described. In this assay, A-10 mammary carcinoma attached to the surface of plastic in microtiter plates were permitted to bind antibody and the bound antibody was detected with purified rabbit /sup 125/I-antimouse-Fab. The bound radioactive material was eluted with glycine-HCl buffer (pH 2.5), and the acid eluates were counted in a gamma counter. This assay can be used to detect cytolic or noncytolic antibody to cell surface antigens in studies with any tumor or normal cell that will adhere to a solid surface.

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells

DEFF Research Database (Denmark)

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla

2009-01-01

: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression...... for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents...... agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates...

Lactobacillus plantarum gene clusters encoding putative cell-surface protein complexes for carbohydrate utilization are conserved in specific gram-positive bacteria

Directory of Open Access Journals (Sweden)

Muscariello Lidia

2006-05-01

Full Text Available Abstract Background Genomes of gram-positive bacteria encode many putative cell-surface proteins, of which the majority has no known function. From the rapidly increasing number of available genome sequences it has become apparent that many cell-surface proteins are conserved, and frequently encoded in gene clusters or operons, suggesting common functions, and interactions of multiple components. Results A novel gene cluster encoding exclusively cell-surface proteins was identified, which is conserved in a subgroup of gram-positive bacteria. Each gene cluster generally has one copy of four new gene families called cscA, cscB, cscC and cscD. Clusters encoding these cell-surface proteins were found only in complete genomes of Lactobacillus plantarum, Lactobacillus sakei, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Lactococcus lactis ssp lactis and Bacillus cereus and in incomplete genomes of L. lactis ssp cremoris, Lactobacillus casei, Enterococcus faecium, Pediococcus pentosaceus, Lactobacillius brevis, Oenococcus oeni, Leuconostoc mesenteroides, and Bacillus thuringiensis. These genes are neither present in the genomes of streptococci, staphylococci and clostridia, nor in the Lactobacillus acidophilus group, suggesting a niche-specific distribution, possibly relating to association with plants. All encoded proteins have a signal peptide for secretion by the Sec-dependent pathway, while some have cell-surface anchors, novel WxL domains, and putative domains for sugar binding and degradation. Transcriptome analysis in L. plantarum shows that the cscA-D genes are co-expressed, supporting their operon organization. Many gene clusters are significantly up-regulated in a glucose-grown, ccpA-mutant derivative of L. plantarum, suggesting catabolite control. This is supported by the presence of predicted CRE-sites upstream or inside the up-regulated cscA-D gene clusters. Conclusion We propose that the CscA, CscB, CscC and Csc

Cell-surface glycoproteins of human sarcomas: differential expression in normal and malignant tissues and cultured cells

International Nuclear Information System (INIS)

Rettig, W.F.; Garin-Chesa, P.; Beresford, H.R.; Oettgen, H.F.; Melamed, M.R.; Old, L.J.

1988-01-01

Normal differentiation and malignant transformation of human cells are characterized by specific changes in surface antigen phenotype. In the present study, the authors have defined six cell-surface antigens of human sarcomas and normal mesenchymal cells, by using mixed hemadsorption assays and immunochemical methods for the analysis of cultured cells and immunohistochemical staining for the analysis of normal tissues and > 200 tumor specimens. Differential patterns of F19, F24, G171, G253, S5, and Thy-1 antigen expression were found to characterize (i) subsets of cultured sarcoma cell lines, (ii) cultured fibroblasts derived from various organs, (iii) normal resting and activated mesenchymal tissues, and (iv) sarcoma and nonmesenchymal tumor tissues. These results provide a basic surface antigenic map for cultured mesenchymal cells and mesenchymal tissues and permit the classification of human sarcomas according to their antigenic phenotypes

Cell-surface proteoglycan in sea urchin primary mesenchyme cell migration

International Nuclear Information System (INIS)

Lane, M.C.

1989-01-01

Early in the development of the sea urchin embryo, the primary mesenchyme cells (PMC) migrate along the basal lamina of the blastocoel. Migration is inhibited in L. pictus embryos cultured in sulfate-free seawater and in S. purpuratus embryos exposed to exogenous β-D-xylosides. An in vitro assay was developed to test the migratory capacity of normal PMC on normal and treated blastocoelic matrix. Sulfate deprivation and exposure to exogenous xyloside render PMC nonmotile on either matrix. Materials removed from the surface of normal PMC by treatment with 1 M urea restored migratory ability to defective cells, whereas a similar preparation isolated from the surface of epithelial cells at the same stage did not. Migration also resumed when cells were removed from the xyloside or returned to normal seawater. The urea extract was partially purified and characterized by radiolabeling, gel electrophoresis, fluorography, ion exchange chromatography, and western blotting. The PMC synthesize a large chondroitin sulfate/dermatan sulfate proteoglycan that is present in an active fraction isolated by chromatography. Chondroitinase ABC digestion of live cells blocked migration reversibly, further supporting the identification of the chondroitin sulfate/dermatan sulfate proteoglycan as the active component in the urea extract. Much of the incorporated sulfate was distributed along the filopodia in 35 SO 4 -labelled PMC by autoradiography. The morphology of normal and treated S. purpuratus PMC was examined by scanning electron microscopy, and differences in spreading, particularly of the extensive filopodia present on the cells, was observed. A model for the role of the chondroitin sulfate/dermatan sulfate proteoglycan in cell detachment during migration is proposed

Cell surface engineering with polyelectrolyte multilayer thin films.

Science.gov (United States)

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

2011-05-11

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

Surface modification of Polycaprolactone (PCL) microcarrier for performance improvement of human skin fibroblast cell culture

Science.gov (United States)

Samsudin, N.; Hashim, Y. Z. H.; Arifin, M. A.; Mel, M.; Salleh, H. Mohd; Sopyan, I.; Hamid, M. Abdul

2018-01-01

Polycaprolactone (PCL) has many advantages for use in biomedical engineering field. In the present work PCL microcarriers of 150-200 μm were fabricated using oil-in-water (o/w) emulsification coupled with solvent evaporation method. The surface charge of PCL microcarrier was then been improved by using ultraviolet/ozone treatment to introduce oxygen functional group. Immobilisation of gelatin onto PCL microspheres using zero-length crosslinker provides a stable protein-support complex, with no diffusional barrier which is ideal for mass processing. The optimum concentration of carboxyl group (COOH) absorbed on the surface was 1495.9 nmol/g and the amount of gelatin immobilized was 1797.3 μg/g on UV/O3 treated microcarriers as compared to the untreated (320 μg/g) microcarriers. The absorption of functional oxygen groups on the surface and the immobilized gelatin was confirmed with Fourier Transformed Infrared spectroscopy and the enhancement of hydrophilicity of the surface was confirmed using water contact angle measurement which decreased (86.93° - 49.34°) after UV/O3 treatment and subsequently after immobilisation of gelatin. The attachment and growth kinetics for human skin fibroblast cell (HSFC) showed that adhesion occurred much more rapidly for gelatin immobilised surface as compared to untreated PCL and UV/O3 PCL microcarrier.

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

Science.gov (United States)

Wheatley Myerson, Jacob; He, Li; Allen, John Stacy; Williams, Todd; Lanza, Gregory; Tollefsen, Douglas; Caruthers, Shelton; Wickline, Samuel

2014-09-01

Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (˜10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes.

Thrombin-inhibiting nanoparticles rapidly constitute versatile and detectable anticlotting surfaces

International Nuclear Information System (INIS)

Myerson, Jacob Wheatley; Lanza, Gregory; Caruthers, Shelton; Wickline, Samuel; He, Li; Allen, John Stacy; Williams, Todd; Tollefsen, Douglas

2014-01-01

Restoring an antithrombotic surface to suppress ongoing thrombosis is an appealing strategy for treatment of acute cardiovascular disorders such as erosion of atherosclerotic plaque. An antithrombotic surface would present an alternative to systemic anticoagulation with attendant risks of bleeding. We have designed thrombin-targeted nanoparticles (NPs) that bind to sites of active clotting to extinguish local thrombin activity and inhibit platelet deposition while exhibiting only transient systemic anticoagulant effects. Perfluorocarbon nanoparticles (PFC NP) were functionalized with thrombin inhibitors (either D-phenylalanyl-L-prolyl-L-arginyl-chloromethyl ketone or bivalirudin) by covalent attachment of more than 15 000 inhibitors to each PFC NP. Fibrinopeptide A (FPA) ELISA demonstrated that thrombin-inhibiting NPs prevented cleavage of fibrinogen by both free and clot-bound thrombin. Magnetic resonance imaging (MRI) confirmed that a layer of thrombin-inhibiting NPs prevented growth of clots in vitro. Thrombin-inhibiting NPs were administered in vivo to C57BL6 mice subjected to laser injury of the carotid artery. NPs significantly delayed thrombotic occlusion of the artery, whereas an equivalent bolus of free inhibitor was ineffective. For thrombin-inhibiting NPs, only a short-lived (∼10 min) systemic effect on bleeding time was observed, despite prolonged clot inhibition. Imaging and quantification of in vivo antithrombotic NP layers was demonstrated by MRI of the PFC NP. 19 F MRI confirmed colocalization of particles with arterial thrombi, and quantitative 19 F spectroscopy demonstrated specific binding and retention of thrombin-inhibiting NPs in injured arteries. The ability to rapidly form and image a new antithrombotic surface in acute vascular syndromes while minimizing risks of bleeding would permit a safer method of passivating active lesions than current systemic anticoagulant regimes. (paper)

Rapid road repair vehicle

Science.gov (United States)

Mara, Leo M.

1998-01-01

Disclosed is a rapid road repair vehicle capable of moving over a surface to be repaired at near normal posted traffic speeds to scan for and find an the high rate of speed, imperfections in the pavement surface, prepare the surface imperfection for repair by air pressure and vacuum cleaning, applying a correct amount of the correct patching material to effect the repair, smooth the resulting repaired surface, and catalog the location and quality of the repairs for maintenance records of the road surface. The rapid road repair vehicle can repair surface imperfections at lower cost, improved quality, at a higher rate of speed than was was heretofor possible, with significantly reduced exposure to safety and health hazards associated with this kind of road repair activities in the past.

Comparing rapid methods for detecting Listeria in seafood and environmental samples using the most probably number (MPN) technique.

Science.gov (United States)

Cruz, Cristina D; Win, Jessicah K; Chantarachoti, Jiraporn; Mutukumira, Anthony N; Fletcher, Graham C

2012-02-15

The standard Bacteriological Analytical Manual (BAM) protocol for detecting Listeria in food and on environmental surfaces takes about 96 h. Some studies indicate that rapid methods, which produce results within 48 h, may be as sensitive and accurate as the culture protocol. As they only give presence/absence results, it can be difficult to compare the accuracy of results generated. We used the Most Probable Number (MPN) technique to evaluate the performance and detection limits of six rapid kits for detecting Listeria in seafood and on an environmental surface compared with the standard protocol. Three seafood products and an environmental surface were inoculated with similar known cell concentrations of Listeria and analyzed according to the manufacturers' instructions. The MPN was estimated using the MPN-BAM spreadsheet. For the seafood products no differences were observed among the rapid kits and efficiency was similar to the BAM method. On the environmental surface the BAM protocol had a higher recovery rate (sensitivity) than any of the rapid kits tested. Clearview™, Reveal®, TECRA® and VIDAS® LDUO detected the cells but only at high concentrations (>10(2) CFU/10 cm(2)). Two kits (VIP™ and Petrifilm™) failed to detect 10(4) CFU/10 cm(2). The MPN method was a useful tool for comparing the results generated by these presence/absence test kits. There remains a need to develop a rapid and sensitive method for detecting Listeria in environmental samples that performs as well as the BAM protocol, since none of the rapid tests used in this study achieved a satisfactory result. Copyright © 2011 Elsevier B.V. All rights reserved.

Rapid detection of bacterial contamination in cell or tissue cultures based on Raman spectroscopy

Science.gov (United States)

Bolwien, Carsten; Sulz, Gerd; Becker, Sebastian; Thielecke, Hagen; Mertsching, Heike; Koch, Steffen

2008-02-01

Monitoring the sterility of cell or tissue cultures is an essential task, particularly in the fields of regenerative medicine and tissue engineering when implanting cells into the human body. We present a system based on a commercially available microscope equipped with a microfluidic cell that prepares the particles found in the solution for analysis, a Raman-spectrometer attachment optimized for non-destructive, rapid recording of Raman spectra, and a data acquisition and analysis tool for identification of the particles. In contrast to conventional sterility testing in which samples are incubated over weeks, our system is able to analyze milliliters of supernatant or cell suspension within hours by filtering relevant particles and placing them on a Raman-friendly substrate in the microfluidic cell. Identification of critical particles via microscopic imaging and subsequent image analysis is carried out before micro-Raman analysis of those particles is then carried out with an excitation wavelength of 785 nm. The potential of this setup is demonstrated by results of artificial contamination of samples with a pool of bacteria, fungi, and spores: single-channel spectra of the critical particles are automatically baseline-corrected without using background data and classified via hierarchical cluster analysis, showing great promise for accurate and rapid detection and identification of contaminants.

Evaluation of Relative Yeast Cell Surface Hydrophobicity Measured by Flow Cytometry

Directory of Open Access Journals (Sweden)

Lisa Colling

2005-01-01

Full Text Available Objective: To develop an efficient method for evaluating cell surface hydrophobicity and to apply the method to demonstrate the effects of fungal growth conditions on cell surface properties.

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

Science.gov (United States)

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

2015-09-30

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

Rapid-mixing studies on the time-scale of radiation damage in cells

International Nuclear Information System (INIS)

Adams, G.E.; Michael, B.D.; Asquith, J.C.; Shenoy, M.A.; Watts, M.E.; Whillans, D.W.

1975-01-01

Rapid mixing studies were performed to determine the time scale of radiation damage in cells. There is evidence that the sensitizing effects of oxygen and other chemical dose-modifying agents on the response of cells to ionizing radiation involve fast free-radical processes. Fast response technique studies in bacterial systems have shown that extremely fast processes occur when the bacteria are exposed to oxygen or other dose-modifying agents during irradiation. The time scales observed were consistent with the involvement of fast free-radical reactions in the expression of these effects

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

Science.gov (United States)

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.

Pure and Oxidized Copper Materials as Potential Antimicrobial Surfaces for Spaceflight Activities

Science.gov (United States)

Hahn, C.; Hans, M.; Hein, C.; Mancinelli, R. L.; Mücklich, F.; Wirth, R.; Rettberg, P.; Hellweg, C. E.; Moeller, R.

2017-12-01

Microbial biofilms can lead to persistent infections and degrade a variety of materials, and they are notorious for their persistence and resistance to eradication. During long-duration space missions, microbial biofilms present a danger to crew health and spacecraft integrity. The use of antimicrobial surfaces provides an alternative strategy for inhibiting microbial growth and biofilm formation to conventional cleaning procedures and the use of disinfectants. Antimicrobial surfaces contain organic or inorganic compounds, such as antimicrobial peptides or copper and silver, that inhibit microbial growth. The efficacy of wetted oxidized copper layers and pure copper surfaces as antimicrobial agents was tested by applying cultures of Escherichia coli and Staphylococcus cohnii to these metallic surfaces. Stainless steel surfaces were used as non-inhibitory control surfaces. The production of reactive oxygen species and membrane damage increased rapidly within 1 h of exposure on pure copper surfaces, but the effect on cell survival was negligible even after 2 h of exposure. However, longer exposure times of up to 4 h led to a rapid decrease in cell survival, whereby the survival of cells was additionally dependent on the exposed cell density. Finally, the release of metal ions was determined to identify a possible correlation between copper ions in suspension and cell survival. These measurements indicated a steady increase of free copper ions, which were released indirectly by cells presumably through excreted complexing agents. These data indicate that the application of antimicrobial surfaces in spaceflight facilities could improve crew health and mitigate material damage caused by microbial contamination and biofilm formation. Furthermore, the results of this study indicate that cuprous oxide layers were superior to pure copper surfaces related to the antimicrobial effect and that cell density is a significant factor that influences the time dependence of

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

Science.gov (United States)

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

2012-01-01

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

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

Science.gov (United States)

Beranek, Maggi Marie

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

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

International Nuclear Information System (INIS)

Hiraiwa, Morgan; Lee, Hyun-Boo; Inoue, Shinnosuke; Chung, Jae-Hyun; Kim, Jong-Hoon; Becker, Annie L; Weigel, Kris M; Cangelosi, Gerard A; Lee, Kyong-Hoon

2015-01-01

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL −1 , comparable to a more labor-intensive fluorescence detection method reported previously. (paper)

Amperometric immunosensor for rapid detection of Mycobacterium tuberculosis

Science.gov (United States)

Hiraiwa, Morgan; Kim, Jong-Hoon; Lee, Hyun-Boo; Inoue, Shinnosuke; Becker, Annie L.; Weigel, Kris M.; Cangelosi, Gerard A.; Lee, Kyong-Hoon; Chung, Jae-Hyun

2015-05-01

Tuberculosis (TB) has been a major public health problem, which can be better controlled by using accurate and rapid diagnosis in low-resource settings. A simple, portable, and sensitive detection method is required for point-of-care (POC) settings. This paper studies an amperometric biosensor using a microtip immunoassay for a rapid and low-cost detection of Mycobacterium tuberculosis (MTB) in sputum. MTB in sputum is specifically captured on the functionalized microtip surface and detected by electric current. According to the numerical study, the current signal on the microtip surface is linearly changed with increasing immersion depth. Using a reference microtip, the immersion depth is compensated for a sensing microtip. On the microtip surface, target bacteria are concentrated and organized by a coffee-ring effect, which amplifies the electric current. To enhance the signal-to-noise ratio, both the sample processing and rinsing steps are presented with the use of deionized water as a medium for the amperometric measurement. When applied to cultured MTB cells spiked into human sputum, the detection limit was 100 CFU mL-1, comparable to a more labor-intensive fluorescence detection method reported previously.

Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers

Energy Technology Data Exchange (ETDEWEB)

Koufaki, Niki; Ranella, Anthi; Barberoglou, Marios; Psycharakis, Stylianos; Fotakis, Costas; Stratakis, Emmanuel [Institute of Electronic Structure and Laser (IESL), Foundation for Research and Technology-Hellas (FORTH), 711 10, Heraklion, Crete (Greece); Aifantis, Katerina E, E-mail: stratak@iesl.forth.gr [Lab of Mechanics and Materials, Aristotle University of Thessaloniki, Thessaloniki (Greece)

2011-12-15

The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity.

Controlling cell adhesion via replication of laser micro/nano-textured surfaces on polymers

International Nuclear Information System (INIS)

Koufaki, Niki; Ranella, Anthi; Barberoglou, Marios; Psycharakis, Stylianos; Fotakis, Costas; Stratakis, Emmanuel; Aifantis, Katerina E

2011-01-01

The aim of this study is to investigate cell adhesion and viability on highly rough polymeric surfaces with gradient roughness ratios and wettabilities prepared by microreplication of laser micro/nano-textured Si surfaces. Negative replicas on polydimethylsiloxane as well as positive ones on a photocurable (organically modified ceramic) and a biodegradable (poly(lactide-co-glycolide)) polymer have been successfully reproduced. The final culture substrates comprised from forests of micron-sized conical spikes exhibiting a range of roughness ratios and wettabilities, was achieved by changing the laser fluence used to fabricate the original template surfaces. Cell culture experiments were performed with the fibroblast NIH/3T3 and PC12 neuronal cell lines in order to investigate how these surfaces are capable of modulating different types of cellular responses including, viability, adhesion and morphology. The results showed a preferential adhesion of both cell types on the microstructured surfaces compared to the unstructured ones. In particular, the fibroblast NIH/3T3 cells show optimal adhesion for small roughness ratios, independent of the surface wettability and polymer type, indicating a non-monotonic dependence of cell adhesion on surface energy. In contrast, the PC12 cells were observed to adhere well to the patterned surfaces independent of the roughness ratio and wettability. These experimental findings are correlated with micromechanical measurements performed on the unstructured and replicated surfaces and discussed on the basis of previous observations describing the relation of cell response to surface energy and rigidity.

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

Science.gov (United States)

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

2011-01-01

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

Rapid identification of bacterial resistance to Ciprofloxacin using surface-enhanced Raman spectroscopy

Science.gov (United States)

Kastanos, Evdokia; Hadjigeorgiou, Katerina; Pitris, Costas

2014-02-01

Due to its effectiveness and broad coverage, Ciprofloxacin is the fifth most prescribed antibiotic in the US. As current methods of infection diagnosis and antibiotic sensitivity testing (i.e. an antibiogram) are very time consuming, physicians prescribe ciprofloxacin before obtaining antibiogram results. In order to avoid increasing resistance to the antibiotic, a method was developed to provide both a rapid diagnosis and the sensitivity to the antibiotic. Using Surface Enhanced Raman Spectroscopy, an antibiogram was obtained after exposing the bacteria to Ciprofloxacin for just two hours. Spectral analysis revealed clear separation between sensitive and resistant bacteria and could also offer some inside into the mechanisms of resistance.

A rapid chemical method for lysing Arabidopsis cells for protein analysis

Directory of Open Access Journals (Sweden)

Takano Tetsuo

2011-07-01

Full Text Available Abstract Background Protein extraction is a frequent procedure in biological research. For preparation of plant cell extracts, plant materials usually have to be ground and homogenized to physically break the robust cell wall, but this step is laborious and time-consuming when a large number of samples are handled at once. Results We developed a chemical method for lysing Arabidopsis cells without grinding. In this method, plants are boiled for just 10 minutes in a solution containing a Ca2+ chelator and detergent. Cell extracts prepared by this method were suitable for SDS-PAGE and immunoblot analysis. This method was also applicable to genomic DNA extraction for PCR analysis. Our method was applied to many other plant species, and worked well for some of them. Conclusions Our method is rapid and economical, and allows many samples to be prepared simultaneously for protein analysis. Our method is useful not only for Arabidopsis research but also research on certain other species.

Development of exosome surface display technology in living human cells

International Nuclear Information System (INIS)

Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

2016-01-01

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

Development of exosome surface display technology in living human cells

Energy Technology Data Exchange (ETDEWEB)

Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu; Zhang, Zhiwen, E-mail: zzhang@scu.edu; Lu, Biao, E-mail: blu2@scu.edu

2016-03-25

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

Rapid and non-enzymatic in vitro retrieval of tumour cells from surgical specimens.

Directory of Open Access Journals (Sweden)

Brigitte Mack

Full Text Available The study of tumourigenesis commonly involves the use of established cell lines or single cell suspensions of primary tumours. Standard methods for the generation of short-term tumour cell cultures include the disintegration of tissue based on enzymatic and mechanical stress. Here, we describe a simple and rapid method for the preparation of single cells from primary carcinomas, which is independent of enzymatic treatment and feeder cells. Tumour biopsies are processed to 1 mm(3 cubes termed explants, which are cultured 1-3 days on agarose-coated well plates in specified medium. Through incisions generated in the explants, single cells are retrieved and collected from the culture supernatant and can be used for further analysis including in vitro and in vivo studies. Collected cells retain tumour-forming capacity in xenotransplantation assays, mimic the phenotype of the primary tumour, and facilitate the generation of cell lines.

Surface characteristics determining the cell compatibility of ionically cross-linked alginate gels

International Nuclear Information System (INIS)

Machida-Sano, Ikuko; Hirakawa, Makoto; Matsumoto, Hiroki; Kamada, Mitsuki; Ogawa, Sakito; Satoh, Nao; Namiki, Hideo

2014-01-01

In this study we investigated differences in the characteristics determining the suitability of five types of ion (Fe 3+ , Al 3+ , Ca 2+ , Ba 2+ and Sr 2+ )-cross-linked alginate films as culture substrates for cells. Human dermal fibroblasts were cultured on each alginate film to examine the cell affinity of the alginates. Since cell behavior on the surface of a material is dependent on the proteins adsorbed to it, we investigated the protein adsorption ability and surface features (wettability, morphology and charge) related to the protein adsorption abilities of alginate films. We observed that ferric, aluminum and barium ion-cross-linked alginate films supported better cell growth and adsorbed higher amounts of serum proteins than other types. Surface wettability analysis demonstrated that ferric and aluminum ion-cross-linked alginates had moderate hydrophilic surfaces, while other types showed highly hydrophilic surfaces. The roughness was exhibited only on barium ion-cross-linked alginate surface. Surface charge measurements revealed that alginate films had negatively charged surfaces, and showed little difference among the five types of gel. These results indicate that the critical factors of ionically cross-linked alginate films determining the protein adsorption ability required for their cell compatibility may be surface wettability and morphology. (paper)

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

Science.gov (United States)

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

2017-09-18

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

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

International Nuclear Information System (INIS)

Zhu, Haiyan; Wang, Lan; Ruan, Yuanyuan; Zhou, Lei; Zhang, Dongmei; Min, Zhihui; Xie, Jianhui; Yu, Min; Gu, Jianxin

2011-01-01

Research highlights: → Hsp60 transported to cell surface through the classical secretory pathway was modified with N-glycosylation. → HSAPB-N18 could efficiently deliver Hsp60 to the cell surface via the unconventional secretory pathway. → Cell surface Hsp60 delivered by HASPB-N18 has a proper conformation. → 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 found 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.

Robotic Patterning a Superhydrophobic Surface for Collective Cell Migration Screening.

Science.gov (United States)

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

Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces.

Science.gov (United States)

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

1985-09-01

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

Mg ion implantation on SLA-treated titanium surface and its effects on the behavior of mesenchymal stem cell

International Nuclear Information System (INIS)

Kim, Beom-Su; Kim, Jin Seong; Park, Young Min; Choi, Bo-Young; Lee, Jun

2013-01-01

Magnesium (Mg) is one of the most important ions associated with bone osseointegration. The aim of this study was to evaluate the cellular effects of Mg implantation in titanium (Ti) surfaces treated with sand blast using large grit and acid etching (SLA). Mg ions were implanted into the surface via vacuum arc source ion implantation. The surface morphology, chemical properties, and the amount of Mg ion release were evaluated by scanning electron microscopy (SEM), Auger electron spectroscopy (AES), Rutherford backscattering spectroscopy (RBS), and inductively coupled plasma-optical emission spectrometer (ICP-OES). Human mesenchymal stem cells (hMSCs) were used to evaluate cellular parameters such as proliferation, cytotoxicity, and adhesion morphology by MTS assay, live/dead assay, and SEM. Furthermore, osteoblast differentiation was determined on the basis of alkaline phosphatase (ALP) activity and the degree of calcium accumulation. In the Mg ion-implanted disk, 2.3 × 10 16 ions/cm 2 was retained. However, after Mg ion implantation, the surface morphology did not change. Implanted Mg ions were rapidly released during the first 7 days in vitro. The MTS assay, live/dead assay, and SEM demonstrated increased cell attachment and growth on the Mg ion-implanted surface. In particular, Mg ion implantation increased the initial cell adhesion, and in an osteoblast differentiation assay, ALP activity and calcium accumulation. These findings suggest that Mg ion implantation using the plasma source ion implantation (PSII) technique may be useful for SLA-treated Ti dental implants to improve their osseointegration capacity. - Highlights: ► Mg ion was coated onto surface of SLA treated titanium via vacuum arc source ion implantation method. ► The morphological characteristics did not change after Mg ion implantation. ► Mg ion implanted SLA Ti is highly cytocompatible. ► Initial cell adhesion of MSCs is improved by Mg ion implantation. ► Mg ion implantation improved

Rapid Surface Sampling and Archival Record (RSSAR) system. Final report, October 1995 - May 1997

International Nuclear Information System (INIS)

1998-01-01

This report describes the results of Phase 2 efforts to develop a Rapid Surface Sampling and Archival Record (RSSAR) System for the detection of semivolatile organic contaminants on concrete, transite, and metal surfaces. The characterization of equipment and building surfaces for the presence of contaminants as part of building decontamination and decommissioning activities is an immensely large task of concern to both government and industry. Because of the high cost of hazardous waste disposal, old, contaminated buildings cannot simply be demolished and scrapped. Contaminated and clean materials must be clearly identified and segregated so that the clean material can be recycled or reused, if possible, or disposed of more cheaply as nonhazardous waste. DOE has a number of sites requiring surface characterization. These sites are large, contain very heterogeneous patterns of contamination (requiring high sampling density), and will thus necessitate an enormous number of samples to be taken and analyzed. Characterization of building and equipment surfaces will be needed during initial investigations, during cleanup operations, and during the final confirmation process, increasing the total number of samples well beyond that needed for initial characterization. This multiplicity of information places a premium on the ability to handle and track data as efficiently as possible

Rapid Surface Sampling and Archival Record (RSSAR) system. Final report, October 1995--May 1997

Energy Technology Data Exchange (ETDEWEB)

NONE

1998-12-31

This report describes the results of Phase 2 efforts to develop a Rapid Surface Sampling and Archival Record (RSSAR) System for the detection of semivolatile organic contaminants on concrete, transite, and metal surfaces. The characterization of equipment and building surfaces for the presence of contaminants as part of building decontamination and decommissioning activities is an immensely large task of concern to both government and industry. Because of the high cost of hazardous waste disposal, old, contaminated buildings cannot simply be demolished and scrapped. Contaminated and clean materials must be clearly identified and segregated so that the clean material can be recycled or reused, if possible, or disposed of more cheaply as nonhazardous waste. DOE has a number of sites requiring surface characterization. These sites are large, contain very heterogeneous patterns of contamination (requiring high sampling density), and will thus necessitate an enormous number of samples to be taken and analyzed. Characterization of building and equipment surfaces will be needed during initial investigations, during cleanup operations, and during the final confirmation process, increasing the total number of samples well beyond that needed for initial characterization. This multiplicity of information places a premium on the ability to handle and track data as efficiently as possible.

Polycarbonate surface cell's adhesion examination after Nd:YAG laser irradiation

Energy Technology Data Exchange (ETDEWEB)

Ramazani, S.A. Ahmad, E-mail: Ramazani@sharif.ir [Polymer Group, Department of Chemical and Petroleum Engineering, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Mousavi, Seyyed Abbas, E-mail: Musavi@che.sharif.ir [Department of Chemistry, Sharif University of Technology, Tehran (Iran, Islamic Republic of); Seyedjafari, Ehsan [Department of Biotechnology, University College of Science, University of Tehran (Iran, Islamic Republic of); Poursalehi, Reza [Department of Physics, University of Shahed, Tehran (Iran, Islamic Republic of); Sareh, Shohreh [Research Center of Iranian Blood Transfusion Organization, Tehran (Iran, Islamic Republic of); Silakhori, Kaveh [Laser Research Center, Atomic Energy Organization, Tehran (Iran, Islamic Republic of); Poorfatollah, Ali Akbar [Research Center of Iranian Blood Transfusion Organization, Tehran (Iran, Islamic Republic of); Shamkhali, Amir Nasser [Department of Chemistry, Sharif University of Technology, Tehran (Iran, Islamic Republic of)

2009-05-05

Nd:YAG laser treatment was used in order to increase surface cell adhesion aspects of polycarbonate (PC) films prepared via melt process. The treatment was carried out under different wavelengths and beam diameters. ATR-FTIR and UV spectra obtained from different samples before and after laser treatment in air showed that laser irradiation has induced some chemical and physical changes in surface properties. The irradiated films were also characterized using scanning electron microscopy (SEM) and contact angle measurements. Effect of pulse numbers on the surface properties was also investigated. Cell culture test was used to evaluate cell adhesion property on the PC films before and after treatment. The results obtained from this test showed that after laser treatment, the cells were attached and proliferated extensively on the Nd:YAG laser treated films in comparison with the unmodified PC. Moreover, it was revealed that a decrease in the laser beam diameter and an increase in the irradiated pulse numbers increased surface wettability and caused a better cell attachment on the polymer surface. The obtained results also showed that a decrease in the laser beam diameter and an increase in the irradiated pulse numbers increased surface wettability and caused a better cell attachment on the polymer surface.

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

Science.gov (United States)

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

2016-09-15

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

Effect of the back surface topography on the efficiency in silicon solar cells

International Nuclear Information System (INIS)

Guo Aijuan; Ye Famin; Feng Shimeng; Guo Lihui; Ji Dong

2009-01-01

Different processes are used on the back surface of silicon wafers to form cells falling into three groups: textured, planar, and sawed-off pyramid back surface. The characteristic parameters of the cells, I SC , V OC , FF, Pm, and E ff , are measured. All these parameters of the planar back surface cells are the best. The FF, Pm, and E ff of sawed-off pyramid back surface cells are superior to textured back surface cells, although I SC and V OC are lower. The parasitic resistance is analyzed to explain the higher FF of the sawed-off pyramid back surface cells. The cross-section scanning electron microscopy (SEM) pictures show the uniformity of the aluminum-silicon alloy, which has an important effect on the back surface recombination velocity and the ohmic contact. The measured value of the aluminum back surface field thickness in the SEM picture is in good agreement with the theoretical value deduced from the Al-Si phase diagram. It is shown in an external quantum efficiency (EQE) diagram that the planar back surface has the best response to a wavelength between 440 and 1000 nm and the sawed-off back surface has a better long wavelength response.

Cell surface glycan alterations in epithelial mesenchymal transition process of Huh7 hepatocellular carcinoma cell.

Directory of Open Access Journals (Sweden)

Shan Li

Full Text Available BACKGROUND AND OBJECTIVE: Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC is high. It is well known that the epithelial mesenchymal transition (EMT and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. METHODOLOGY: HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. RESULTS: After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. CONCLUSIONS: The findings of this study systematically clarify the alterations of cell surface

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

International Nuclear Information System (INIS)

Zhang, Xiaojun; Chen, Yuan; Chen, Yong

2014-01-01

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

Display of wasp venom allergens on the cell surface of Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Borodina, Irina; Jensen, B. M.; Søndergaard, Ib

2010-01-01

Background: Yeast surface display is a technique, where the proteins of interest are expressed as fusions with yeast surface proteins and thus remain attached to the yeast cell wall after expression. Our purpose was to study whether allergens expressed on the cell surface of baker's yeast...... were expressed on the surface as fusions with a-agglutinin complex protein AGA2. The expression was confirmed by fluorescent cytometry (FACS) after staining the cells with antibody against a C-tag attached to the C-terminal end of the allergens. Phospholipase A1 and hyaluronidase retained...... their enzymatic activities. Phospholipase A1 severely inhibited the growth of the yeast cells. Antigen 5 - expressing yeast cells bound IgE antibodies from wasp venom allergic patient sera but not from control sera as demonstrated by FACS. Moreover, antigen 5 - expressing yeast cells were capable of mediating...

Rapid outer-surface protein C DNA tattoo vaccination protects against Borrelia afzelii infection.

Science.gov (United States)

Wagemakers, A; Mason, L M K; Oei, A; de Wever, B; van der Poll, T; Bins, A D; Hovius, J W R

2014-12-01

Borrelia afzelii is the predominant Borrelia species causing Lyme borreliosis in Europe. Currently there is no human vaccine against Lyme borreliosis, and most research focuses on recombinant protein vaccines against Borrelia burgdorferi sensu stricto. DNA tattooing is a novel vaccination method that can be applied in a rapid vaccination schedule. We vaccinated C3H/HeN mice with B. afzelii strain PKo OspC (outer-surface protein C) using a codon-optimized DNA vaccine tattoo and compared this with recombinant protein vaccination in a 0-2-4 week vaccination schedule. We also assessed protection by DNA tattoo in a 0-3-6 day schedule. DNA tattoo and recombinant OspC vaccination induced comparable total IgG responses, with a lower IgG1/IgG2a ratio after DNA tattoo. Two weeks after syringe-challenge with 5 × 10(5) B. afzelii spirochetes most vaccinated mice had negative B. afzelii tissue DNA loads and all were culture negative. Furthermore, DNA tattoo vaccination in a 0-3-6 day regimen also resulted in negative Borrelia loads and cultures after challenge. To conclude, DNA vaccination by tattoo was fully protective against B. afzelii challenge in mice in a rapid vaccination protocol, and induces a favorable humoral immunity compared to recombinant protein vaccination. Rapid DNA tattoo is a promising vaccination strategy against spirochetes.

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

Science.gov (United States)

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

2015-01-01

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

The rapid evolution of CT findings in pulmonary langerhans cell histiocytosis: a case report

International Nuclear Information System (INIS)

Kang, Tae Wook; Lee, Kyung Soo; Cho, Eun Yoon

2007-01-01

Imaging findings of pulmonary Langerhans cell histiocytosis (PLCH) demonstrate evolving changes over time, and the radiological transitions shown by imaging tools may allow a prediction of histopathological activity in PLCH. However, there are no reports describing how rapidly CT findings change with time. We describe a case of PLCH that showed a rapid evolutional change of the pulmonary lesions in a 48-year-old man, in which the nodular lesions showed cystic changes within two-month follow-up periods on chest CT scans

Surface plasmon resonance based biosensor: A new platform for rapid diagnosis of livestock diseases

Directory of Open Access Journals (Sweden)

Pravas Ranjan Sahoo

2016-12-01

Full Text Available Surface plasmon resonance (SPR based biosensors are the most advanced and developed optical label-free biosensor technique used for powerful detection with vast applications in environmental protection, biotechnology, medical diagnostics, drug screening, food safety, and security as well in livestock sector. The livestock sector which contributes the largest economy of India, harbors many bacterial, viral, and fungal diseases impacting a great loss to the production and productive potential which is a major concern in both small and large ruminants. Hence, an accurate, sensitive, and rapid diagnosis is required for prevention of these above-mentioned diseases. SPR based biosensor assay may fulfill the above characteristics which lead to a greater platform for rapid diagnosis of different livestock diseases. Hence, this review may give a detail idea about the principle, recent development of SPR based biosensor techniques and its application in livestock sector.

Surface determinants of low density lipoprotein uptake by endothelial cells

International Nuclear Information System (INIS)

Goeroeg, P.; Pearson, J.D.

1984-01-01

The surface sialic acid content of aortic endothelial cells in vitro was substantially lower in sparse cultures than at confluence. Binding of LDL to endothelial cells did not change at different culture densities and was unaffected by brief pretreatment with neuraminidase to partially remove surface sialic acid residues. In contrast, internalisation of LDL declined by a factor of 3 between low density cell cultures and confluent monolayers; neuraminidase pretreatment increased LDL uptake and the effect was most marked (>10-fold) at confluence. Pretreatment with cationised ferritin, which removed most of the surface sialic acid residues as well as glycosaminoglycans, increased LDL internalisation by up to 20-fold, again with most effect on confluent monolayers. Thus LDL uptake is inversely correlated with sialic acid content. We conclude that changes in the surface density of sialic acid (and possibly other charged) residues significantly modulate endothelial LDL uptake, and suggest that focal increases in LDL accumulation during atherogenesis may be related to alterations in endothelial endocytic properties at sites of increased cell turnover or damage. (author)

Synthesis of laser beam rapidly solidified novel surfaces on D2 tool steel

International Nuclear Information System (INIS)

Ahmed, B.A.; Rizwan, K.F.; Minhas, J.A.; Waheed-ul-Haq, S.; Shahid, M.

2011-01-01

Surface layer of D2 tool steel was subjected to laser surface melting using continuous wave 2.5 kW CO/sub 2/ laser in point source melting mode. The processing parameters were varied to achieve a uniform depth of around 2 mm. Microstructural study revealed epitaxial growth of fine dendritic structure with secondary dendrite arm spacing in the range of 20-25 mu m. The phases in the parent annealed sample were BCC ferrite and chromium rich M7C3 carbide. The major phase after laser treatment was austenite and M7C3. The average hardness of annealed sample was 195 HV which increased to 410 HV after laser melting. Corrosion studies in 2% HCl solution exhibited a drastic improvement in corrosion resistance in laser treated samples. Improvement in properties is attributed to the refinement and uniformity of microstructure in the rapidly solidified surface. The case of a moving heat source was subjected to computer aided simulation to predict the melt depth at different processing conditions in point source melting mode. The calculated depths using the model, in ABAQUS software was found in good agreement with the experimental data. (author)

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells

International Nuclear Information System (INIS)

Amacher, S.L.; Goodman, L.J.; Bravo, D.A.; Wong, K.Y.; Goldfine, I.D.; Hawley, D.M.; Firestone, G.L.

1989-01-01

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways

Rapid fabricating technique for multi-layered human hepatic cell sheets by forceful contraction of the fibroblast monolayer.

Directory of Open Access Journals (Sweden)

Yusuke Sakai

Full Text Available Cell sheet engineering is attracting attention from investigators in various fields, from basic research scientists to clinicians focused on regenerative medicine. However, hepatocytes have a limited proliferation potential in vitro, and it generally takes a several days to form a sheet morphology and multi-layered sheets. We herein report our rapid and efficient technique for generating multi-layered human hepatic cell (HepaRG® cell sheets using pre-cultured fibroblast monolayers derived from human skin (TIG-118 cells as a feeder layer on a temperature-responsive culture dish. Multi-layered TIG-118/HepaRG cell sheets with a thick morphology were harvested on day 4 of culturing HepaRG cells by forceful contraction of the TIG-118 cells, and the resulting sheet could be easily handled. In addition, the human albumin and alpha 1-antitrypsin synthesis activities of TIG-118/HepaRG cells were approximately 1.2 and 1.3 times higher than those of HepaRG cells, respectively. Therefore, this technique is considered to be a promising modality for rapidly fabricating multi-layered human hepatocyte sheets from cells with limited proliferation potential, and the engineered cell sheet could be used for cell transplantation with highly specific functions.

Enhancing dye-sensitized solar cell efficiency by anode surface treatments

International Nuclear Information System (INIS)

Chang, Chao-Hsuan; Lin, Hsin-Han; Chen, Chin-Cheng; Hong, Franklin C.-N.

2014-01-01

In this study, titanium substrates treated with HF solution and KOH solution sequentially forming micro- and nano-structures were used for the fabrication of flexible dye-sensitized solar cells (DSSCs). After wet etching treatments, the titanium substrates were then exposed to the O 2 plasma treatment and further immersed in titanium tetrachloride (TiCl 4 ) solution. The process conditions for producing a very thin TiO 2 blocking layer were studied, in order to avoid solar cell current leakage for increasing the solar cell efficiency. Subsequently, TiO 2 nanoparticles were spin-coated on Ti substrates with varied thickness. The dye-sensitized solar cells on the titanium substrates were subjected to simulate AM 1.5 G irradiation of 100 mW/cm 2 using backside illumination mode. Surface treatments of Ti substrate and TiO 2 anode were found to play a significant role in improving the efficiency of DSSC. The efficiencies of the backside illumination solar cells were raised from 4.6% to 7.8% by integrating these surface treatments. - Highlights: • The flexible dye-sensitized solar cell (DSSC) device can be fabricated. • Many effective surface treatment methods to improve DSSC efficiency are elucidated. • The efficiency is dramatically enhanced by integrating surface treatment methods. • The back-illuminated DSSC efficiency was raised from 4.6% to 7.8%

Tracking Traction Force Changes of Single Cells on the Liquid Crystal Surface

Directory of Open Access Journals (Sweden)

Chin Fhong Soon

2015-01-01

Full Text Available Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT system can be used in conjunction with a bespoke cell traction force mapping (CTFM software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.

Surface grafting of carboxylic groups onto thermoplastic polyurethanes to reduce cell adhesion

Energy Technology Data Exchange (ETDEWEB)

Alves, P., E-mail: palves@eq.uc.pt [CIEPQPF, Departamento de Engenharia Química, Universidade de Coimbra, Polo II, Pinhal de Marrocos, 3030-790 Coimbra (Portugal); Ferreira, P. [CIEPQPF, Departamento de Engenharia Química, Universidade de Coimbra, Polo II, Pinhal de Marrocos, 3030-790 Coimbra (Portugal); Kaiser, Jean-Pierre [EMPA, St. Gallen, Lerchenfeldstrasse 5, CH-9014 St. Gallen (Switzerland); Salk, Natalie [Mikrofertigung – Micro Engineering, Fraunhofer IFAM, Wiener Strasse 12, D-288359 Bremen (Germany); Bruinink, Arie [EMPA, St. Gallen, Lerchenfeldstrasse 5, CH-9014 St. Gallen (Switzerland); Sousa, Hermínio C. de; Gil, M.H. [CIEPQPF, Departamento de Engenharia Química, Universidade de Coimbra, Polo II, Pinhal de Marrocos, 3030-790 Coimbra (Portugal)

2013-10-15

The interaction of polymers with other materials is an important issue, being their surface properties clearly crucial. For some important polymer applications, their surfaces have to be modified. Surface modification aims to tailor the surface characteristics of a material for a specific application without affecting its bulk properties. Materials can be surface modified by using biological, chemical or physical methods. The aim of this work was to improve the reactivity of the thermoplastic polyurethane (TPU) material (Elastollan{sup ®}) surface and to make its surface cell repellent by grafting carboxylic groups onto its surface. Two TPU materials were studied: a polyether-based TPU and a polyester-based TPU. The grafting efficiency was evaluated by contact angle measurements and by analytical determination of the COOH groups. Scanning electron microscopy (SEM) of the membranes surface was performed as well as cell adhesion tests. It was proved that the surfaces of the TPUs membranes were successfully modified and that cell adhesion was remarkably reduced.

Finite element analysis (FEA) of dental implant fixture for mechanical stability and rapid osseointegration

Science.gov (United States)

Tabassum, Shafia; Murtaza, Ahmar; Ali, Hasan; Uddin, Zia Mohy; Zehra, Syedah Sadaf

2017-10-01

For rapid osseointegration of dental implant fixtures, various surface treatments including plasma spraying, hydroxyapatite coating, acid-etching, and surface grooving are used. However undesirable effects such as chemical modifications, loss of mechanical properties, prolonged processing times and post production treatment steps are often associated with these techniques. The osseointegration rate of the dental implants can be promoted by increasing the surface area of the dental implant, thus increasing the bone cells - implant material contact and allow bone tissues to grow rapidly. Additive Manufacturing (AM) techniques can be used to fabricate dental implant fixtures with desirable surface area in a single step manufacturing process. AM allows the use of Computer Aided Designing (CAD) for customised rapid prototyping of components with precise control over geometry. In this study, the dental implant fixture that replaces the tooth root was designed on commercially available software COMSOL. Nickel - titanium alloy was selected as build materials for dental implant. The geometry of the dental fixture was varied by changing the interspacing distance (thread pitch) and number of threads to increase the total surface area. Three different microstructures were introduced on the surface of dental implant. The designed models were used to examine the effect of changing geometries on the total surface area. Finite Element Analysis (FEA) was performed to investigate the effect of changing geometries on the mechanical properties of the dental implant fixtures using stress analysis.

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

Energy Technology Data Exchange (ETDEWEB)

Makarova, Olga V.; Adams, Daniel L.; Divan, Ralu; Rosenmann, Daniel; Zhu, Peixuan; Li, Shuhong; Amstutz, Platte; Tang, Cha-Mei

2016-09-01

There is a critical need to improve the accuracy of drug screening and testing through the development of in vitro culture systems that more effectively mimic the in vivo environment. Surface topographical features on the nanoscale level, in short nanotopography, effect the cell growth patterns, and hence affect cell function in culture. We report the preliminary results on the fabrication, and subsequent cellular growth, of nanoscale surface topography on polymer microfilters using cell lines as a precursor to circulating tumor cells (CTCs). To create various nanoscale features on the microfilter surface, we used reactive ion etching (RIE) with and without an etching mask. An anodized aluminum oxide (AAO) membrane fabricated directly on the polymer surface served as an etching mask. Polymer filters with a variety of modified surfaces were used to compare the effects on the culture of cancer cell lines in blank culture wells, with untreated microfilters or with RIE-treated microfilters. We then report the differences of cell shape, phenotype and growth patterns of bladder and glioblastoma cancer cell lines after isolation on the various types of material modifications. Our data suggest that RIE modified polymer filters can isolate model cell lines while retaining ell viability, and that the RIE filter modification allows T24 monolayering cells to proliferate as a structured cluster. Copyright 2016 The Authors. Published by Elsevier B.V. All rights reserved.

HOS cell adhesion on Ti6Al4V surfaces texturized by laser engraving

Science.gov (United States)

Sandoval Amador, A.; Carreño Garcia, H.; Escobar Rivero, P.; Peña Ballesteros, D. Y.; Estupiñán Duran, H. A.

2016-02-01

The cell adhesion of the implant is determinate by the chemical composition, topography, wettability, surface energy and biocompatibility of the biomaterial. In this work the interaction between human osteosarcoma HOS cells and textured Ti6Al4V surfaces were evaluated. Ti6Al4V surfaces were textured using a CO2 laser in order to obtain circular spots on the surfaces. Test surfaces were uncoated (C1) used as a control surface, and surfaces with points obtained by laser engraving, with 1mm spacing (C2) and 0.5mm (C3). The HOS cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% antibiotics. No cells toxicity after one month incubation time occurred. The increased cell adhesion and cell spreading was observed after 1, 3 and 5 days without significant differences between the sample surfaces (C2 and C3) and control (uncoated) at the end of the experiment.

HOS cell adhesion on Ti6Al4V surfaces texturized by laser engraving

International Nuclear Information System (INIS)

Sandoval Amador, A; Carreño Garcia, H; Escobar Rivero, P; Peña Ballesteros, D Y; Estupiñán Duran, H A

2016-01-01

The cell adhesion of the implant is determinate by the chemical composition, topography, wettability, surface energy and biocompatibility of the biomaterial. In this work the interaction between human osteosarcoma HOS cells and textured Ti 6 Al 4 V surfaces were evaluated. Ti 6 Al 4 V surfaces were textured using a CO 2 laser in order to obtain circular spots on the surfaces. Test surfaces were uncoated (C1) used as a control surface, and surfaces with points obtained by laser engraving, with 1mm spacing (C2) and 0.5mm (C3). The HOS cells were cultured in RPMI-1640 medium with 10% fetal bovine serum and 1% antibiotics. No cells toxicity after one month incubation time occurred. The increased cell adhesion and cell spreading was observed after 1, 3 and 5 days without significant differences between the sample surfaces (C2 and C3) and control (uncoated) at the end of the experiment. (paper)

Rapid selection of escape mutants by the first CD8 T cell responses in acute HIV-1 infection

Energy Technology Data Exchange (ETDEWEB)

Korber, Bette Tina Marie [Los Alamos National Laboratory

2008-01-01

The recent failure of a vaccine that primes T cell responses to control primary HIV-1 infection has raised doubts about the role of CD8+ T cells in early HIV-1 infection. We studied four patients who were identified shortly after HIV-1 infection and before seroconversion. In each patient there was very rapid selection of multiple HIV-1 escape mutants in the transmitted virus by CD8 T cells, including examples of complete fixation of non-synonymous substitutions within 2 weeks. Sequencing by single genome amplification suggested that the high rate of virus replication in acute infection gave a selective advantage to virus molecules that contained simultaneous and gained sequential T cell escape mutations. These observations show that whilst early HIV-1 specific CD8 T cells can act against virus, rapid escape means that these T cell responses are unlikely to benefit the patient and may in part explain why current HIV-1 T cell vaccines may not be protective.

Rapid generation of mitochondrial superoxide induces mitochondrion-dependent but caspase-independent cell death in hippocampal neuronal cells that morphologically resembles necroptosis☆

Science.gov (United States)

Fukui, Masayuki; Choi, Hye Joung; Zhu, Bao Ting

2013-01-01

Studies in recent years have revealed that excess mitochondrial superoxide production is an important etiological factor in neurodegenerative diseases, resulting from oxidative modifications of cellular lipids, proteins, and nucleic acids. Hence, it is important to understand the mechanism by which mitochondrial oxidative stress causes neuronal death. In this study, the immortalized mouse hippocampal neuronal cells (HT22) in culture were used as a model and they were exposed to menadione (also known as vitamin K3) to increase intracellular superoxide production. We found that menadione causes preferential accumulation of superoxide in the mitochondria of these cells, along with the rapid development of mitochondrial dysfunction and cellular ATP depletion. Neuronal death induced by menadione is independent of the activation of the MAPK signaling pathways and caspases. The lack of caspase activation is due to the rapid depletion of cellular ATP. It was observed that two ATP-independent mitochondrial nucleases, namely, AIF and Endo G, are released following menadione exposure. Silencing of their expression using specific siRNAs results in transient suppression (for ~12 h) of mitochondrial superoxide-induced neuronal death. While suppression of the mitochondrial superoxide dismutase expression markedly sensitizes neuronal cells to mitochondrial superoxide-induced cytotoxicity, its over-expression confers strong protection. Collectively, these findings showed that many of the observed features associated with mitochondrial superoxide-induced cell death, including caspase independency, rapid depletion of ATP level, mitochondrial release of AIF and Endo G, and mitochondrial swelling, are distinctly different from those of apoptosis; instead they resemble some of the known features of necroptosis. PMID:22575170

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.

Science.gov (United States)

Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung

2013-08-06

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

ASSESSMENT OF SURFACE FINISH AND DIMENSIONAL ACCURACY OF TOOLS MANUFACTURED BY METAL CASTING IN RAPID PROTOTYPING SAND MOULDS

OpenAIRE

Nyembwe, K.; De Beer, D. J.; Van der Walt, J. G.; Bhero, S.

2012-01-01

In this paper, an initial assessment of the quality parameters of the surface finish and dimensional accuracy of tools made by metal casting in rapid prototyping (RP) sand moulds is undertaken. A case study from a local tool room, dealing with the manufacturing of an aluminium die for the lost wax process, is employed. Modern techniques, including surface roughness analysis and three dimensional scanning, are used to determine and understand how each manufacturing step influences the final qu...

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation.

Science.gov (United States)

Graessel, Anke; Hauck, Stefanie M; von Toerne, Christine; Kloppmann, Edda; Goldberg, Tatyana; Koppensteiner, Herwig; Schindler, Michael; Knapp, Bettina; Krause, Linda; Dietz, Katharina; Schmidt-Weber, Carsten B; Suttner, Kathrin

2015-08-01

Naive CD4(+) T cells are the common precursors of multiple effector and memory T-cell subsets and possess a high plasticity in terms of differentiation potential. This stem-cell-like character is important for cell therapies aiming at regeneration of specific immunity. Cell surface proteins are crucial for recognition and response to signals mediated by other cells or environmental changes. Knowledge of cell surface proteins of human naive CD4(+) T cells and their changes during the early phase of T-cell activation is urgently needed for a guided differentiation of naive T cells and may support the selection of pluripotent cells for cell therapy. Periodate oxidation and aniline-catalyzed oxime ligation technology was applied with subsequent quantitative liquid chromatography-tandem MS to generate a data set describing the surface proteome of primary human naive CD4(+) T cells and to monitor dynamic changes during the early phase of activation. This led to the identification of 173 N-glycosylated surface proteins. To independently confirm the proteomic data set and to analyze the cell surface by an alternative technique a systematic phenotypic expression analysis of surface antigens via flow cytometry was performed. This screening expanded the previous data set, resulting in 229 surface proteins, which were expressed on naive unstimulated and activated CD4(+) T cells. Furthermore, we generated a surface expression atlas based on transcriptome data, experimental annotation, and predicted subcellular localization, and correlated the proteomics result with this transcriptional data set. This extensive surface atlas provides an overall naive CD4(+) T cell surface resource and will enable future studies aiming at a deeper understanding of mechanisms of T-cell biology allowing the identification of novel immune targets usable for the development of therapeutic treatments. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Surface strategies for control of neuronal cell adhesion: A review

Science.gov (United States)

Roach, P.; Parker, T.; Gadegaard, N.; Alexander, M. R.

2010-06-01

Material engineering methods have been used for many years to develop biomedical devices for use within the body to augment, repair or replace damaged tissues ranging from contact lenses to heart valves. Here we review the findings gathered from the wide and varied surface analytical approaches applied to study the interaction between biology and man-made materials. The key material characteristics identified to be important for biological recognition are surface chemistry, topography and compliance. Model surfaces with controlled chemistry and topography have provided insight into biological response to various types of topographical features over a wide range of length scales from nano to micrometres, along with 3D matrices that have been used as scaffolds to support cells for tissue formation. The cellular response to surfaces with localised areas of patterned chemistry and to those presenting gradually changing chemistry are discussed. Where previous reviews have been structured around specific classes of surface modification, e.g. self-assembly, or have broadly examined the response of various cells to numerous surfaces, we aim in this article to focus in particular on the tissues involved in the nervous system whilst providing a broad overview of key issues from the field of cell and protein surface interactions with surfaces. The goal of repair and treatment of diseases related to the central and peripheral nervous systems rely on understanding the local interfacial environment and controlling responses at the cellular level. The role of the protein layer deposited from serum containing media onto man-made surfaces is discussed. We highlight the particular problems associated with the repair of the nervous system, and review how neuronal attachment and axon guidance can be accomplished using various surface cues when cultured with single and multiple cell types. We include a brief glossary of techniques discussed in the body of this article aimed at the

Role of prostate apoptosis response 4 in translocation of GRP78 from the endoplasmic reticulum to the cell surface of trophoblastic cells.

Directory of Open Access Journals (Sweden)

Marie Cohen