In vitro biocompatibility of magnesium-incorporated submicro-porous titanium oxide surface produced by hydrothermal treatment

NASA Astrophysics Data System (ADS)

Park, Jin-Woo; Kim, Youn-Jeong; Jang, Je-Hee; An, Chang-Hyeon

2010-11-01

This study investigated the surface characteristics and in vitro biocompatibility of titanium (Ti) oxide surface incorporating magnesium ions (Mg), produced by hydrothermal treatment using an alkaline Mg-containing solution, for future biomedical applications. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, X-ray photoelectron spectroscopy, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) and optical profilometry. Mouse calvaria-derived osteoblastic cell (MC3T3-E1) attachment, spreading, proliferation, alkaline phosphatase (ALP) activity, and osteoblastic gene expression on Mg-containing surfaces were compared with untreated Ti surfaces. Hydrothermal treatment resulted in Mg-incorporated Ti oxide layer with submicro-porous surface structures approximately 2 μm in thickness. ICP-AES analysis revealed Mg ions release from treated surfaces into the solution. The Mg-incorporated surface displayed significantly increased cellular attachment and ALP activity compared with untreated surface ( p < 0.05), and supported better cell spreading. Real-time polymerase chain reaction analysis showed notably higher mRNA expression of the osteoblast transcription factor genes (Dlx5, Runx2) and the osteoblast phenotype genes (ALP, bone sialoprotein and osteocalcin) in cells grown on the Mg-incorporated surfaces than untreated surfaces. These results demonstrate that the Mg-incorporated submicro-porous Ti oxide surface produced by hydrothermal treatment may improve implant osseointegration by enhancing the attachment, spreading and differentiation of osteoblastic cells.

Simultaneous dynamic optical and electrical properties of endothelial cell attachment on indium tin oxide bioelectrodes.

PubMed

Choi, Chang K; English, Anthony E; Kihm, Kenneth D; Margraves, Charles H

2007-01-01

This study quantifies the dynamic attachment and spreading of porcine pulmonary artery endothelial cells (PPAECs) on optically thin, indium tin oxide (ITO) biosensors using simultaneous differential interference contrast microscopy (DICM) and electrical microimpedance spectroscopy. A lock-in amplifier circuit monitored the impedance of PPAECs cultivated on the transparent ITO bioelectrodes as a function of frequency between 10 Hz and 100 kHz and as a function of time, while DICM images were simultaneously acquired. A digital image processing algorithm quantified the cell-covered electrode area as a function of time. The results of this study show that the fraction of the cell-covered electrode area is in qualitative agreement with the electrical impedance during the attachment phase following the cell settling on the electrode surface. The possibility of several distinctly different states of electrode coverage and cellular attachment giving rise to similar impedance signals is discussed.

Substrate porosity enhances chondrocyte attachment, spreading, and cartilage tissue formation in vitro.

PubMed

Spiteri, C G; Pilliar, R M; Kandel, R A

2006-09-15

Tissue engineering is being explored as a new approach to treat damaged cartilage. As the biomaterial used may influence tissue formation, the effects of substrate geometry on chondrocyte behavior in vitro were examined. Articular chondrocytes were isolated and cultured on the surface of smooth, rough, porous-coated, and fully porous Ti-6Al-4V substrates. The percentage of chondrocytes that attached to each substrate at 24 h was determined. After 24 and 72 h, chondrocytes were visualized by scanning electron microscopy and cell areas were measured. Collagen and proteoglycan accumulation within the first 24 h was determined by incorporation with [3H]-proline and [35S]-SO4, respectively. Chondrocyte attachment as well as matrix accumulation was enhanced as substrate surface area increased. Cell areas on the fully porous substrate were over four times greater than on any other substrate by 72 h in culture. After 8 weeks in culture, a continuous layer of cartilaginous tissue formed only on the surface of the fully porous substrate. This suggests that fully porous Ti-6Al-4V substrates provide the conditions that favor cartilage tissue formation by influencing cell attachment and extent of cell spreading. Understanding how substrate porosity influences chondrocyte behavior may help identify methods to further enhance cartilage tissue formation in vitro. 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.

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

PubMed

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

2018-06-18

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

Impaired Integrin-mediated Adhesion and Signaling in Fibroblasts Expressing a Dominant-negative Mutant PTP1B

PubMed Central

Arregui, Carlos O.; Balsamo, Janne; Lilien, Jack

1998-01-01

To investigate the role of nonreceptor protein tyrosine phosphatase 1B (PTP1B) in β1-integrin– mediated adhesion and signaling, we transfected mouse L cells with normal and catalytically inactive forms of the phosphatase. Parental cells and cells expressing the wild-type or mutant PTP1B were assayed for (a) adhesion, (b) spreading, (c) presence of focal adhesions and stress fibers, and (d) tyrosine phosphorylation. Parental cells and cells expressing wild-type PTP1B show similar morphology, are able to attach and spread on fibronectin, and form focal adhesions and stress fibers. In contrast, cells expressing the inactive PTP1B have a spindle-shaped morphology, reduced adhesion and spreading on fibronectin, and almost a complete absence of focal adhesions and stress fibers. Attachment to fibronectin induces tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin in parental cells and cells transfected with the wild-type PTP1B, while in cells transfected with the mutant PTP1B, such induction is not observed. Additionally, in cells expressing the mutant PTP1B, tyrosine phosphorylation of Src is enhanced and activity is reduced. Lysophosphatidic acid temporarily reverses the effects of the mutant PTP1B, suggesting the existence of a signaling pathway triggering focal adhesion assembly that bypasses the need for active PTP1B. PTP1B coimmunoprecipitates with β1-integrin from nonionic detergent extracts and colocalizes with vinculin and the ends of actin stress fibers in focal adhesions. Our data suggest that PTP1B is a critical regulatory component of integrin signaling pathways, which is essential for adhesion, spreading, and formation of focal adhesions. PMID:9813103

Effect of Micro- and Nanoscale Topography on the Adhesion of Bacterial Cells to Solid Surfaces

PubMed Central

Hsu, Lillian C.; Fang, Jean; Borca-Tasciuc, Diana A.; Worobo, Randy W.

2013-01-01

Attachment and biofilm formation by bacterial pathogens on surfaces in natural, industrial, and hospital settings lead to infections and illnesses and even death. Minimizing bacterial attachment to surfaces using controlled topography could reduce the spreading of pathogens and, thus, the incidence of illnesses and subsequent human and financial losses. In this context, the attachment of key microorganisms, including Escherichia coli, Listeria innocua, and Pseudomonas fluorescens, to silica and alumina surfaces with micron and nanoscale topography was investigated. The results suggest that orientation of the attached cells occurs preferentially such as to maximize their contact area with the surface. Moreover, the bacterial cells exhibited different morphologies, including different number and size of cellular appendages, depending on the topographical details of the surface to which they attached. This suggests that bacteria may utilize different mechanisms of attachment in response to surface topography. These results are important for the design of novel microbe-repellant materials. PMID:23416997

High-content profiling of cell responsiveness to graded substrates based on combinyatorially variant polymers.

PubMed

Liu, Er; Treiser, Matthew D; Patel, Hiral; Sung, Hak-Joon; Roskov, Kristen E; Kohn, Joachim; Becker, Matthew L; Moghe, Prabhas V

2009-08-01

We have developed a novel approach combining high information and high throughput analysis to characterize cell adhesive responses to biomaterial substrates possessing gradients in surface topography. These gradients were fabricated by subjecting thin film blends of tyrosine-derived polycarbonates, i.e. poly(DTE carbonate) and poly(DTO carbonate) to a gradient temperature annealing protocol. Saos-2 cells engineered with a green fluorescent protein (GFP) reporter for farnesylation (GFP-f) were cultured on the gradient substrates to assess the effects of nanoscale surface topology and roughness that arise during the phase separation process on cell attachment and adhesion strength. The high throughput imaging approach allowed us to rapidly identify the "global" and "high content" structure-property relationships between cell adhesion and biomaterial properties such as polymer chemistry and topography. This study found that cell attachment and spreading increased monotonically with DTE content and were significantly elevated at the position with intermediate regions corresponding to the highest "gradient" of surface roughness, while GFP-f farnesylation intensity descriptors were sensitively altered by surface roughness, even in cells with comparable levels of spreading.

Reversible changes in cell morphology due to cytoskeletal rearrangements measured in real-time by QCM-D.

PubMed

Tymchenko, Nina; Nilebäck, Erik; Voinova, Marina V; Gold, Julie; Kasemo, Bengt; Svedhem, Sofia

2012-12-01

The mechanical properties and responses of cells to external stimuli (including drugs) are closely connected to important phenomena such as cell spreading, motility, activity, and potentially even differentiation. Here, reversible changes in the viscoelastic properties of surface-attached fibroblasts were induced by the cytoskeleton-perturbing agent cytochalasin D, and studied in real-time by the quartz crystal microbalance with dissipation (QCM-D) technique. QCM-D is a surface sensitive technique that measures changes in (dynamically coupled) mass and viscoelastic properties close to the sensor surface, within a distance into the cell that is usually only a fraction of its size. In this work, QCM-D was combined with light microscopy to study in situ cell attachment and spreading. Overtone-dependent changes of the QCM-D responses (frequency and dissipation shifts) were first recorded, as fibroblast cells attached to protein-coated sensors in a window equipped flow module. Then, as the cell layer had stabilised, morphological changes were induced in the cells by injecting cytochalasin D. This caused changes in the QCM-D signals that were reversible in the sense that they disappeared upon removal of cytochalasin D. These results are compared to other cell QCM-D studies. Our results stress the combination of QCM-D and light microscopy to help interpret QCM-D results obtained in cell assays and thus suggests a direction to develop the QCM-D technique as an even more useful tool for real-time cell studies.

Mouse embryo attachment to substratum and interaction of trophoblast with cultured cells

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

Glass, R.H.; Spindle, A.I.; Pedersen, R.A.

1979-06-01

Hatching, attachment, and trophoblast outgrowth of mouse embryos in vitro were examined as a model for implantation. Mouse embryos attached and grew out on glass cover slips that were partially covered with cultured mouse cells (L cells, liver cells, transformed JLS-V11 cells, and teratocarcinoma cells). Scanning electron microscopy showed that processes of these cells made contact with trophoblast, but there was no evidence of cell lysis or of phagocytosis of the cells by trophoblast. Time-lapse cinematography showed that after contact the cultured mouse cells retracted from the trophoblast, which then spread into the areas vacated by those cells. This suggestsmore » a means by which the trophoblast gains entry into the endometrium without destruction of maternal cells. Neuraminidase (100 or 250 units/ml) had no effect on attachment of mouse embryos to glass. However, attachment was inhibited by trypsin at concentrations of 0.25%, 0.025%, and 0.0025%. Treatment of early blastocysts with diazooxo-norleucine, an inhibitor of glycoprotein synthesis, decreased the number of embryos hatching from the zona pellucida; treatment at the late blastocyst stage decreased hatching to a lesser extent. Among the late blastocysts that did hatch, the number forming trophoblast outgrowths was lower than in controls. These results suggest that glycoproteins may be of importance for embryo hatching, attachment, and outgrowth.« less

Effect of bioactive extruded PLA/HA composite films on focal adhesion formation of preosteoblastic cells.

PubMed

Persson, Maria; Lorite, Gabriela S; Kokkonen, Hanna E; Cho, Sung-Woo; Lehenkari, Petri P; Skrifvars, Mikael; Tuukkanen, Juha

2014-09-01

The quality of the initial cell attachment to a biomaterial will influence any further cell function, including spreading, proliferation, differentiation and viability. Cell attachment is influenced by the material's ability to adsorb proteins, which is related to the surface chemistry and topography of the material. In this study, we incorporated hydroxyapatite (HA) particles into a poly(lactic acid) (PLA) composite and evaluated the surface structure and the effects of HA density on the initial cell attachment in vitro of murine calvarial preosteoblasts (MC3T3-EI). Scanning electron microscopy (SEM), atomic force microscopy (AFM) and infrared spectroscopy (FTIR) showed that the HA particles were successfully incorporated into the PLA matrix and located at the surface which is of importance in order to maintain the bioactive effect of the HA particles. SEM and AFM investigation revealed that the HA density (particles/area) as well as surface roughness increased with HA loading concentration (i.e. 5, 10, 15 and 20wt%), which promoted protein adsorption. Furthermore, the presence of HA on the surface enhanced cell spreading, increased the formation of actin stress fibers and significantly improved the expression of vinculin in MC3T3-E1 cells which is a key player in the regulation of cell adhesion. These results suggest the potential utility of PLA/HA composites as biomaterials for use as a bone substitute material and in tissue engineering applications. Copyright © 2014 Elsevier B.V. All rights reserved.

A fraction of methylene chloride from Geum japonicum Thunberg inhibits tumor metastatic and angiogenic potential.

PubMed

Heo, Jin-Chul; Son, Minsik; Woo, Sang-Uk; Kweon, Mi-Ae; Yoon, Eun Kyung; Lee, Hee Kyung; Choi, Won-Sik; Cho, Kang-Jin; Lee, Sang-Han

2008-06-01

The plant Geum japonicum Thunberg (GjT) has been used as a diuretic in traditional medicine. Herein, we report that the GjT extract blocks both the spread of human umbilical vein endothelial cells (HUVECs) on matrigel and the migration of B16 cells. We used various assays to test for cell attachment, spreading, wound healing and angiogenesis. A reverse transcription-polymerase chain reaction (RT-PCR) and a mitogen-activated protein kinase (MAPK) assay were also carried out for the mechanistic study of GjT. Our results showed that a fraction of methylene chloride fraction from GjT inhibited B16 cells during cell attachment and migration and suppressed tube formation in a dose-dependent manner. An RT-PCR analysis showed that the methylene chloride extract decreased the mRNA expression of CD44 and TIMP-2. A Western blot analysis of the phosphorylation of MAPK kinases (ERK, JNK and p38) showed that the GjT fraction increased the expression of phospho-JNK, suggesting that GjT has the potential to alleviate metastatic and angiogenic activity, via a phospho-JNK signaling pathway.

Controlling Cell Function with Geometry

NASA Astrophysics Data System (ADS)

Mrksich, Milan

2012-02-01

This presentation will describe the use of patterned substrates to control cell shape with examples that illustrate the ways in which cell shape can regulate cell function. Most cells are adherent and must attach to and spread on a surface in order to survive, proliferate and function. In tissue, this surface is the extracellular matrix (ECM), an insoluble scaffold formed by the assembly of several large proteins---including fibronectin, the laminins and collagens and others---but in the laboratory, the surface is prepared by adsorbing protein to glass slides. To pattern cells, gold-coated slides are patterned with microcontact printing to create geometric features that promote cell attachment and that are surrounded by inert regions. Cells attach to these substrates and spread to adopt the shape defined by the underlying pattern and remain stable in culture for several days. Examples will be described that used a series of shapes to reveal the relationship between the shape of the cell and the structure of its cytoskeleton. These geometric cues were used to control cell polarity and the tension, or contractility, present in the cytoskeleton. These rules were further used to control the shapes of mesenchymal stem cells and in turn to control the differentiation of these cells into specialized cell types. For example, stem cells that were patterned into a ``star'' shape preferentially differentiated into bone cells whereas those that were patterned into a ``flower'' shape preferred a fat cell fate. These influences of shape on differentiation depend on the mechanical properties of the cytoskeleton. These examples, and others, reveal that shape is an important cue that informs cell function and that can be combined with the more common soluble cues to direct and study cell function.

UV laser-ablated surface textures as potential regulator of cellular response.

PubMed

Chandra, Prafulla; Lai, Karen; Sung, Hak-Joon; Murthy, N Sanjeeva; Kohn, Joachim

2010-06-01

Textured surfaces obtained by UV laser ablation of poly(ethylene terephthalate) films were used to study the effect of shape and spacing of surface features on cellular response. Two distinct patterns, cones and ripples with spacing from 2 to 25 μm, were produced. Surface features with different shapes and spacings were produced by varying pulse repetition rate, laser fluence, and exposure time. The effects of the surface texture parameters, i.e., shape and spacing, on cell attachment, proliferation, and morphology of neonatal human dermal fibroblasts and mouse fibroblasts were studied. Cell attachment was the highest in the regions with cones at ∼4 μm spacing. As feature spacing increased, cell spreading decreased, and the fibroblasts became more circular, indicating a stress-mediated cell shrinkage. This study shows that UV laser ablation is a useful alternative to lithographic techniques to produce surface patterns for controlling cell attachment and growth on biomaterial surfaces.

Cell attachment properties of Portland cement-based endodontic materials: biological and methodological considerations.

PubMed

Ahmed, Hany Mohamed Aly; Luddin, Norhayati; Kannan, Thirumulu Ponnuraj; Mokhtar, Khairani Idah; Ahmad, Azlina

2014-10-01

The attachment and spreading of mammalian cells on endodontic biomaterials are an area of active research. The purpose of this review is to discuss the cell attachment properties of Portland cement (PC)-based materials by using scanning electron microscope (SEM). In addition, methodological aspects and technical challenges are discussed. A PubMed electronic search was conducted by using appropriate key words to identify the available investigations on the cell attachment properties of PC-based endodontic materials. After retrieving the full text of related articles, the cross citations were also identified. A total of 23 articles published between January 1993 and October 2013 were identified. This review summarizes the cell attachment properties of commercial and experimental PC-based materials on different cell cultures by using SEM. Methodological procedures, technical challenges, and relevance of SEM in determining the biological profile of PC-based materials are discussed. SEM observations demonstrate that commercial MTA formulations show favorable cell attachment properties, which is consistent with their successful clinical outcomes. The favorable cell attachment properties of PC and its modified formulations support its potential use as a substitute for mineral trioxide aggregate. However, researchers should carefully select cell types for their SEM investigations that would be in contact with the proposed PC-based combinations in the clinical situation. Despite being a technical challenge, SEM provides useful information on the cell attachment properties of PC-based materials; however, other assays for cell proliferation and viability are essential to come up with an accurate in vitro biological profile of any given PC-based formulation. Copyright © 2014 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

PubMed Central

Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

2012-01-01

The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0 nm) and sandblasting (Ra: approximately 1.0 μm), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

Broad-spectrum antiviral activity of chebulagic acid and punicalagin against viruses that use glycosaminoglycans for entry

PubMed Central

2013-01-01

Background We previously identified two hydrolyzable tannins, chebulagic acid (CHLA) and punicalagin (PUG) that blocked herpes simplex virus type 1 (HSV-1) entry and spread. These compounds inhibited viral glycoprotein interactions with cell surface glycosaminoglycans (GAGs). Based on this property, we evaluated their antiviral efficacy against several different viruses known to employ GAGs for host cell entry. Results Extensive analysis of the tannins’ mechanism of action was performed on a panel of viruses during the attachment and entry steps of infection. Virus-specific binding assays and the analysis of viral spread during treatment with these compounds were also conducted. CHLA and PUG were effective in abrogating infection by human cytomegalovirus (HCMV), hepatitis C virus (HCV), dengue virus (DENV), measles virus (MV), and respiratory syncytial virus (RSV), at μM concentrations and in dose-dependent manners without significant cytotoxicity. Moreover, the natural compounds inhibited viral attachment, penetration, and spread, to different degrees for each virus. Specifically, the tannins blocked all these steps of infection for HCMV, HCV, and MV, but had little effect on the post-fusion spread of DENV and RSV, which could suggest intriguing differences in the roles of GAG-interactions for these viruses. Conclusions CHLA and PUG may be of value as broad-spectrum antivirals for limiting emerging/recurring viruses known to engage host cell GAGs for entry. Further studies testing the efficacy of these tannins in vivo against certain viruses are justified. PMID:23924316

Physical Properties and Cellular Responses to Crosslinkable Poly(Propylene Fumarate)/Hydroxyapatite Nanocomposites

PubMed Central

Lee, Kee-Won; Wang, Shanfeng; Yaszemski, Michael J.; Lu, Lichun

2008-01-01

A series of crosslinkable nanocomposites has been developed using hydroxyapatite (HA) nanoparticles and poly(propylene fumarate) (PPF). PPF/HA nanocomposites with four different weight fractions of HA nanoparticles have been characterized in terms of thermal and mechanical properties. To assess surface chemistry of crosslinked PPF/HA nanocomposites, their hydrophilicity and capability of adsorbing proteins have been determined using static contact angle measurement and MicroBCA protein assay kit after incubation with 10% fetal bovine serum (FBS), respectively. In vitro cell studies have been performed using MC3T3-E1 mouse pre-osteoblast cells to investigate the ability of PPF/HA nanocomposites to support cell attachment, spreading, and proliferation after 1, 4, and 7 days. By adding HA nanoparticles to PPF, the mechanical properties of crosslinked PPF/HA nanocomposites have not been increased due to the initially high modulus of crosslinked PPF. However, hydrophilicity and serum protein adsorption on the surface of nanocomposites have been significantly increased, resulting in enhanced cell attachment, spreading, and proliferation after 4 days of cell seeding. These results indicate that crosslinkable PPF/HA nanocomposites are useful for hard tissue replacement because of excellent mechanical strength and osteoconductivity. PMID:18403013

A tailored three-dimensionally printable agarose-collagen blend allows encapsulation, spreading, and attachment of human umbilical artery smooth muscle cells.

PubMed

Köpf, Marius; Campos, Daniela F Duarte; Blaeser, Andreas; Sen, Kshama S; Fischer, Horst

2016-05-20

In recent years, novel biofabrication technologies have enabled the rapid manufacture of hydrogel-cell suspensions into tissue-imitating constructs. The development of novel materials for biofabrication still remains a challenge due to a gap between contradicting requirements such as three-dimensional printability and optimal cytocompatibility. We hypothesise that blending of different hydrogels could lead to a novel material with favourable biological and printing properties. In our work, we combined agarose and type I collagen in order to develop a hydrogel blend capable of long-term cell encapsulation of human umbilical artery smooth muscle cells (HUASMCs) and 3D drop-on-demand printing. Different blends were prepared with 0.25%, 0.5%, 0.75%, and 1.5% agarose and 0.2% type I collagen. The cell morphology of HUASMCs and the printing accuracy were assessed for each agarose-collagen combination, keeping the content of collagen constant. The hydrogel blend which displayed sufficient cell spreading and printing accuracy (0.5% agarose, 0.2% type I collagen, AGR0.5COLL0.2) was then characterised based on swelling and degradation over 21 days and mechanical stiffness. The cellular response regarding cell attachment of HUASMCs embedded in the hydrogel blend was further studied using SEM, TEM, and TPLSM. Printing trials were fabricated in a drop-on-demand printing process. The swelling and degradation evaluation showed an average of 20% mass loss and less than 10% swelling. AGR0.5COLL0.2 exhibited significant increase in stiffness compared to pure agarose and type I collagen. In addition, columns of AGR0.5COLL0.2 three centimeters in height were successfully printed submerged in cooled perfluorocarbon, proving the intrinsic printability of the hydrogel blend. Ultimately, a promising novel hydrogel blend showing cell spreading and attachment as well as suitability for bioprinting was identified and could, for example, serve in the manufacture of in vitro 3D models to capture more complex features of disease and drug discovery.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

PubMed

Williamson, Matthew R; Shuttleworth, Adrian; Canfield, Ann E; Black, Richard A; Kielty, Cay M

2007-12-01

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

[Stimulation of cell cultures recovery after cryopreservation by the cattle cord blood FRACTION (below 5 kDa) or Actovegin].

PubMed

Gulevskiĭ, A K; Trifonova, A V; Lavrik, A A

2013-01-01

The capacities of the cattle cord blood low-molecular fraction (below 5 kDa) and Actovegin (the vealer blood fraction (below 5 kDa)) for recovering functions of cell cultures after cryopreservation compared. Their influence proliferation of the flozen-thawed cell cultures, certain stages of their growth, cell attachment, rate of cell spreading, and mitotic regiment has been studied. Both the cord blood low-molecular fraction and Actovegin were shown to stimulate growth of the cell cultures after cryopreservation more efficiently at the concentration of 224 μg/ml. However, despite the stimulating effect discovered, their application did not bring proliferative indices on the 1st passage after cryopreservation to the values of the native culture. The effects of the cord blood low-molecular fraction and Actovegin on the human fibroblast culture were identical by the following parameters: cell attachment, rates of cell spreading and proliferation. In culture BHK-21 clone 13/04 the efficiency of Actovegin was low, while the cord blood low-molecular fraction has a conspicuous stimulating effect on its adhesion and proliferation. The investigations carried out can serve as a basis for the development of regenerative media containing the cattle cord blood low-molecular fraction (below 5 kDa) or Actovegin as active components at the concentration of 224 μg/ml with the purpose of fast recovery of culture prolifetative properties after cryopreservation.

An adaptor role for cytoplasmic Sam68 in modulating Src activity during cell polarization.

PubMed

Huot, Marc-Etienne; Brown, Claire M; Lamarche-Vane, Nathalie; Richard, Stéphane

2009-04-01

The Src-associated substrate during mitosis with a molecular mass of 68 kDa (Sam68) is predominantly nuclear and is known to associate with proteins containing the Src homology 3 (SH3) and SH2 domains. Although Sam68 is a Src substrate, little is known about the signaling pathway that link them. Src is known to be activated transiently after cell spreading, where it modulates the activity of small Rho GTPases. Herein we report that Sam68-deficient cells exhibit loss of cell polarity and cell migration. Interestingly, Sam68-deficient cells exhibited sustained Src activity after cell attachment, resulting in the constitutive tyrosine phosphorylation and activation of p190RhoGAP and its association with p120rasGAP. Consistently, we observed that Sam68-deficient cells exhibited deregulated RhoA and Rac1 activity. By using total internal reflection fluorescence microscopy, we observed Sam68 near the plasma membrane after cell attachment coinciding with phosphorylation of its C-terminal tyrosines and association with Csk. These findings show that Sam68 localizes near the plasma membrane during cell attachment and serves as an adaptor protein to modulate Src activity for proper signaling to small Rho GTPases.

Fibroblastic interactions with high-porosity Ti-6Al-4V metal foam.

PubMed

Cheung, Serene; Gauthier, Maxime; Lefebvre, Louis-Philippe; Dunbar, Michael; Filiaggi, Mark

2007-08-01

A novel metallic Ti-6Al-4V foam in development at the National Research Council of Canada was investigated for its ability to foster cell attachment and growth using a fibroblast cell culture model. The foam was manufactured via a powder metallurgical process that could produce interconnected porosity greater than 70%. Cell attachment was assessed after 6 and 24 h, while proliferation was examined after 3 and 7 days. Ingrown fibroblasts displayed a number of different morphologies; some fibroblasts were spread thinly in close apposition with the irregular surface, or more often had several anchorage points and extended in three dimensions as they spanned pore space. It was also demonstrated that fibroblasts were actively migrating through the porous scaffold over a 14-day period. In a 60-day extended culture, fibroblasts were bridging and filling macropores and had extensively infiltrated the foams. Overall, it was established that this foam was supportive of cell attachment and proliferation, migration through the porous network, and that it was capable of sustaining a large cell population.

JC Polyomavirus Attachment, Entry, and Trafficking: Unlocking the Keys to a Fatal Infection

PubMed Central

Maginnis, Melissa S.; Nelson, Christian D.S.; Atwood, Walter J.

2014-01-01

The human JC polyomavirus (JCPyV) causes a lifelong persistent infection in the reno-urinary tract in the majority of the adult population worldwide. In healthy individuals infection is asymptomatic, while in immunocompromised individuals the virus can spread to the central nervous system and cause a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). There are currently very few treatment options for this rapidly progressing and devastating disease. Understanding the basic biology of JCPyV-host cell interactions is critical for the development of therapeutic strategies to prevent or treat PML. Research in our laboratory has focused on gaining a detailed mechanistic understanding of the initial steps in the JCPyV life cycle in order to define how JCPyV selectively targets cells in the kidney and brain. JCPyV requires sialic acids to attach to host cells and initiate infection, and JCPyV demonstrates specificity for the oligosaccharide lactoseries tetrasaccharide c (LSTc) with an α2,6-linked sialic acid. Following viral attachment, JCPyV entry is facilitated by the 5-hydroxytryptamine (5-HT)2 family of serotonin receptors via clathrin-dependent endocytosis. JCPyV then undergoes retrograde transport to the endoplasmic reticulum (ER) where viral disassembly begins. A novel retrograde transport inhibitor termed Retro-2cycl prevents trafficking of JCPyV to the ER and inhibits both initial virus infection and infectious spread in cell culture. Understanding the molecular mechanisms by which JCPyV establishes infection will open up new avenues for the prevention or treatment of virus-induced disease. PMID:25078361

Effect of Atmospheric Plasma Treatment to Titanium Surface on Initial Osteoblast-Like Cell Spreading. .

PubMed

Kim, In-Hye; Son, Jun-Sik; Kwon, Tae-Yub; Kim, Kyo-Han

2015-01-01

Plasma treatments are becoming a popular method for modifying the characteristics of a range of substrate surfaces. Atmospheric pressure plasma is cost-efficient, safe and simple compared to high-pressure plasma. This study examined the effects of atmospheric pressure plasma to a titanium (Ti) surface on osteoblast-like cell (osteoblast) spreading and cellular networks. The characteristics of the Ti surface before and after the atmospheric plasma treatment were analyzed by X-ray photoemission spectroscopy (XPS), scanning electron microscopy (SEM), contact angle measurements, and an optical 3D profiling system. The morphology of osteoblasts attached to the Ti surfaces was observed by SEM and confocal laser scanning microscopy. The atmospheric pressure plasma made the Ti surfaces more hydrophilic. The osteoblasts that adhered to the untreated surface were round and spherical, whereas the cells covered a larger surface area on the plasma-treated surface. The plasma-treated Ti surface showed enhanced cell spreading and migration with more developed cellular networks. In conclusion, an atmospheric plasma treatment is a potential surface modifying method that can enhance the initial the cell affinity at the early stages in vitro.

Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

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

Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.

1987-05-01

Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cellsmore » (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.« less

A Single Regulator Mediates Strategic Switching between Attachment/Spread and Growth/Virulence in the Plant Pathogen Ralstonia solanacearum

PubMed Central

Khokhani, Devanshi; Lowe-Power, Tiffany M.; Tran, Tuan Minh

2017-01-01

ABSTRACT The PhcA virulence regulator in the vascular wilt pathogen Ralstonia solanacearum responds to cell density via quorum sensing. To understand the timing of traits that enable R. solanacearum to establish itself inside host plants, we created a ΔphcA mutant that is genetically locked in a low-cell-density condition. Comparing levels of gene expression of wild-type R. solanacearum and the ΔphcA mutant during tomato colonization revealed that the PhcA transcriptome includes an impressive 620 genes (>2-fold differentially expressed; false-discovery rate [FDR], ≤0.005). Many core metabolic pathways and nutrient transporters were upregulated in the ΔphcA mutant, which grew faster than the wild-type strain in tomato xylem sap and on dozens of specific metabolites, including 36 found in xylem. This suggests that PhcA helps R. solanacearum to survive in nutrient-poor environmental habitats and to grow rapidly during early pathogenesis. However, after R. solanacearum reaches high cell densities in planta, PhcA mediates a trade-off from maximizing growth to producing costly virulence factors. R. solanacearum infects through roots, and low-cell-density-mode-mimicking ΔphcA cells attached to tomato roots better than the wild-type cells, consistent with their increased expression of several adhesins. Inside xylem vessels, ΔphcA cells formed aberrantly dense mats. Possibly as a result, the mutant could not spread up or down tomato stems as well as the wild type. This suggests that aggregating improves R. solanacearum survival in soil and facilitates infection and that it reduces pathogenic fitness later in disease. Thus, PhcA mediates a second strategic switch between initial pathogen attachment and subsequent dispersal inside the host. PhcA helps R. solanacearum optimally invest resources and correctly sequence multiple steps in the bacterial wilt disease cycle. PMID:28951474

Spread of Hepatitis B Viruses In Vitro Requires Extracellular Progeny and May Be Codetermined by Polarized Egress

PubMed Central

Funk, A.; Hohenberg, H.; Mhamdi, M.; Will, H.; Sirma, H.

2004-01-01

Viruses can spread by different mechanisms: via intracellular particles through cell junctions to neighboring cells or via secreted virions to adjacent or remote cells. The observation of clusters of hepadnavirus-infected cells both in vivo and in primary hepatocytes neither proves the first mechanism nor excludes the second. In order to test which mechanism, if not both, is used by hepatitis B viruses in order to spread, we used primary duck hepatocytes and duck hepatitis B virus (DHBV) as an infection model. If extracellular progeny virus alone determines spreading, neutralizing antisera or drugs blocking virus binding to hepatocytes should abolish secondary infection. In order to test this, we used DHBV envelope-specific neutralizing antisera, as well as suramin, a known inhibitor of infection. Both reagents strongly reduced hepatocellular attachment of viral particles and almost completely abolished primary infection, whereas an ongoing intracellular infection was not affected as long as no progeny virus was released. In contrast, incubation of infected primary hepatocytes with these reagents during release of progeny virus completely prevented secondary infection. Moreover, the combination of electron and immunofluorescence microscopy analyses revealed the residence of viral particles in cytoplasmic vesicles preferentially located near the basolateral membrane of infected hepatocytes. Taken together, these data strongly suggest that hepatitis B viruses mainly spread by secreted, extracellular progeny and point to polarized egress of viral particles into intercellular compartments, which restricts their diffusion and favors transmission of virus to adjacent cells. PMID:15047813

Functionalized coatings by cold spray: An in vitro study of micro- and nanocrystalline hydroxyapatite compared to porous titanium.

PubMed

Vilardell, A M; Cinca, N; Garcia-Giralt, N; Dosta, S; Cano, I G; Nogués, X; Guilemany, J M

2018-06-01

Three different surface treatments on a Ti6Al4V alloy have been in vitro tested for possible application in cementless joint prosthesis. All of them involve the novelty of using the Cold Spray technology for their deposition: (i) an as-sprayed highly rough titanium and, followed by the deposition of a thin hydroxyapatite layer with (ii) microcrystalline or (iii) nanocrystalline structure. Primary human osteoblasts were extracted from knee and seeded onto the three different surfaces. Cell viability was tested by MTS and LIVE/DEAD assays, cell differentiation by alkaline phosphatase (ALP) quantification and cell morphology by Phalloidin staining. All tests were carried out at 1, 7 and 14 days of cell culture. Different cell morphologies between titanium and hydroxyapatite surfaces were exhibited. At 1 day of cell culture, cells on the titanium coating were spread and flattened, expanding the filopodia actin filaments in all directions, while cells on the hydroxyapatite coatings showed round like-shape morphology due to slower attachment. Higher cell viability was detected at all times of cell culture on titanium coating due to a better attachment at 1 day. However, from 7 days of cell culture, cells on hydroxyapatite showed good attachment onto surfaces and highly increased their proliferation, mostly on nanocrystalline, achieving similar cell viability levels than titanium coatings. ALP levels were significantly higher in titanium, in part, because of greatest cell number. Overall, the best cell functional results were obtained on titanium coatings whereas microcrystalline hydroxyapatite presented the worst cellular parameters. However, results indicate that nanocrystalline hydroxyapatite coatings may achieve promising results for the faster cell proliferation once cells are attached on the surface. Copyright © 2018 Elsevier B.V. All rights reserved.

The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

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

Delpeut, Sebastien; Noyce, Ryan S.; IWK Health Centre, Canadian Center for Vaccinology, Goldbloom Pavilion, Halifax, Nova Scotia, Canada B3H 1X5

The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4more » (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.« less

Synchronized cell attachment triggered by photo-activatable adhesive ligands allows QCM-based detection of early integrin binding

PubMed Central

Iturri, Jagoba; García-Fernández, Luis; Reuning, Ute; García, Andrés J.; Campo, Aránzazu del; Salierno, Marcelo J.

2015-01-01

The Quartz Crystal Microbalance with dissipation (QCM-D) technique was applied to monitor and quantify integrin-RGD recognition during the early stages of cell adhesion. Using QCM-D crystals modified with a photo-activatable RGD peptide, the time point of presentation of adhesive ligand at the surface of the QCM-D crystal could be accurately controlled. This allowed temporal resolution of early integrin-RGD binding and the subsequent cell spreading process, and their separate detection by QCM-D. The specificity of the integrin-RGD binding event was corroborated by performing the experiments in the presence of soluble cyclicRGD as a competitor, and cytochalasin D as inhibitor of cell spreading. Larger frequency change in the QCM-D signal was observed for cells with larger spread area, and for cells overexpressing integrin αvβ3 upon stable transfection. This strategy enables quantification of integrin activity which, in turn, may allow discrimination among different cell types displaying distinct integrin subtypes and expression levels thereof. On the basis of these findings, we believe the strategy can be extended to other photoactivatable ligands to characterize cell membrane receptors activity, a relevant issue for cancer diagnosis (and prognosis) as other several pathologies. PMID:25825012

Directional cell elongation through filopodia-steered lamellipodial extension on patterned silk fibroin films.

PubMed

You, Renchuan; Li, Xiufang; Luo, Zuwei; Qu, Jing; Li, Mingzhong

2015-03-05

Micropatterned biomaterials have been used to direct cell alignment for specific tissue engineering applications. However, the understanding of how cells respond to guidance cues remains limited. Plasticity in protrusion formation has been proposed to enable cells to adapt their motility mode to microenvironment. In this study, the authors investigated the key role of protrusion response in cell guidance on patterned silk fibroin films. The results revealed that the ability to transform between filopodia and small lamellipodia played important roles in directional cell guidance. Filopodia did not show directional extension on patterned substrates prior to spreading, but they transduced topographical cues to the cell to trigger the formation of small lamellipodia along the direction of a microgrooved or parallel nanofiber pattern. The polar lamellipodia formation provided not only a path with directionality, but a driving force for directional cell elongation. Moreover, aligned nanofibers coating provided better mechanical support for the traction of filopodia and lamellipodia, promoting cell attachment, spreading, and migration. This study provides new insight into how cells respond to guidance cues and how filopodia and lamellipodia control cell contact guidance on micropatterned biomaterial surfaces.

Biomimetic surface patterning for long-term transmembrane access

PubMed Central

VanDersarl, Jules J.; Renaud, Philippe

2016-01-01

Here we present a planar patch clamp chip based on biomimetic cell membrane fusion. This architecture uses nanometer length-scale surface patterning to replicate the structure and function of membrane proteins, creating a gigaohm seal between the cell and a planar electrode array. The seal is generated passively during cell spreading, without the application of a vacuum to the cell surface. This interface can enable cell-attached and whole-cell recordings that are stable to 72 hours, and generates no visible damage to the cell. The electrodes can be very small (<5 μm) and closely packed, offering a high density platform for cellular measurement. PMID:27577519

Biomimetic surface patterning for long-term transmembrane access.

PubMed

VanDersarl, Jules J; Renaud, Philippe

2016-08-31

Here we present a planar patch clamp chip based on biomimetic cell membrane fusion. This architecture uses nanometer length-scale surface patterning to replicate the structure and function of membrane proteins, creating a gigaohm seal between the cell and a planar electrode array. The seal is generated passively during cell spreading, without the application of a vacuum to the cell surface. This interface can enable cell-attached and whole-cell recordings that are stable to 72 hours, and generates no visible damage to the cell. The electrodes can be very small (<5 μm) and closely packed, offering a high density platform for cellular measurement.

Highly specific detection of H2O2-dependent luminol chemiluminescence in stimulated human leukocytes using polyvinyl films.

PubMed

Moriguchi, K; Ohno, N; Ogawa, T; Hirai, K

1999-01-01

When human polymorphonuclear leukocytes (PMN) were attached to glass coverslips, cells always spread and formed reactive oxygen species prior to any experimental stimulation. To avoid this, a polyvinylidine chloride film was used as an inactive substance to place the cells. Cells engaged in phagocytosis on the film exhibited a specific H2O2-mediated luminol chemiluminescence (LCL) at the cell-particle interface; the cells stimulated with 12-O-tetradecanoylphorbol-13-acetate became aggregated and the LCL was observed at the cell-cell contact. These results corresponded well with those obtained by an electron microscopic H2O2-demonstration method.

Biofunctionalization of a “Clickable” Organic Layer Photochemically Grafted on Titanium Substrates

PubMed Central

Li, Yan; Zhao, Meirong; Wang, Jun; Liu, Kai; Cai, Chengzhi

2011-01-01

We have developed a general method combining photochemical grafting and copper-catalyzed click chemistry for biofunctionalization of titanium substrates. The UV-activated grafting of an α,ω-alkenyne onto TiO2/Ti substrates provided a “clickable” thin film platform. The selective attachment of the vinyl end of the molecule to the surface was achieved by masking the alkynyl end with a trimethylgermanyl (TMG) protecting group. Subsequently, various oligo(ethylene glycol) (OEG) derivatives terminated with an azido group were attached to the TMG-alkynyl modified titanium surface via a one-pot deprotection/click reaction. The films were characterized by X-ray photoelectron spectroscopy (XPS), contact angle goniometry, ellipsometry, and atomic force microscopy (AFM). We showed that the titanium surface presenting click-immobilized OEG substantially suppressed the nonspecific attachment of protein and cells as compared to the unmodified titanium substrate. Furthermore, glycine-arginine-glycine-aspartate (GRGD), a cell adhesion peptide, was coimmobilized with OEG on the platform. We demonstrated that the resultant GRGD-presenting thin film on Ti substrates can promote the specific adhesion and spreading of AsPC-1 cells. PMID:21417429

Gelatin/chondroitin sulfate nanofibrous scaffolds for stimulation of wound healing: In-vitro and in-vivo study.

PubMed

Pezeshki-Modaress, Mohamad; Mirzadeh, Hamid; Zandi, Mojgan; Rajabi-Zeleti, Sareh; Sodeifi, Niloofar; Aghdami, Nasser; Mofrad, Mohammad R K

2017-07-01

In this research, fabrication of gelatin/chondroitin sulfate (GAG) nanofibrous scaffolds using electrospinning technique for skin tissue engineering was studied. The influence of GAG content on chemical, physical, mechanical and biological properties of the scaffolds were investigated. Human dermal fibroblast (HDF) cells were cultured and bioactivity of electrospun gelatin/GAG scaffolds for skin tissue engineering was assayed. Biological results illustrated that HDF cells attached and spread well on gelatin/GAG nanofibrous scaffolds displaying spindle-like shapes and stretching. MTS assay was performed to evaluate the cell proliferation on electrospun gelatin/GAG scaffolds. The results confirmed the influence of GAG content as well as the nanofibrous structure on cell proliferation and attachment of substrates. The gelatin/GAG nanofibrous scaffolds with the desired thickness for in-vivo evaluations were used on the full-thickness wounds. Pathobiological results showed that cell loaded gelatin/GAG scaffolds significantly accelerated wounds healing. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2020-2034, 2017. © 2017 Wiley Periodicals, Inc.

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

NASA Technical Reports Server (NTRS)

Fong, J. H.; Ingber, D. E.

1996-01-01

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

Microgravity

NASA Image and Video Library

2004-04-15

The loss of productivity due to flu is staggering. Costs range as much as $20 billio a year. High mutation rates of the flu virus have hindered development of new drugs or vaccines. The secret lies in a small molecule which is attached to the host cell's surface. Each flu virus, no matter what strain, must remove this small molecule to escape the host cell to spread infection. Using data from space and earth grown crystals, researchers from the Center of Macromolecular Crystallography (CMC) are desining drugs to bind with this protein's active site. This lock and key fit reduces the spread of flu in the body by blocking its escape route. In collaboration with its corporate partner, the CMC has refined drug structure in preparation for clinical trials. Tested and approved relief is expected to reach drugstores by year 2004.

Age-associated reduction of cellular spreading/mechanical force up-regulates matrix metalloproteinase-1 expression and collagen fibril fragmentation via c-Jun/AP-1 in human dermal fibroblasts.

PubMed

Qin, Zhaoping; Voorhees, John J; Fisher, Gary J; Quan, Taihao

2014-12-01

The dermal compartment of human skin is largely composed of dense collagen-rich fibrils, which provide structural and mechanical support. Skin dermal fibroblasts, the major collagen-producing cells, are interact with collagen fibrils to maintain cell spreading and mechanical force for function. A characteristic feature of aged human skin is fragmentation of collagen fibrils, which is initiated by matrix metalloproteinase 1 (MMP-1). Fragmentation impairs fibroblast attachment and thereby reduces spreading. Here, we investigated the relationship among fibroblast spreading, mechanical force, MMP-1 expression, and collagen fibril fragmentation. Reduced fibroblast spreading due to cytoskeletal disruption was associated with reduced cellular mechanical force, as determined by atomic force microscopy. These reductions substantially induced MMP-1 expression, which led to collagen fibril fragmentation and disorganization in three-dimensional collagen lattices. Constraining fibroblast size by culturing on slides coated with collagen micropatterns also significantly induced MMP-1 expression. Reduced spreading/mechanical force induced transcription factor c-Jun and its binding to a canonical AP-1 binding site in the MMP-1 proximal promoter. Blocking c-Jun function with dominant negative mutant c-Jun significantly reduced induction of MMP-1 expression in response to reduced spreading/mechanical force. Furthermore, restoration of fibroblast spreading/mechanical force led to decline of c-Jun and MMP-1 levels and eliminated collagen fibril fragmentation and disorganization. These data reveal a novel mechanism by which alteration of fibroblast shape/mechanical force regulates c-Jun/AP-1-dependent expression of MMP-1 and consequent collagen fibril fragmentation. This mechanism provides a foundation for understanding the cellular and molecular basis of age-related collagen fragmentation in human skin. © 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Bioactivity of Y2O3 and CeO2 doped SiO2-SrO-Na2O glass-ceramics.

PubMed

Placek, L M; Keenan, T J; Wren, A W

2016-08-01

The bioactivity of yttrium and cerium are investigated when substituted for Sodium (Na) in a 0.52SiO2-0.24SrO-0.24-xNa2O-xMO glass-ceramics (where x = 0.08 and MO = Y2O3 or CeO2). Bioactivity is monitored through pH and inductively coupled plasma-optical emission spectrometry where pH of simulated body fluid ranged from 7.5 to 7.6 and increased between 8.2 and 10.0 after 14-day incubation with the glass-ceramic disks. Calcium (Ca) and phosphorus (P) levels in simulated body fluid after incubation with yttrium and cerium containing disks show a continual decline over the 14-day period. In contrast, Con disks (not containing yttrium or cerium) caused the elimination of Ca in solution after 1 day and throughout the incubation period, and initially showed a decline in P levels followed by an increase at 14 days. Scanning electron microscopy and energy dispersive spectroscopy confirmed the presence of Ca and P on the surface of the simulated body fluid-incubated disks and showed precipitates on Con and HCe (8 mol% cerium) samples. Cell viability of MC3T3 osteoblasts was not significantly affected at a 9% extract concentration. Optical microscopy after 24 h cell incubation with disks showed that Con samples do not support osteoblast or Schwann cell growth, while all yttrium and cerium containing disks have direct contact with osteoblasts spread across the wells. Schwann cells attached in all wells, but only showed spreading with the HY-S (8 mol% yttrium, heated to sintering temperature) and YCe (4 mol% yttrium and cerium) disks. Scanning electron microscopy of the compatible disks shows osteoblast and sNF96.2 Schwann cells attachment and spreading directly on the disk surfaces. © The Author(s) 2016.

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

PubMed

Saitakis, Michael; Gizeli, Electra

2012-02-01

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

Monitoring biofilm attachment on medical devices surfaces using hyperspectral imaging

NASA Astrophysics Data System (ADS)

Le, Hanh N. D.; Hitchins, Victoria M.; Ilev, Ilko K.; Kim, Do-Hyun

2014-02-01

Microbial biofilm is a colony of single bacteria cells (planktonic) that attached to surfaces, attract other microorganisms to attach and grow, and together they build an extracellular matrix composed of polysaccharides, protein, and DNA. Eventually, some cells will detach and spread to other surface. Biofilm on medical devices can cause severe infection to all age ranges from infant to adult. Therefore, it is important to detect biofilm in a fast and efficient manner. Hyperspectral imaging was utilized for distinguishing wide area of biofilm coverage on various materials and on different textures of stainless steeltest coupons. Not only is the coverage of biofilm important, but also the shear stress of biofilm on the attached surfaces is significant. This study investigates the effects of shear stress on the adhesion of biofilms on common medical device surfaces such as glass, polycarbonate, polytetrafluoroethylene, and stainless steel with different textures. Biofilm was grown using Ps. aeruginosa and growth was monitored after 24 and 48 hours at 37° C. The coupons covered with biofilm were tilted at 45 degrees and 90 degrees for 30 seconds to induce shear stress and Hyperspectral images were taken. We hypothesize that stronger attachment on rough surface would be able to withstand greater shear stress compared to smooth surface.

Towards an ideal polymer scaffold for tendon/ligament tissue engineering

NASA Astrophysics Data System (ADS)

Sahoo, Sambit; Ouyang, Hong Wei; Goh, James Cho-Hong; Tay, Tong-Earn; Toh, Siew Lok

2005-04-01

Tissue engineering holds promise in treating injured tendons and ligaments by replacing the injured tissues with "engineered tissues" with identical mechanical and functional characteristics. A biocompatible, biodegradable, porous scaffold with optimized architecture, sufficient surface area for cell attachment, growth and proliferation, faborable mechanical properties, and suitable degradation rate is a pre-requisite to achieve success with this aproach. Knitted poly(lactide-co-glycolide) (PLGA) scaffolds comprising of microfibers of 25 micron diameter were coated with PLGA nanofibers on their surfaces by electrospinning technique. A cell suspension of pig bone marrow stromal cells (BMSC) was seeded on the scaffolds by pipetting, and the cell-scaffold constructs were cultured in a CO2 incubator, at 37°C for 1-2 weeks. The "engineered tissues" were then assessed for cell attachment and proliferation, tissue formation, and mechanical properties. Nanofibers, of diameter 300-900 nm, were spread randomly over the knitted scaffold. The reduction in pore-size from about 1 mm (in the knitted scaffold) to a few micrometers (in the nano-microscaffold) allowed cell seeding by direct pipetting, and eliminated the need of a cell-delivery system like fibrin gel. BMSCs were seen to attach and proliferate well on the nano-microscaffold, producing abundant extracellular matrix. Mechanical testing revealed that the cell-seeded nano-microscaffolds possessed slightly higher values of failure load, elastic-region stiffness and toe-region stiffness, than the unseeded scaffolds. The combination of superior mechanical strength and integrity of knitted microfibers, with the large surface area and improved hydrophilicity of the electrospun nanofibers facilitated cell attachment and new tissue formation. This holds promise in tissue engineering of tendon/ligament.

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

PubMed Central

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

2009-01-01

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

3-Dimensional Protein Structure of Influenza

NASA Technical Reports Server (NTRS)

2004-01-01

The loss of productivity due to flu is staggering. Costs range as much as $20 billio a year. High mutation rates of the flu virus have hindered development of new drugs or vaccines. The secret lies in a small molecule which is attached to the host cell's surface. Each flu virus, no matter what strain, must remove this small molecule to escape the host cell to spread infection. Using data from space and earth grown crystals, researchers from the Center of Macromolecular Crystallography (CMC) are desining drugs to bind with this protein's active site. This lock and key fit reduces the spread of flu in the body by blocking its escape route. In collaboration with its corporate partner, the CMC has refined drug structure in preparation for clinical trials. Tested and approved relief is expected to reach drugstores by year 2004.

Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions▿ †

PubMed Central

Sherer, Nathan M.; Jin, Jing; Mothes, Walther

2010-01-01

The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions. PMID:20089647

The role of endocytic Rab GTPases in regulation of growth factor signaling and the migration and invasion of tumor cells

PubMed Central

Porther, N; Barbieri, MA

2015-01-01

Metastasis is characterized pathologically by uncontrolled cell invasion, proliferation, migration and angiogenesis. It is a multistep process that encompasses the modulation of membrane permeability and invasion, cell spreading, cell migration and proliferation of the extracellular matrix, increase in cell adhesion molecules and interaction, decrease in cell attachment and induced survival signals and propagation of nutrient supplies (blood vessels). In cancer, a solid tumor cannot expand and spread without a series of synchronized events. Changes in cell adhesion receptor molecules (e.g., integrins, cadherin-catenins) and protease expressions have been linked to tumor invasion and metastasis. It has also been determined that ligand-growth factor receptor interactions have been associated with cancer development and metastasis via the endocytic pathway. Specifically, growth factors, which include IGF-1 and IGF-2 therapy, have been associated with most if not all of the features of metastasis. In this review, we will revisit some of the key findings on perhaps one of the most important hallmarks of cancer metastasis: cell migration and cell invasion and the role of the endocytic pathway in mediating this phenomenon PMID:26317377

Recombinant Spider Silk Functionalized with a Motif from Fibronectin Mediates Cell Adhesion and Growth on Polymeric Substrates by Entrapping Cells During Self-Assembly.

PubMed

Tasiopoulos, Christos Panagiotis; Widhe, Mona; Hedhammar, My

2018-05-02

In vitro endothelialization of synthetic grafts or engineered vascular constructs is considered a promising alternative to overcome shortcomings in the availability of autologous vessels and in-graft complications with synthetics. A number of cell-seeding techniques have been implemented to render vascular grafts accessible for cells to attach, proliferate, and spread over the surface area. Nonetheless, seeding efficiency and the time needed for cells to adhere varies dramatically. Herein, we investigated a novel cell-seeding approach (denoted co-seeding) that enables cells to bind to a motif from fibronectin included in a recombinant spider silk protein. Entrapment of cells occurs at the same time as the silk assembles into a nanofibrillar coating on various substrates. Cell adhesion analysis showed that the technique can markedly improve cell-seeding efficiency to nonfunctionalized polystyrene surfaces, as well as establish cell attachment and growth of human dermal microvascular endothelial cells on bare polyethylene terephthalate and polytetrafluoroethylene (PTFE) substrates. Scanning electron microscopy images revealed a uniform endothelial cell layer and cell-substratum compliance with the functionalized silk protein to PTFE surfaces. The co-seeding technique holds a great promise as a method to reliably and quickly cellularize engineered vascular constructs as well as to in vitro endothelialize commercially available cardiovascular grafts.

Herpes Simplex Virus Type 2 Glycoprotein G Is Targeted by the Sulfated Oligo- and Polysaccharide Inhibitors of Virus Attachment to Cells▿

PubMed Central

Adamiak, Beata; Ekblad, Maria; Bergström, Tomas; Ferro, Vito; Trybala, Edward

2007-01-01

Variants of herpes simplex virus type 2 (HSV-2) generated by virus passage in GMK-AH1 cells in the presence of the sulfated oligosaccharide PI-88 were analyzed. Many of these variants were substantially resistant to PI-88 in their initial infection of cells and/or their cell-to-cell spread. The major alteration detected in all variants resistant to PI-88 in the initial infection of cells was a frameshift mutation(s) in the glycoprotein G (gG) gene that resulted in the lack of protein expression. Molecular transfer of the altered gG gene into the wild-type background confirmed that the gG-deficient recombinants were resistant to PI-88. In addition to PI-88, all gG-deficient variants of HSV-2 were resistant to the sulfated polysaccharide heparin. The gG-deficient virions were capable of attaching to cells, and this activity was relatively resistant to PI-88. In addition to having a drug-resistant phenotype, the gG-deficient variants were inefficiently released from infected cells. Purified gG bound to heparin and showed the cell-binding activity which was inhibited by PI-88. Many PI-88 variants produced syncytia in cultured cells and contained alterations in gB, including the syncytium-inducing L792P amino acid substitution. Although this phenotype can enhance the lateral spread of HSV in cells, it conferred no virus resistance to PI-88. Some PI-88 variants also contained occasional alterations in gC, gD, gE, gK, and UL24. In conclusion, we found that glycoprotein gG, a mucin-like component of the HSV-2 envelope, was targeted by sulfated oligo- and polysaccharides. This is a novel finding that suggests the involvement of HSV-2 gG in interactions with sulfated polysaccharides, including cell surface glycosaminoglycans. PMID:17928351

Towards understanding of Nipah virus attachment protein assembly and the role of protein affinity and crowding for membrane curvature events.

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

Stachowiak, Jeanne C.; Hayden, Carl C.; Negrete, Oscar.

2013-10-01

Pathogenic viruses are a primary threat to our national security and to the health and economy of our world. Effective defense strategies to combat viral infection and spread require the development of understanding of the mechanisms that these pathogens use to invade the host cell. We present in this report results of our research into viral particle recognition and fusion to cell membranes and the role that protein affinity and confinement in lipid domains plays in membrane curvature in cellular fusion and fission events. Herein, we describe 1) the assembly of the G attachment protein of Nipah virus using pointmore » mutation studies to define its role in viral particle fusion to the cell membrane, 2) how lateral pressure of membrane bound proteins induce curvature in model membrane systems, and 3) the role of membrane curvature in the selective partitioning of molecular receptors and specific affinity of associated proteins.« less

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

PubMed Central

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

2017-01-01

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

Using self-assembled monolayers to pattern ECM proteins and cells on substrates.

PubMed

Ostuni, Emanuele; Whitesides, George M; Ingber, Donald E; Chen, Christopher S

2009-01-01

We present a method that uses microcontact printing of alkanethiols on gold to generate patterned substrates presenting "islands" of extracellular matrix (ECM) surrounded by nonadhesive regions such that single cells attach and spread only on the adhesive regions. We have used this micropatterning technology to demonstrate that mammalian cells can be switched between growth and apoptosis programs in the presence of saturating concentrations of growth factors by either promoting or preventing cell spreading (Science 276:1425-1428, 1997). From the perspective of fundamental cell biology, these results suggested that the local differentials in growth and viability that are critical for the formation of complex tissue patterns may be generated by local changes in cell-ECM interactions. In the context of cell culture technologies, such as bioreactors and cellular engineering applications, the regulation of cell function by cell shape indicates that the adhesive microenvironment around cells can be carefully optimized by patterning a substrate in addition to using soluble factors (Biotech. Prog. 14:356-363, 1998). Micropatterning technology is playing a central role both in our understanding how ECM and cell shape regulate cell physiology and in facilitating the development of cellular biosensor and tissue engineering applications (Science 264:696-698, 1994; J. Neurosci. Res. 13:213-20, 1985; Biotech. Bioeng. 43:792-800, 1994).

Comparison of osteoblast-like cell responses to calcium silicate and tricalcium phosphate ceramics in vitro.

PubMed

Ni, Siyu; Chang, Jiang; Chou, Lee; Zhai, Wanyin

2007-01-01

Calcium silicate ceramics have been proposed as new bone repair biomaterials, since they have proved to be bioactive, degradable, and biocompatible. Beta-tricalcium phosphate ceramic is a well-known degradable material for bone repair. This study compared the effects of CaSiO3 (alpha-, and beta-CaSiO3) and beta-Ca3(PO4)2 (beta-TCP) ceramics on the early stages of rat osteoblast-like cell attachment, proliferation, and differentiation. Osteoblast-like cells were cultured directly on CaSiO3 (alpha-, and beta-CaSiO3) and beta-TCP ceramics. Attachment of a greater number of cells was observed on CaSiO3 (alpha-, and beta-CaSiO3) ceramics compared with beta-TCP ceramics after incubation for 6 h. SEM observations showed an intimate contact between cells and the substrates, significant cells adhesion, and that the cells spread and grew on the surfaces of all the materials. In addition, the proliferation rate and alkaline phosphatase (ALP) activity of the cells on the CaSiO3 (alpha-, and beta-CaSiO3) ceramics were improved when compared with the beta-TCP ceramics. In the presence of CaSiO3, elevated levels of calcium and silicon in the culture medium were observed throughout the 7-day culture period. In conclusion, the results of the present study revealed that CaSiO3 ceramics showed greater ability to support cell attachment, proliferation, and differentiation than beta-TCP ceramic. 2006 Wiley Periodicals, Inc.

The effect of Si nano-columns in 2-D and 3-D on cellular behaviour: nanotopography-induced CaP deposition from differentiating mesenchymal stem cells.

PubMed

Guvendik, S; Trabzon, L; Ramazanoglu, M

2011-10-01

Si nano-columns were deposited in 2-D and 3-D in the form of well-defined geometries by physical vapor deposition. The films were grown by e-beam evaporation with an angle between source and substrate. The Si nano-columns were deposited in the shape of spiral with two different incoming atomic flux angle so that the manipulation of nano-columns in 3-D (out-of-plane) was obtained. The Si nano-columns were also grown as vertical stick with square, triangle and linear cross sections in 2D (in-plane). Rat bone marrow mesenchymal stem cells (MSCs) were cultured on these different Si nanosurfaces. MTS assay was carried out to determine the cell proliferation and viability based on different nanotopographies. For the evaluation of cell distribution and morphology, a SEM (Scanning Electron Microscopy) analysis was performed. Any CaP deposition on Si nanosurfaces was observed using energy dispersive X-Ray spectroscopy in SEM (SEM-EDX). After 4 days of culture, there was a higher value of cell proliferation on square columns and spiral Si nano-columns grown with 85 degrees of incoming atomic flux. The cell attachment and spreading was also affected by the geometry of Si nano-columns. While there were still cells showing round/spherical morphology with minimal spreading on conventional Si surfaces, most of the cells cultured on different Si nanotopographies attached on the surface and displayed flattened morphology, especially on the square columns surface. Moreover, CaP deposition was discovered on square columns and spiral films with 85 degrees substrate angle. So, it can be concluded that there is a clear correlation between cell responses and nano-sized geometry on Si surface and it is possible to induce cellular differentiation and CaP formation in certain geometrical constraints.

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

PubMed

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

2017-10-12

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

Development of magnesium calcium phosphate biocement for bone regeneration.

PubMed

Jia, Junfeng; Zhou, Huanjun; Wei, Jie; Jiang, Xin; Hua, Hong; Chen, Fangping; Wei, Shicheng; Shin, Jung-Woog; Liu, Changsheng

2010-08-06

Magnesium calcium phosphate biocement (MCPB) with rapid-setting characteristics was fabricated by using the mixed powders of magnesium oxide (MgO) and calcium dihydrogen phosphate (Ca(H(2)PO(4))(2).H(2)O). The results revealed that the MCPB hardened after mixing the powders with water for about 7 min, and the compressive strength reached 43 MPa after setting for 1 h, indicating that the MCPB had a short setting time and high initial mechanical strength. After the acid-base reaction of MCPB containing MgO and Ca(H(2)PO(4))(2).H(2)O in a molar ratio of 2 : 1, the final hydrated products were Mg(3)(PO(4))(2) and Ca(3)(PO(4))(2). The MCPB was degradable in Tris-HCl solution and the degradation ratio was obviously higher than calcium phosphate biocement (CPB) because of its fast dissolution. The attachment and proliferation of the MG(63) cells on the MCPB were significantly enhanced in comparison with CPB, and the alkaline phosphatase activity of MG(63) cells on the MCPB was significantly higher than on the CPB at 7 and 14 days. The MG(63) cells with normal phenotype spread well on the MCPB surfaces, and were attached in close proximity to the substrate, as seen by scanning electron microscopy (SEM). The results demonstrated that the MCPB had a good ability to support cell attachment, proliferation and differentiation, and exhibited good cytocompatibility.

Establishment of Epithelial Attachment on Titanium Surface Coated with Platelet Activating Peptide

PubMed Central

Sugawara, Shiho; Maeno, Masahiko; Lee, Cliff; Nagai, Shigemi; Kim, David M.; Da Silva, John; Kondo, Hisatomo

2016-01-01

The aim of this study was to produce epithelial attachment on a typical implant abutment surface of smooth titanium. A challenging complication that hinders the success of dental implants is peri-implantitis. A common cause of peri-implantitis may results from the lack of epithelial sealing at the peri-implant collar. Histologically, epithelial sealing is recognized as the attachment of the basement membrane (BM). BM-attachment is promoted by activated platelet aggregates at surgical wound sites. On the other hand, platelets did not aggregate on smooth titanium, the surface typical of the implant abutment. We then hypothesized that epithelial BM-attachment was produced when titanium surface was modified to allow platelet aggregation. Titanium surfaces were coated with a protease activated receptor 4-activating peptide (PAR4-AP). PAR4-AP coating yielded rapid aggregation of platelets on the titanium surface. Platelet aggregates released robust amount of epithelial chemoattractants (IGF-I, TGF-β) and growth factors (EGF, VEGF) on the titanium surface. Human gingival epithelial cells, when they were co-cultured on the platelet aggregates, successfully attached to the PAR4-AP coated titanium surface with spread laminin5 positive BM and consecutive staining of the epithelial tight junction component ZO1, indicating the formation of complete epithelial sheet. These in-vitro results indicate the establishment of epithelial BM-attachment to the titanium surface. PMID:27741287

The complex leaves of the monkey's comb (Amphilophium crucigerum, Bignoniaceae): a climbing strategy without glue.

PubMed

Seidelmann, Katrin; Melzer, Björn; Speck, Thomas

2012-11-01

Monkey's comb (Amphilophium crucigerum) is a widely spread neotropical leaf climber that develops attachment pads for anchorage. A single complex leaf of the species comprises a basal pair of foliate, assimilating leaflets and apical, attaching leaflet tendrils. This study aims to analyze these leaves and their ontogenetic development for a better understanding of the attachment process, the form-structure-function relationships involved, and the overall maturation of the leaves. Thorough morphometrical, morphological, and anatomical analyses incorporated high-resolution microscopy, various staining techniques, SEM, and photographic recordings over the entire ontogenetic course of leaf development. The foliate, assimilating leaflets and the anchorage of the more apical leaflet tendrils acted independently of each other. Attachment was achieved by coiling of the leaflet tendrils and/or development of attachment pads at the tendril apices that grow opportunistically into gaps and fissures of the substrate. In contact zones with the substrate, the cells of the pads differentiate into a vessel element-like tissue. During the entire attachment process of the plant, no glue was excreted. The complex leaves of monkey's comb are highly differentiated organs with specialized leaf parts whose functions-photosynthesis or attachment-work independently of each other. The function of attachment includes coiling and maturation process of the leaflet tendrils and the formation of attachment pads, resulting in a biomechanically sound and persistent anchorage of the plant without the need of glue excretion. This kind of glue-less attachment is not only of interest in the framework of analyzing the functional variety of attachment structures evolved in climbing plants, but also for the development of innovative biomimetic attachment structures for manifold technical applications.

Nanoporous metals for biodegradable implants: Initial bone mesenchymal stem cell adhesion and degradation behavior.

PubMed

Heiden, Michael; Huang, Sabrina; Nauman, Eric; Johnson, David; Stanciu, Lia

2016-07-01

Nanostructured Fe-Mn and Fe-Mn-Zn metal scaffolds were generated through a well-controlled selective leaching process in order to fulfill the growing demand for adjustable degradation rates and improved cellular response of resorbable materials. Mouse bone marrow mesenchymal stem cells (D1 ORL UVA) were seeded onto eleven, carefully chosen nanoporous surfaces for 24 h in vitro. Using a combination of fluorescence microscopy, scanning electron microscopy (SEM), and an MTS assay, it was discovered that scaffolds with nanoscale roughened surfaces had increased cell attachment by up to 123% compared to polished smooth Fe-Mn surfaces. Significant cell spreading and construction of cell multilayers were also apparent after 24 h, suggesting better adhesion. Additionally, static electrochemical polarization experiments revealed an improvement of up to 26% in the actual rate of biodegradation for Fe-Mn surface-modified materials. However, any residual concentration of zinc after leaching was shown to slightly increase corrosion resistance. The results demonstrate that selectively leached, nanostructured Fe-Mn surfaces have the potential of being tailored to a diverse set of transient implant scenarios, while also effectively boosting overall biocompatibility, initial cell attachment, and degradation rate. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1747-1758, 2016. © 2016 Wiley Periodicals, Inc.

Cellular response of preosteoblasts to nanograined/ultrafine-grained structures.

PubMed

Misra, R D K; Thein-Han, W W; Pesacreta, T C; Hasenstein, K H; Somani, M C; Karjalainen, L P

2009-06-01

Metallic materials with submicron- to nanometer-sized grains provide surfaces that are different from conventional polycrystalline materials because of the large proportion of grain boundaries with high free energy. In the study described here, the combination of cellular and molecular biology, materials science and engineering advances our understanding of cell-substrate interactions, especially the cellular activity between preosteoblasts and nanostructured metallic surfaces. Experiments on the effect of nano-/ultrafine grains have shown that cell attachment, proliferation, viability, morphology and spread are favorably modulated and significantly different from conventional coarse-grained structures. Additionally, immunofluorescence studies demonstrated stronger vinculin signals associated with actin stress fibers in the outer regions of the cells and cellular extensions on nanograined/ultrafine-grained substrate. These observations suggest enhanced cell-substrate interaction and activity. The differences in the cellular response on nanograined/ultrafine-grained and coarse-grained substrates are attributed to grain size and degree of hydrophilicity. The outcomes of the study are expected to reduce challenges to engineer bulk nanostructured materials with specific physical and surface properties for medical devices with improved cellular attachment and response. The data lay the foundation for a new branch of nanostructured materials for biomedical applications.

Improved Mesenchymal Stem Cells Attachment and In Vitro Cartilage Tissue Formation on Chitosan-Modified Poly(l-Lactide-co-Epsilon-Caprolactone) Scaffold

PubMed Central

Wu, Yingnan; Li, Chao; Zhang, Tianting; Zou, Yu; Hui, James H.P.; Lee, Eng Hin

2012-01-01

Considering the load-bearing physiological requirement of articular cartilage, scaffold for cartilage tissue engineering should exhibit appropriate mechanical responses as natural cartilage undergoing temporary deformation on loading with little structural collapse, and recovering to the original geometry on unloading. A porous elastomeric poly l-lactide-co-ɛ-caprolactone (PLCL) was generated and crosslinked at the surface to chitosan to improve its wettability. Human bone marrow derived mesenchymal stem cells (MSC) attachment, morphological change, proliferation and in vitro cartilage tissue formation on the chitosan-modified PLCL scaffold were compared with the unmodified PLCL scaffold. Chitosan surface promoted more consistent and even distribution of the seeded MSC within the scaffold. MSC rapidly adopted a distinct spread-up morphology on attachment on the chitosan-modified PLCL scaffold with the formation of F-actin stress fiber which proceeded to cell aggregation; an event much delayed in the unmodified PLCL. Enhanced cartilage formation on the chitosan-modified PLCL was shown by real-time PCR analysis, histological and immunochemistry staining and biochemical assays of the cartilage extracellular matrix components. The Young's modulus of the derived cartilage tissues on the chitosan-modified PLCL scaffold was significantly increased and doubled that of the unmodified PLCL. Our results show that chitosan modification of the PLCL scaffold improved the cell compatibility of the PLCL scaffold without significant alteration of the physical elastomeric properties of PLCL and resulted in the formation of cartilage tissue of better quality. PMID:21902611

Mesenchymal stem cells cultured on magnetic nanowire substrates

NASA Astrophysics Data System (ADS)

Perez, Jose E.; Ravasi, Timothy; Kosel, Jürgen

2017-02-01

Stem cells have been shown to respond to extracellular mechanical stimuli by regulating their fate through the activation of specific signaling pathways. In this work, an array of iron nanowires (NWs) aligned perpendicularly to the surface was fabricated by pulsed electrodepositon in porous alumina templates followed by a partial removal of the alumina to reveal 2-3 μm of the NWs. This resulted in alumina substrates with densely arranged NWs of 33 nm in diameter separated by 100 nm. The substrates were characterized by scanning electron microscopy (SEM) energy dispersive x-ray analysis and vibrating sample magnetometer. The NW array was then used as a platform for the culture of human mesenchymal stem cells (hMSCs). The cells were stained for the cell nucleus and actin filaments, as well as immuno-stained for the focal adhesion protein vinculin, and then observed by fluorescence microscopy in order to characterize their spreading behavior. Calcein AM/ethidium homodimer-1 staining allowed the determination of cell viability. The interface between the cells and the NWs was studied using SEM. Results showed that hMSCs underwent a re-organization of actin filaments that translated into a change from an elongated to a spherical cell shape. Actin filaments and vinculin accumulated in bundles, suggesting the attachment and formation of focal adhesion points of the cells on the NWs. Though the overall number of cells attached on the NWs was lower compared to the control, the attached cells maintained a high viability (>90%) for up to 6 d. Analysis of the interface between the NWs and the cells confirmed the re-organization of F-actin and revealed the adhesion points of the cells on the NWs. Additionally, a net of filopodia surrounded each cell, suggesting the probing of the array to find additional adhesion points. The cells maintained their round shape for up to 6 d of culture. Overall, the NW array is a promising nanostructured platform for studying and influencing hMSCs differentiation.

Impaired hair follicle morphogenesis and polarized keratinocyte movement upon conditional inactivation of integrin-linked kinase in the epidermis.

PubMed

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St Arnaud, René; Dedhar, Shoukat; D'Souza, Sudhir J A; Dagnino, Lina

2008-04-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function.

Impaired Hair Follicle Morphogenesis and Polarized Keratinocyte Movement upon Conditional Inactivation of Integrin-linked Kinase in the Epidermis

PubMed Central

Nakrieko, Kerry-Ann; Welch, Ian; Dupuis, Holly; Bryce, Dawn; Pajak, Agnieszka; St. Arnaud, René; Dedhar, Shoukat

2008-01-01

Integrin-linked kinase (ILK) is key for cell survival, migration, and adhesion, but little is known about its role in epidermal development and homeostasis in vivo. We generated mice with conditional inactivation of the Ilk gene in squamous epithelia. These mice die perinatally and exhibit skin blistering and severe defects in hair follicle morphogenesis, including greatly reduced follicle numbers, failure to progress beyond very early developmental stages, and pronounced defects in follicular keratinocyte proliferation. ILK-deficient epidermis shows abnormalities in adhesion to the basement membrane and in differentiation. ILK-deficient cultured keratinocytes fail to attach and spread efficiently and exhibit multiple abnormalities in actin cytoskeletal organization. Ilk gene inactivation in cultured keratinocytes causes impaired ability to form stable lamellipodia, to directionally migrate, and to polarize. These defects are accompanied by abnormal distribution of active Cdc42 to cell protrusions, as well as reduced activation of Rac1 upon induction of cell migration in scraped keratinocyte monolayers. Significantly, alterations in cell spreading and forward movement in single cells can be rescued by expression of constitutively active Rac1 or RhoG. Our studies underscore a central and distinct role for ILK in hair follicle development and in polarized cell movements, two key aspects of epithelial morphogenesis and function. PMID:18234842

EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces.

PubMed

Saeki, Noritaka; Nishino, Shingo; Shimizu, Tomohiro; Ogawa, Kazushige

2015-01-01

Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the β2 integrin/ICAM1 and β2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.

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

NASA Astrophysics Data System (ADS)

Chen, Jianbo

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

Mutant Fusion Proteins with Enhanced Fusion Activity Promote Measles Virus Spread in Human Neuronal Cells and Brains of Suckling Hamsters

PubMed Central

Shirogane, Yuta; Suzuki, Satoshi O.; Ikegame, Satoshi; Koga, Ritsuko

2013-01-01

Subacute sclerosing panencephalitis (SSPE) is a fatal degenerative disease caused by persistent measles virus (MV) infection in the central nervous system (CNS). From the genetic study of MV isolates obtained from SSPE patients, it is thought that defects of the matrix (M) protein play a crucial role in MV pathogenicity in the CNS. In this study, we report several notable mutations in the extracellular domain of the MV fusion (F) protein, including those found in multiple SSPE strains. The F proteins with these mutations induced syncytium formation in cells lacking SLAM and nectin 4 (receptors used by wild-type MV), including human neuronal cell lines, when expressed together with the attachment protein hemagglutinin. Moreover, recombinant viruses with these mutations exhibited neurovirulence in suckling hamsters, unlike the parental wild-type MV, and the mortality correlated with their fusion activity. In contrast, the recombinant MV lacking the M protein did not induce syncytia in cells lacking SLAM and nectin 4, although it formed larger syncytia in cells with either of the receptors. Since human neuronal cells are mainly SLAM and nectin 4 negative, fusion-enhancing mutations in the extracellular domain of the F protein may greatly contribute to MV spread via cell-to-cell fusion in the CNS, regardless of defects of the M protein. PMID:23255801

Role of c-Src in cellular events associated with colony-stimulating factor-1-induced spreading in osteoclasts.

PubMed

Insogna, K; Tanaka, S; Neff, L; Horne, W; Levy, J; Baron, R

1997-01-01

We and others have observed that in response to treatment with Colony Stimulating Factor-1 (CSF-1) neonatal rat osteoclasts demonstrate rapid cytoplasmic spreading. The receptor for CSF-1, c-Fms, is expressed in osteoclasts, possesses intrinsic tyrosine-kinase activity, and signals via rapid phosphorylation of selected proteins. It has been reported previously that c-Src becomes tyrosine phosphorylated following CSF-1 treatment of fibroblasts overexpressing c-Fms. We therefore examined the cellular events associated with CSF-1-induced spreading in osteoclasts and what role, if any, c-Src played in these processes. Confocal microscopic studies using phosphotyrosine (P-tyr) monoclonal antibodies demonstrated that CSF-1 induced a significant dose- and time-dependent increase in P-tyr labeling of neonatal rat osteoclasts. Phalloidin staining was consistent with partial to complete disassembly of the actin attachment ring with redistribution of actin to the spreading cytoplasmic edge of the cell. Quantitation of cellular F-actin using NBD-phallicidin confirmed a decrease in polymerized actin following exposure to CSF-1. In contrast, CSF-1 failed to induce any cytoplasmic spreading in osteoclasts isolated from mice with targeted disruption of the src gene. Further, in src- osteoclasts no well defined attachment ring could be identified. To investigate cell-signaling events associated with osteoclast spreading, detergent lysates were made from purified multinucleated osteoclast-like cells (OCLs) obtained by coculturing murine bone marrow and osteoblasts with calcitriol. Western blot analyses of lysates from control and CSF-1-treated normal cells indicated that several proteins were specifically phosphorylated in response to CSF-1, most notably proteins of 165, 60, and 85-90 kDa. Immunoprecipitation studies revealed that the 165 and 60 kDa proteins were, respectively, c-Fms and c-Src. The c-Src kinase activity was increased 2.9-fold following CSF-1 treatment. The 85-90 kDa protein is as yet unidentified. Since activated receptor tyrosine kinases may induce spreading in part by reducing phosphoinositol 4,5-bisphosphate (PIP2) binding to actin-associated proteins, a monoclonal antibody to PIP2 was used to assess the nature of PIP2 binding proteins in OCLs. Proteins of 85-90 kDa, 43 kDa, and 30 kDa were consistently demonstrated to bind PIP2. Further, the PIP2 content of the 85-90 kDa protein appeared to decrease with CSF-1 treatment. Whether this protein represents the phosphoprotein of the same M.W. is unclear. We also examined the effect of CSF-1 on the PIP2 content of alpha-actinin. Alpha-actinin showed low-level PIP2 binding, which was demonstrable only after immuno-precipitation and did not change with CSF-1 treatment. However, CSF-1 did cause a significant decline in the phosphotyrosine content of alpha-actinin. In contrast, in src- OCLs, CSF-1 induced more prolonged phosphorylation of c-Fms, and the 85-90 kDa protein was markedly hypophosphorylated. Further, alpha-actinin did not dephosphorylate in src- cells. We conclude that CSF-1-induced osteoclast spreading is accompanied by rapid reorganization of the actin cytoskeleton and phosphorylation of several cellular substrates, including c-Fms and c-Src. PIP2 binding to at least one protein appears to decrease with CSF-1 treatment, which may favor actin depolymerization. The reduced tyrosine phosphorylation of alpha-actinin could effect its ability to bind to actin. Thus c-Src may play an important role in these cellular events since in its absence, osteoclasts do not spread and signaling events downstream are altered. Whether these changes relate in part to the basal abnormalities in the cytoskeletal organization of src- osteoclasts remains to be determined.

Recombinant fibronectin fragment III8-10/polylactic acid hybrid nanofibers enhance the bioactivity of titanium surface.

PubMed

Guillem-Marti, Jordi; Boix-Lemonche, Gerard; Gugutkov, Dencho; Ginebra, Maria-Pau; Altankov, George; Manero, Jose M

2018-04-01

To develop a nanofiber (NF)-based biomimetic coating on titanium (Ti) that mimics the complex spatiotemporal organization of the extracellular matrix (ECM). Recombinant cell attachment site (CAS) of fibronectin type III8-10 domain was co-electrospun with polylactic acid (PLA) and covalently bound on polished Ti discs. Osteoblast-like SaOS-2 cells were used to evaluate their complex bioactivity. A significant increase of cell spreading was found on CAS/PLA hybrid NFs, followed by control pure PLA NFs and bare Ti discs. Cell proliferation showed similar trend being about twice higher on CAS/PLA NFs. The significantly increased ALP activity at day 21 indicated an enhanced differentiation of SaOS-2 cells. Coating of Ti implants with hybrid CAS/PLA NFs may improve significantly their osseointegration potential.

MTA-enriched nanocomposite TiO(2)-polymeric powder coatings support human mesenchymal cell attachment and growth.

PubMed

Shi, Wen; Mozumder, Mohammad Sayem; Zhang, Hui; Zhu, Jesse; Perinpanayagam, Hiran

2012-10-01

The objective of the study described in this paper was the development of novel polymer/ceramic nanocomposite coatings for implants through the application of ultrafine powder coating technology. Polyester resins were combined with µm-sized TiO(2) (25%) as the biocompatibility agent, nTiO(2) (0.5%) as the flow additive and mineral trioxide aggregates (ProRoot® MTA, 5%) as bioactive ceramics. Ultrafine powders were prepared and applied to titanium to create continuous polymeric powder coatings (PPCs) through the application of electrostatic ultrafine powder coating technology. Energy dispersive x-ray analysis confirmed that MTA had been incorporated into the PPCs, and elemental mapping showed that it had formed small clusters that were evenly distributed across the surface. Scanning electron microscopy (SEM) revealed continuous and smooth, but highly textured surface coatings that contrasted with the scalloped appearance of commercially pure titanium (cpTi) controls. Atomic force microscopy revealed intricate nano-topographies with an abundance of submicron-sized pits and nano-projections, evenly dispersed across their surfaces. Inverted fluorescence microscopy, SEM and cell counts showed that human embryonic palatal mesenchymal cells attached and spread out onto PPC and MTA-enriched PPCs within 24 h. Mitochondrial enzyme activity measured viable and metabolically active cells on all of the surfaces. After 72 h of growth, cell counts and metabolic activity were significantly higher (P < 0.05) on the grey-MTA enriched PPC surfaces, than on unmodified PPC and cpTi. The novel polymer/ceramic nanocomposites that were created with ultrafine powder coating technology were continuous, homogenous and nano-rough coatings that enhanced human mesenchymal cell attachment and growth.

Maize leaf trichomes represent an entry point of infection for Fusarium species.

PubMed

Nguyen, Thi Thanh Xuan; Dehne, Heinz-Wilhelm; Steiner, Ulrike

2016-08-01

Fifteen day old maize seedlings were inoculated with Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides. More than 90 % F. proliferatum and F. verticillioides conidia and 50 % of F. graminearum formed one germ tube whereas the other 50 % of F. graminearum conidia formed two to three germ tubes. The germ tubes of F. graminearum conidia were longer than those of F. proliferatum and F. verticillioides. The three species of Fusarium infected bi-cellular trichomes by adhering and growing along the trichomes or by attaching to the cap cell of the trichomes 48 h after inoculation. Hyphae penetrated into the trichomes at the base, the side or at the top of the cap cells. The hyphae colonized the cap cells and then spread to base cells. Prickle trichomes were infected 72 h after inoculation. The hyphae either wrapped around prickle trichomes or formed a mass of hyphae around the top of prickle trichomes or formed appressorium. Macro trichomes were infected by F. graminearum 7 d after inoculation. Following penetration, the fungus spread to adjacent epidermal cells and to the subcuticle. This investigation provides the first assessment of F. graminearum, F. proliferatum, and F. verticillioides infection via trichomes of maize leaves. Copyright © 2016 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Early osteoblast responses to orthopedic implants: Synergy of surface roughness and chemistry of bioactive ceramic coating.

PubMed

Aniket; Reid, Robert; Hall, Benika; Marriott, Ian; El-Ghannam, Ahmed

2015-06-01

Pro-osteogenic stimulation of bone cells by bioactive ceramic-coated orthopedic implants is influenced by both surface roughness and material chemistry; however, their concomitant impact on osteoblast behavior is not well understood. The aim of this study is to investigate the effects of nano-scale roughness and chemistry of bioactive silica-calcium phosphate nanocomposite (SCPC50) coated Ti-6Al-4V on modulating early bone cell responses. Cell attachment was higher on SCPC50-coated substrates compared to the uncoated controls; however, cells on the uncoated substrate exhibited greater spreading and superior quality of F-actin filaments than cells on the SCPC50-coated substrates. The poor F-actin filament organization on SCPC50-coated substrates is thought to be due to the enhanced calcium uptake by the ceramic surface. Dissolution analyses showed that an increase in surface roughness was accompanied by increased calcium uptake, and increased phosphorous and silicon release, all of which appear to interfere with F-actin assembly and osteoblast morphology. Moreover, cell attachment onto the SCPC50-coated substrates correlated with the known adsorption of fibronectin, and was independent of surface roughness. High-throughput genome sequencing showed enhanced expression of extracellular matrix and cell differentiation related genes. These results demonstrate a synergistic relationship between bioactive ceramic coating roughness and material chemistry resulting in a phenotype that leads to early osteoblast differentiation. © 2014 Wiley Periodicals, Inc.

Combining Dynamic Stretch and Tunable Stiffness to Probe Cell Mechanobiology In Vitro

PubMed Central

Throm Quinlan, Angela M.; Sierad, Leslie N.; Capulli, Andrew K.; Firstenberg, Laura E.; Billiar, Kristen L.

2011-01-01

Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G′ = 0.3 kPa) to stiff (G′ = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch. PMID:21858051

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces.

PubMed

Ionescu, Michael; Zaini, Paulo A; Baccari, Clelia; Tran, Sophia; da Silva, Aline M; Lindow, Steven E

2014-09-16

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an "exploratory" lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents.

Immobilization of Cell-Adhesive Laminin Peptides in Degradable PEGDA Hydrogels Influences Endothelial Cell Tubulogenesis

PubMed Central

Ali, Saniya; Saik, Jennifer E.; Gould, Dan J.; Dickinson, Mary E.

2013-01-01

Abstract Attachment, spreading, and organization of endothelial cells into tubule networks are mediated by interactions between cells in the extracellular microenvironment. Laminins are key extracellular matrix components and regulators of cell adhesion, migration, and proliferation. In this study, laminin-derived peptides were conjugated to poly(ethylene glycol) (PEG) monoacrylate and covalently incorporated into degradable PEG diacrylate (PEGDA) hydrogels to investigate the influence of these peptides on endothelial cellular adhesion and function in organizing into tubule networks. Degradable PEGDA hydrogels were synthesized by incorporating a matrix metalloproteinase (MMP)–sensitive peptide, GGGPQGIWGQGK (abbreviated PQ), into the polymer backbone. The secretion of MMP-2 and MMP-9 by endothelial cells promotes polymer degradation and consequently cell migration. We demonstrate the formation of extensive networks of tubule-like structures by encapsulated human umbilical vein endothelial cells in hydrogels with immobilized synthetic peptides. The resulting structures were stabilized by pericyte precursor cells (10T1/2s) in vitro. During tubule formation and stabilization, extracellular matrix proteins such as collagen IV and laminin were deposited. Tubules formed in the matrix of metalloproteinase sensitive hydrogels were visualized from 7 days to 4 weeks in response to different combination of peptides. Moreover, hydrogels functionalized with laminin peptides and transplanted in a mouse cornea supported the ingrowth and attachment of endothelial cells to the hydrogel during angiogenesis. Results of this study illustrate the use of laminin-derived peptides as potential candidates for modification of biomaterials to support angiogenesis. PMID:23914330

Superior in vitro biological response and mechanical properties of an implantable nanostructured biomaterial: Nanohydroxyapatite-silicone rubber composite.

PubMed

Thein-Han, W W; Shah, J; Misra, R D K

2009-09-01

A potential approach to achieving the objective of favorably modulating the biological response of implantable biopolymers combined with good mechanical properties is to consider compounding the biopolymer with a bioactive nanocrystalline ceramic biomimetic material with high surface area. The processing of silicone rubber (SR)-nanohydroxyapatite (nHA) composite involved uniform dispersion of nHA via shear mixing and ultrasonication, followed by compounding at sub-ambient temperature, and high-pressure solidification when the final curing reaction occurs. The high-pressure solidification approach enabled the elastomer to retain the high elongation of SR even in the presence of the reinforcement material, nHA. The biological response of the nanostructured composite in terms of initial cell attachment, cell viability and proliferation was consistently greater on SR-5wt.% nHA composite surface compared to pure SR. Furthermore, in the nanocomposite, cell spreading, morphology and density were distinctly different from that of pure SR. Pre-osteoblasts grown on SR-nHA were well spread, flat, large in size with a rough cell surface, and appeared as a group. In contrast, these features were less pronounced in SR (e.g. smooth cell surface, not well spread). Interestingly, an immunofluorescence study illustrated distinct fibronectin expression level, and stronger vinculin focal adhesion contacts associated with abundant actin stress fibers in pre-osteoblasts grown on the nanocomposite compared to SR, implying enhanced cell-substrate interaction. This finding was consistent with the total protein content and SDS-PAGE analysis. The study leads us to believe that further increase in nHA content in the SR matrix beyond 5wt.% will encourage even greater cellular response. The integration of cellular and molecular biology with materials science and engineering described herein provides a direction for the development of a new generation of nanostructured materials.

Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells.

PubMed

Sangani, Rajnikumar; Pandya, Chirayu D; Bhattacharyya, Maryka H; Periyasamy-Thandavan, Sudharsan; Chutkan, Norman; Markand, Shanu; Hill, William D; Hamrick, Mark; Isales, Carlos; Fulzele, Sadanand

2014-03-01

Bone marrow stromal cell (BMSC) adhesion and migration are fundamental to a number of pathophysiologic processes, including fracture and wound healing. Vitamin C is beneficial for bone formation, fracture repair and wound healing. However, the role of the vitamin C transporter in BMSC adhesion, migration and wound healing is not known. In this study, we knocked-down the sodium-dependent vitamin C transporter, SVCT2, the only known transporter of vitamin C in BMSCs, and performed cell adhesion, migration, in-vitro scratch wound healing and F-actin re-arrangement studies. We also investigated the role of oxidative stress on the above processes. Our results demonstrate that both oxidative stress and down-regulation of SVCT2 decreased cell attachment and spreading. A trans-well cell migration assay showed that vitamin C helped in BMSC migration and that knockdown of SVCT2 decreased cell migration. In the in-vitro scratch wound healing studies, we established that oxidative stress dose-dependently impairs wound healing. Furthermore, the supplementation of vitamin C significantly rescued the BMSCs from oxidative stress and increased wound closing. The knockdown of SVCT2 in BMSCs strikingly decreased wound healing, and supplementing with vitamin C failed to rescue cells efficiently. The knockdown of SVCT2 and induction of oxidative stress in cells produced an alteration in cytoskeletal dynamics. Signaling studies showed that oxidative stress phosphorylated members of the MAP kinase family (p38) and that vitamin C inhibited their phosphorylation. Taken together, these results indicate that both the SVCT2 transporter and oxidative stress play a vital role in BMSC attachment, migration and cytoskeletal re-arrangement. BMSC-based cell therapy and modulation of SVCT2 could lead to a novel therapeutic approach that enhances bone remodeling, fracture repair and wound healing in chronic disease conditions. Published by Elsevier B.V.

Covalent Functionalization of NiTi Surfaces with Bioactive Peptide Amphiphile Nanofibers

PubMed Central

Sargeant, Timothy D.; Rao, Mukti S.; Koh, Chung-Yan

2009-01-01

Surface modification enables the creation of bioactive implants using traditional material substrates without altering the mechanical properties of the bulk material. For applications such as bone plates and stents, it is desirable to modify the surface of metal alloy substrates to facilitate cellular attachment, proliferation, and possibly differentiation. In this work we present a general strategy for altering the surface chemistry of nickel-titanium shape memory alloy (NiTi) in order to covalently attach self-assembled peptide amphiphile (PA) nanofibers with bioactive functions. Bioactivity in the systems studied here includes biological adhesion and proliferation of osteoblast and endothelial cell types. The optimized surface treatment creates a uniform TiO2 layer with low levels of Ni on the NiTi surface, which is subsequently covered with an aminopropylsilane coating using a novel, lower temperature vapor deposition method. This method produces an aminated surface suitable for covalent attachment of PA molecules containing terminal carboxylic acid groups. The functionalized NiTi surfaces have been characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectroscopy (ToF-SIMS), and atomic force microscopy (AFM). These techniques offer evidence that the treated metal surfaces consist primarily of TiO2 with very little Ni, and also confirm the presence of the aminopropylsilane overlayer. Self-assembled PA nanofibers presenting the biological peptide adhesion sequence Arg-Gly-Asp-Ser are capable of covalently anchoring to the treated substrate, as demonstrated by spectrofluorimetry and AFM. Cell culture and scanning electron microscopy (SEM) demonstrate cellular adhesion, spreading, and proliferation on these functionalized metal surfaces. Furthermore, these experiments demonstrate that covalent attachment is crucial for creating robust PA nanofiber coatings, leading to confluent cell monolayers. PMID:18083225

Biological effects of functionalizing copolymer scaffolds with nanodiamond particles.

PubMed

Xing, Zhe; Pedersen, Torbjorn O; Wu, Xujun; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Kloss, Frank R; Waag, Thilo; Krueger, Anke; Steinmüller-Nethl, Doris; Mustafa, Kamal

2013-08-01

Significant evidence has indicated that poly(L-lactide)-co-(ɛ-caprolactone) [(poly(LLA-co-CL)] scaffolds could be one of the suitable candidates for bone tissue engineering. Oxygen-terminated nanodiamond particles (n-DP) were combined with poly(LLA-co-CL) and revealed to be positive for cell growth. In this study, we evaluated the influence of poly(LLA-co-CL) scaffolds modified by n-DP on attachment, proliferation, differentiation of bone marrow stromal cells (BMSCs) in vitro, and on bone formation using a sheep calvarial defect model. BMSCs were seeded on either poly(LLA-co-CL)- or n-DP-coated scaffolds and incubated for 1 h. Scanning electron microscopy (SEM) and fluorescence microscopy were used in addition to protein and DNA measurements to evaluate cellular attachment on the scaffolds. To determine the effect of n-DP on proliferation of BMSCs, cell/scaffold constructs were harvested after 3 days and evaluated by Bicinchoninic Acid (BCA) protein assay and SEM. In addition, the osteogenic differentiation of cells grown for 2 weeks on the various scaffolds and in a dynamic culture condition was evaluated by real-time RT-PCR. Unmodified and modified scaffolds were implanted into the calvaria of six-year-old sheep. The expression of collagen type I (COL I) and bone morphogenetic protein-2 (BMP-2) after 4 weeks as well as the formation of new bone after 12 and 24 weeks were analyzed by immunohistochemistry and histology. Scaffolds modified with n-DP supported increased cell attachment and the mRNA expression of osteopontin (OPN), bone sialoprotein (BSP), and BMP-2 were significantly increased after 2 weeks of culture. The BMSCs had spread well on the various scaffolds investigated after 3 days in the study with no significant difference in cell proliferation. Furthermore, the in vivo data revealed more positive staining of COL I and BMP-2 in relation to the n-DP-coated scaffolds after 4 weeks and presented more bone formation after 12 and 24 weeks. n-DP modification significantly increased cell attachment and differentiation of BMSCs on poly(LLA-co-CL) scaffolds in vitro and enhanced bone formation in vivo.

A comparative study of proliferation and osteogenic differentiation of adipose-derived stem cells on akermanite and beta-TCP ceramics.

PubMed

Liu, Qihai; Cen, Lian; Yin, Shuo; Chen, Lei; Liu, Guangpeng; Chang, Jiang; Cui, Lei

2008-12-01

This study investigated the in vitro effects of akermanite, a new kind of Ca-, Mg-, Si-containing bioceramic, on the attachment, proliferation and osteogenic differentiation of human adipose-derived stem cells (hASCs). Parallel comparison of the cellular behaviors of hASCs on the akermanite was made with those on beta-tricalcium phosphate (beta-TCP). Scanning electron microscope (SEM) observation and fluorescent DiO labeling were carried out to reveal the attachment and growth of hASCs on the two ceramic surfaces, while the quantitative assay of cell proliferation with time was detected by DNA assay. Osteogenic differentiation of hASCs cultured on the akermanite and beta-TCP was assayed by ALP expression and osteocalcin (OCN) deposition, which was further confirmed by Real-time PCR analysis for markers of osteogenic differentiation. It was shown that hASCs attached and spread well on the akermanite as those on beta-TCP, and similar proliferation behaviors of hASCs were observed on the two ceramics. Both of them exhibited good compatibility to hASCs with only minor cytotoxicity as compared with the tissue culture plates. Interestingly, the osteogenic differentiation of hASCs could be enhanced on the akermanite compared with that on the beta-TCP when the culture time was extended to approximately 10 days. Thus, it can be ascertained that akermanite ceramics may serve as a potential scaffold for bone tissue engineering.

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion.

PubMed

Veazey, Ronald S; Klasse, Per Johan; Schader, Susan M; Hu, Qinxue; Ketas, Thomas J; Lu, Min; Marx, Preston A; Dufour, Jason; Colonno, Richard J; Shattock, Robin J; Springer, Martin S; Moore, John P

2005-11-03

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.

Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

PubMed

Shenoy, Anitha K; Lu, Jianrong

2016-10-01

Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Biodegradable polyester-based microcarriers with modified surface tailored for tissue engineering.

PubMed

Privalova, A; Markvicheva, E; Sevrin, Ch; Drozdova, M; Kottgen, C; Gilbert, B; Ortiz, M; Grandfils, Ch

2015-03-01

Microcarriers have been proposed in tissue engineering, namely for bone, cartilage, skin, vascular, and central nervous system. Although polyester-based microcarriers have been already used for this purpose, their surface properties should be improved to provide better cell growth. The goal of this study was to prepare microbeads based on poly(D,L-lactide) acid, poly(L-lactide) acid, and to study cell behavior (adhesion, spreading, growth, and proliferation) in function of microbead topography and surface chemistry. To improve L-929 fibroblasts adhesion, microbead surface has been modified with three polycations: chitosan, poly(2-dimethylamino ethylmethacrylate) (PDMAEMA), or chitosan-g-oligolactide copolymer (chit-g-OLA). Although modification of the microbead surface with chitosan and PDMAEMA was performed through physical adsorption on the previously prepared microbeads, chit-g-OLA copolymer was introduced directly during microbead processing. This simple approach (1) bypass the use of an emulsifier (polyvinyl alcohol, PVA); (2) avoid surface "contamination" with PVA molecules limiting a control of the surface characteristics. In vitro study of the growth of mouse fibroblasts on the microbeads showed that both surface topography and chemistry affected cell attachment, spreading, and proliferation. Cultivation of L-929 fibroblasts for 7 days resulted in the formation of a 3D cell-scaffold network. © 2014 Wiley Periodicals, Inc.

Effects of topographical and mechanical property alterations induced by oxygen plasma modification on stem cell behavior.

PubMed

Yang, Yong; Kulangara, Karina; Lam, Ruby T S; Dharmawan, Rena; Leong, Kam W

2012-10-23

Polymeric substrates intended for cell culture and tissue engineering are often surface-modified to facilitate cell attachment of most anchorage-dependent cell types. The modification alters the surface chemistry and possibly topography. However, scant attention has been paid to other surface property alterations. In studying oxygen plasma treatment of polydimethylsiloxane (PDMS), we show that oxygen plasma treatment alters the surface chemistry and, consequently, the topography and elasticity of PDMS at the nanoscale level. The elasticity factor has the predominant effect, compared with the chemical and topographical factors, on cell adhesions of human mesenchymal stem cells (hMSCs). The enhanced focal adhesions favor cell spreading and osteogenesis of hMSCs. Given the prevalent use of PDMS in biomedical device construction and cell culture experiments, this study highlights the importance of understanding how oxygen plasma treatment would impact subsequent cell-substrate interactions. It helps explain inconsistency in the literature and guides preparation of PDMS-based biomedical devices in the future.

Vertically aligned carbon nanotubes as cytocompatible material for enhanced adhesion and proliferation of osteoblast-like cells.

PubMed

Giannona, Suna; Firkowska, Izabela; Rojas-Chapana, José; Giersig, Michael

2007-01-01

In this study, we describe the spatial organization of CAL-72 osteoblast-like cells on arrays of vertically aligned multi-walled carbon nanotubes (VACNTs). It was observed that, unlike cell growth on non-patterned surfaces, the cell attachment and spreading process on VACNTs was significantly enhanced. Additionally, since carbon nanotubes are known to possess resilient mechanical properties and are chemically stable, the effect of periodic arrays of VACNTs on CAL-72 osteoblast-like cells was also studied. The periodicity and alignment of VACNTs considerably influenced growth, shape and orientation of the cells by steering toward the nanopattern. This situation is of great interest for the potential application of VACNTs in bone bioenginnering. This data provides evidence that CAL-72 osteoblast-like cells can sense physical features at the nanoscale. These results give a fascinating insight into the ways in which cell growth can be influenced by man-made nanostructures and could provide a framework for achieving controlled cell guidance with controlled organization and special physical properties.

Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts.

PubMed

Glogauer, M; Ferrier, J; McCulloch, C A

1995-11-01

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

PubMed Central

Reh, Lucia; Magnus, Carsten; Schanz, Merle; Weber, Jacqueline; Uhr, Therese; Rusert, Peter; Trkola, Alexandra

2015-01-01

An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the transmission mode and differed depending on the window of action during the entry process, underscoring that powerful combinations of bnAbs are needed for in vivo application. PMID:26158270

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

PubMed

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

2007-01-01

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

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

PubMed Central

Gardner, Thomas J.

2016-01-01

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

The Spread of English: A Worldwide Factor in the Making and Breaking of Bilingualism. CATESOL Occasional Papers, No. 4.

ERIC Educational Resources Information Center

Fishman, Joshua

English is spreading throughout the world far more rapidly than any other language; however, the level of sentimental attachment or genuine liking for English falls far short of the level of necessity-based desire to learn it. There is a possibility that the spread of English is decreasing: given some rapid economic or military change around the…

Xylella fastidiosa outer membrane vesicles modulate plant colonization by blocking attachment to surfaces

PubMed Central

Ionescu, Michael; Zaini, Paulo A.; Baccari, Clelia; Tran, Sophia; da Silva, Aline M.; Lindow, Steven E.

2014-01-01

Outer membrane vesicles (OMVs) of Gram-negative bacteria have been studied intensively in recent years, primarily in their role in delivering virulence factors and antigens during pathogenesis. However, the near ubiquity of their production suggests that they may play other roles, such as responding to envelope stress or trafficking various cargoes to prevent dilution or degradation by other bacterial species. Here we show that OMVs produced by Xylella fastidiosa, a xylem-colonizing plant pathogenic bacterium, block its interaction with various surfaces such as the walls of xylem vessels in host plants. The release of OMVs was suppressed by the diffusible signal factor-dependent quorum-sensing system, and a X. fastidiosa ΔrpfF mutant in which quorum signaling was disrupted was both much more virulent to plants and less adhesive to glass and plant surfaces than the WT strain. The higher virulence of the ΔrpfF mutant was associated with fivefold higher numbers of OMVs recovered from xylem sap of infected plants. The frequency of attachment of X. fastidiosa to xylem vessels was 20-fold lower in the presence of OMVs than in their absence. OMV production thus is a strategy used by X. fastidiosa cells to adjust attachment to surfaces in its transition from adhesive cells capable of insect transmission to an “exploratory” lifestyle for systemic spread within the plant host which would be hindered by attachment. OMV production may contribute to the movement of other bacteria in porous environments by similarly reducing their contact with environmental constituents. PMID:25197068

Enhanced cell adhesion on severe peened-plasma nitrided 316L stainless steel

NASA Astrophysics Data System (ADS)

Jayalakshmi, M.; Bhat, Badekai Ramachandra; Bhat, K. Udaya

2018-04-01

Plasma nitriding is an effective technique to enhance the wear resistance of austenitic stainless steels. Recently, severe surface deformation techniques are extensively used prior to nitriding to enhance diffusion kinetics. In the present study, AISI 316L austenitic stainless steel is subjected to peening-nitriding duplex treatment and biocompatibility of treated surfaces is assessed through adhesion of the fibroblast cells. Three-fold increase in the surface microhardness is observed from the un-peened sample to the peened-nitrided sample; with severe peened sample showing intermediate hardness. Similar trend is observed in the number of the fibroblast cells attached to the sample surface. Spreading of some of the fibroblast cells is observed on the sample subjected to duplex treatment; while the other two samples showed only the spindle shaped fibroblasts. Combined influence of surface nanocrystallization and presence of nitride layer is responsible for the improved biocompatibility.

Improved attachment of mesenchymal stem cells on super-hydrophobic TiO2 nanotubes.

PubMed

Bauer, Sebastian; Park, Jung; von der Mark, Klaus; Schmuki, Patrik

2008-09-01

Self-organized layers of vertically orientated TiO(2) nanotubes providing defined diameters ranging from 15 up to 100nm were grown on titanium by anodic oxidation. These TiO(2) nanotube layers show super-hydrophilic behavior. After coating TiO(2) nanotube layers with a self-assembled monolayer (octadecylphosphonic acid) they showed a diameter-dependent wetting behavior ranging from hydrophobic (108+/-2 degrees ) up to super-hydrophobic (167+/-2 degrees ). Cell adhesion, spreading and growth of mesenchymal stem cells on the unmodified and modified nanotube layers were investigated and compared. We show that cell adhesion and proliferation are strongly affected in the super-hydrophobic range. Adsorption of extracellular matrix proteins as fibronectin, type I collagen and laminin, as well as bovine serum albumin, on the coated and uncoated surfaces showed a strong influence on wetting behavior and dependence on tube diameter.

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal pattern, is involved in tissue formation and in key processes of tumour spread.

Titania-polymeric powder coatings with nano-topography support enhanced human mesenchymal cell responses.

PubMed

Mozumder, Mohammad Sayem; Zhu, Jesse; Perinpanayagam, Hiran

2012-10-01

Titanium implant osseointegration is dependent on the cellular response to surface modifications and coatings. Titania-enriched nanocomposite polymeric resin coatings were prepared through the application of advanced ultrafine powder coating technology. Their surfaces were readily modified to create nano-rough (<100 nm) surface nano-topographies that supported human embryonic palatal mesenchymal cell responses. Energy dispersive x-ray spectroscopy confirmed continuous and homogenous coatings with a similar composition and even distribution of titanium. Scanning electron microscopy (SEM) showed complex micro-topographies, and atomic force microscopy revealed intricate nanofeatures and surface roughness. Cell counts, mitochondrial enzyme activity reduction of yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to dark purple, SEM, and inverted fluorescence microscopy showed a marked increase in cell attachment, spreading, proliferation, and metabolic activity on the nanostructured surfaces. Reverse Transcription- Polymerase Chain Reaction (RT-PCR) analysis showed that type I collagen and Runx2 expression were induced, and Alizarin red staining showed that mineral deposits were abundant in the cell cultures grown on nanosurfaces. This enhancement in human mesenchymal cell attachment, growth, and osteogenesis were attributed to the nanosized surface topographies, roughness, and moderate wetting characteristics of the coatings. Their dimensional similarity to naturally occurring matrix proteins and crystals, coupled with their increased surface area for protein adsorption, may have facilitated the response. Therefore, this application of ultrafine powder coating technology affords highly biocompatible surfaces that can be readily modified to accentuate the cellular response. Copyright © 2012 Wiley Periodicals, Inc.

ICAM-1 and AMPK regulate cell detachment and apoptosis by N-methyl-N Prime -nitro-N-nitrosoguanidine, a widely spread environmental chemical, in human hormone-refractory prostate cancers

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

Chen, Yi-Cheng; Lu, Pin-Hsuan; Hsu, Jui-Ling

2011-12-15

Poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, plays a crucial role in the regulation of DNA repair. PARP-1 hyperactivation causes DNA damage and cell death. The underlying mechanism is complicated and is through diverse pathways. The understanding of responsible signaling pathways may offer implications for effective therapies. After concentration-response determination of N-Methyl-N Prime -Nitro-N-Nitrosoguanidine (MNNG, a PARP-1 activating agent and an environmental mutagen) in human hormone-refractory prostate cancers, the data showed that concentrations below 5 {mu}M did not change cell survival but cause a time-dependent up-regulation of intracellular adhesion molecule-1 (ICAM-1) in mRNA, total protein and cell surface levels.more » Detection of phosphorylation and degradation of I{kappa}B-{alpha} and nuclear translocation of NF-{kappa}B showed that MNNG induced the activation of NF-{kappa}B that was responsible for the ICAM-1 up-regulation since PDTC (a NF-{kappa}B inhibitor) significantly abolished this effect. However, higher concentrations (e.g., 10 {mu}M) of MNNG induced a 61% detachment of the cells which were apoptosis associated with the activation of AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Further identification showed that both AMPK and JNK other than p38 MAPK functionally contributed to cell death. The remaining 39% attached cells were survival associated with high ICAM-1 expression. In conclusion, the data suggest that NF-{kappa}B-dependent up-regulation of ICAM-1 plays a key role on cell attachment and survival; whereas, activation of AMPK and JNK participates in cytotoxic signaling pathways in detached cells caused by PARP-1 activation. Highlights: Black-Right-Pointing-Pointer Low level of DNA damage helps cell attachment and survival via ICAM-1 upregulation. Black-Right-Pointing-Pointer High level of DNA damage causes AMPK- and JNK-involved cell detachment and death. Black-Right-Pointing-Pointer The study provides an anticancer approach targeting PARP-1 and DNA damage response.« less

Computer Virus Protection

ERIC Educational Resources Information Center

Rajala, Judith B.

2004-01-01

A computer virus is a program--a piece of executable code--that has the unique ability to replicate. Like biological viruses, computer viruses can spread quickly and are often difficult to eradicate. They can attach themselves to just about any type of file, and are spread by replicating and being sent from one individual to another. Simply having…

Immobilization of cross linked Col-I-OPN bone matrix protein on aminolysed PCL surfaces enhances initial biocompatibility of human adipogenic mesenchymal stem cells (hADMSC)

NASA Astrophysics Data System (ADS)

Kim, Young-Hee; Jyoti, Md. Anirban; Song, Ho-Yeon

2014-06-01

In bone tissue engineering surface modification is considered as one of the important ways of fabricating successful biocompatible material. Addition of biologically active functionality on the surfaces has been tried for improving the overall biocompatibility of the system. In this study poly-ɛ-caprolactone film surfaces have been modified through aminolysis and immobilization process. Collagen type I (COL-I) and osteopontin (OPN), which play an important role in osteogenesis, was immobilized onto PCL films followed by aminolysis treatment using 1,6-hexanediamine. Characterization of animolysed and immobilized surfaces were done by a number techniques using scanning electron microscopy (SEM), FT-IR, XPS, ninhydrin staining, SDS-PAGE and confocal microscopy and compared between the modified and un-modified surfaces. Results of the successive experiments showed that aminolysis treatment was homogeneously achieved which helped to entrap or immobilize Col-I-OPN proteins on surfaces of PCL film. In vitro studies with human adipogenic mesenchymal stem cells (hADMSC) also confirmed the attachment and proliferation of cells was better in modified PCL surfaces than the unmodified surfaces. SEM, confocal microscopy and MTT assay showed a significant increase in cell spreading, attachment and proliferations on the biofunctionalized surfaces compared to the unmodified PCL surfaces at all-time points indicating the success of surface biofunctionalization.

Spatial Periodicity of Escherichia coli K-12 Biofilm Microstructure Initiates during a Reversible, Polar Attachment Phase of Development and Requires the Polysaccharide Adhesin PGA

PubMed Central

Agladze, Konstantin; Wang, Xin; Romeo, Tony

2005-01-01

Using fast Fourier transform (FFT) analysis, we previously observed that cells within Escherichia coli biofilm are organized in nonrandom or periodic spatial patterns (K. Agladze et al., J. Bacteriol. 185:5632-5638, 2003). Here, we developed a gravity displacement assay for examining cell adherence and used it to quantitatively monitor the formation of two distinct forms of cell attachment, temporary and permanent, during early biofilm development. Temporarily attached cells were mainly surface associated by a cell pole; permanent attachments were via the lateral cell surface. While temporary attachment precedes permanent attachment, both forms can coexist in a population. Exposure of attached cells to gravity liberated an unattached population capable of rapidly reassembling a new monolayer, composed of temporarily attached cells, and possessing periodicity. A csrA mutant, which forms biofilm more vigorously than its wild-type parent, exhibited an increased proportion of permanently attached cells and a form of attachment that was not apparent in the parent strain, permanent polar attachment. Nevertheless, it formed periodic attachment patterns. In contrast, biofilm mutants with altered lipopolysaccharide synthesis (waaG) exhibited increased cell-cell interactions, bypassed the polar attachment step, and produced FFT spectra characteristic of aperiodic cell distribution. Mutants lacking the polysaccharide adhesin β-1,6-N-acetyl-d-glucosamine (ΔpgaC) also exhibited aperiodic cell distribution, but without apparent cell-cell interactions, and were defective in forming permanent attachments. Thus, spatial periodicity of biofilm microstructure is genetically determined and evident during the formation of temporary cell surface attachments. PMID:16321928

Dependence of corneal keratocyte adhesion, spreading, and integrin β1 expression on deacetylated chitosan coating.

PubMed

Sun, Chi-Chin; Chou, Shih-Feng; Lai, Jui-Yang; Cho, Ching-Hsien; Lee, Chih-Hung

2016-06-01

This study reports, for the first time, the regulation of corneal keratocyte adhesion, spreading, morphology, and integrin gene expression on chitosan coating due to the effects of deacetylation. The degree of deacetylation (DD) in chitosan materials was confirmed by elemental analysis, gel permeation chromatography, and Fourier transform infrared spectroscopy. In this study, chitosan samples with the same molecular weight level but varying DD (74.1 ± 0.5%, 84.4 ± 0.7%, and 94.2 ± 0.5%) were obtained by heat-alkaline treatment under a nitrogen atmosphere. For higher DD groups, the biopolymer carried abundant amino groups since the deacetylation process removed larger amount of acetyl groups from the chitosan molecules. Results showed that the mechanical stability and crystallinity of the chitosan coatings significantly increased with increasing DD value. Fibronectin adsorption, keratocyte adhesion, and cell spreading exhibited a positive correlation with DD due to the chemical functionality of polysaccharides (bearing acetyl and amino groups) and increase of substrate stiffness and crystallinity. In particular, when adhered to chitosan coatings with a DD value of 74.1%, the keratocytes appeared to be fibroblastic, elongated, and spindle shape, indicating a loss of their characteristic dendritic morphology. Furthermore, the gene expression of integrin β1 (i.e., a cell-matrix adhesion molecule) was significantly up-regulated on the chitosan coatings with higher DD, which supports favorable attachment of corneal keratocytes. Our findings suggest that DD-mediated physicochemical properties of chitosan coatings greatly affect cell-substrate crosstalk during corneal keratocyte cultivation. Copyright © 2016 Elsevier B.V. All rights reserved.

Embryonic stem cell trials for macular degeneration: a preliminary report.

PubMed

Schwartz, Steven D; Hubschman, Jean-Pierre; Heilwell, Gad; Franco-Cardenas, Valentina; Pan, Carolyn K; Ostrick, Rosaleen M; Mickunas, Edmund; Gay, Roger; Klimanskaya, Irina; Lanza, Robert

2012-02-25

It has been 13 years since the discovery of human embryonic stem cells (hESCs). Our report provides the first description of hESC-derived cells transplanted into human patients. We started two prospective clinical studies to establish the safety and tolerability of subretinal transplantation of hESC-derived retinal pigment epithelium (RPE) in patients with Stargardt's macular dystrophy and dry age-related macular degeneration--the leading cause of blindness in the developed world. Preoperative and postoperative ophthalmic examinations included visual acuity, fluorescein angiography, optical coherence tomography, and visual field testing. These studies are registered with ClinicalTrials.gov, numbers NCT01345006 and NCT01344993. Controlled hESC differentiation resulted in greater than 99% pure RPE. The cells displayed typical RPE behaviour and integrated into the host RPE layer forming mature quiescent monolayers after transplantation in animals. The stage of differentiation substantially affected attachment and survival of the cells in vitro after clinical formulation. Lightly pigmented cells attached and spread in a substantially greater proportion (>90%) than more darkly pigmented cells after culture. After surgery, structural evidence confirmed cells had attached and continued to persist during our study. We did not identify signs of hyperproliferation, abnormal growth, or immune mediated transplant rejection in either patient during the first 4 months. Although there is little agreement between investigators on visual endpoints in patients with low vision, it is encouraging that during the observation period neither patient lost vision. Best corrected visual acuity improved from hand motions to 20/800 (and improved from 0 to 5 letters on the Early Treatment Diabetic Retinopathy Study [ETDRS] visual acuity chart) in the study eye of the patient with Stargardt's macular dystrophy, and vision also seemed to improve in the patient with dry age-related macular degeneration (from 21 ETDRS letters to 28). The hESC-derived RPE cells showed no signs of hyperproliferation, tumorigenicity, ectopic tissue formation, or apparent rejection after 4 months. The future therapeutic goal will be to treat patients earlier in the disease processes, potentially increasing the likelihood of photoreceptor and central visual rescue. Advanced Cell Technology. Copyright © 2012 Elsevier Ltd. All rights reserved.

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications.

PubMed

Ribeiro, A R; Oliveira, F; Boldrini, L C; Leite, P E; Falagan-Lotsch, P; Linhares, A B R; Zambuzzi, W F; Fragneaud, B; Campos, A P C; Gouvêa, C P; Archanjo, B S; Achete, C A; Marcantonio, E; Rocha, L A; Granjeiro, J M

2015-09-01

Titanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration. Copyright © 2015 Elsevier B.V. All rights reserved.

Femtosecond laser-induced cell-cell surgical attachment.

PubMed

Katchinskiy, Nir; Godbout, Roseline; Goez, Helly R; Elezzabi, Abdulhakem Y

2014-04-01

Laser-induced cell-cell surgical attachment using femtosecond laser pulses is reported. We have demonstrated the ability to attach single cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength delivered from a Ti:Sapphire laser. To check that the cells did not go through a cell-fusion process, a fluorescent dye Calcein AM was used to verify that the fluorescent dye did not migrate from a dyed cell to a non-dyed cell. The mechanical integrity of the attached joint was assessed using an optical tweezer. Attachment of cells was performed without the induction of cell-cell fusion, with attachment efficiency of 95%, and while preserving the cells' viability. Cell-cell attachment was achieved by delivery of one to two trains of femtosecond laser pulses lasting 15 ms each. Laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane. The inner cell membrane remained intact during the attachment procedure, and isolation of the cells' cytoplasm from the surrounding medium was obtained. A strong physical attachment between the cells was obtained due to the bonding of the membranes' ionized phospholipid molecules and the formation of a joint cellular membrane at the connection point. The cellular attachment technique, femtosecond laser-induced cell-cell surgical attachment, can potentially provide a platform for the creation of engineered tissue and cell cultures. © 2014 Wiley Periodicals, Inc.

Biocompatibility evaluation of cigarette and carbon papers used in repair of traumatic tympanic membrane perforations: experimental study.

PubMed

Altuntaş, Emine Elif; Sümer, Zeynep

2013-01-01

The purposes of this study were to investigate the biocompatibility of two different paper patches (carbon and cigarette papers) and compare the adhesion and proliferation features of L929 fibroblast cells by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT Test) test and scanning electron microscopy (SEM). In this study, time-dependent cytotoxic effects of cigarette and carbon papers used in repairing small traumatic TM perforations were investigated in vitro by using MTT test. And also adhesion and spreading of cells over disk surface were observed by SEM. Cytotoxicity test carried out by MTT analysis on leakage products collected from two types of paper patches at the end of 24 and 48 h revealed no cytotoxicity (P > 0.05). In SEM studies, it was observed that cells started to proliferate over disk surface as a result of 48-h incubation, and SEM revealed that the cell proliferation over cigarette paper was more compared to the one over carbon paper. We believe that this is the first study where biocompatibility and adhesion features of carbon and cigarette paper have been studied by using L929 fibroblast cell culture. As a result, biocompatibility of cigarette paper and also whether cigarette paper was superior to carbon paper in cell attachment and biocompatibility were studied. It was found, by MTT test and SEM test, that cigarette paper had a higher biocompatibility and cell attachment, and thus cigarette paper should be the patch to be preferred in cases where TM perforations are repaired by paper-patch method.

Genipin-crosslinked microcarriers mediating hepatocellular aggregates formation and functionalities.

PubMed

Lau, Ting Ting; Wang, Chunming; Png, Sze Wei; Su, Kai; Wang, Dong-An

2011-01-01

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery. Copyright © 2010 Wiley Periodicals, Inc.

In vitro response to alkaline phosphatase coatings immobilized onto titanium implants using electrospray deposition or polydopamine-assisted deposition.

PubMed

Nijhuis, Arnold W G; van den Beucken, Jeroen J J P; Jansen, John A; Leeuwenburgh, Sander C G

2014-04-01

Immobilization of biomolecules onto implant surfaces is one of the most straightforward strategies to control the interaction between an implant and its biological environment. Recently, it was shown that the enzyme alkaline phosphatase (ALP) could be efficiently immobilized onto titanium implants in a single step using polydopamine. We hypothesized that such polydopamine-ALP coatings can enhance the early attachment of cells and increase mineralization. Therefore, the current study aimed at immobilization of ALP onto titanium by means of either one- or two-step polydopamine-assisted immobilization or electrospray deposition, the comparative characterization of these experimental substrates and subsequent cell behavioral analysis using primary osteoblast-like cells. Uncoated titanium and ALP-free polydopamine coatings served as controls. Despite significant ALP surface activity and lower water contact for angles ALP-containing surface modifications, only marginal effects on early cell behavior (i.e., cell spreading) and osteogenic differentiation (i.e., proliferation, differentiation and mineralization) were observed in comparison to uncoated titanium. Copyright © 2013 Wiley Periodicals, Inc.

Optoelectric biosensor using indium-tin-oxide electrodes.

PubMed

Choi, Chang Kyoung; Kihm, Kenneth D; English, Anthony E

2007-06-01

The use of an optically thin indium-tin-oxide (ITO) electrode is presented for an optoelectric biosensor simultaneously recording optical images and microimpedance to examine time-dependent cellular growth. The transmittance of a 100 nm thick ITO electrode layer is approximately the same as the transmittance of a clean glass substrate, whereas the industry-standard Au(47.5 nm)/Ti(2.5 nm) electrode layer drops the transmittance to less than 10% of that of the glass substrate. The simultaneous optoelectric measurements permit determining the correlation of the cell-covered area increase with the microimpedance increase, and the example results obtained for live porcine pulmonary artery endothelial cells delineate the quantitative and comprehensive nature of cellular attachment and spreading to the substrate, which has not been clearly perceived before.

AFFINITY OF ANIMAL CELL NUCLEOLI FOR NORMAL SERUM

PubMed Central

Maisel, John C.; Lytle, Ralph I.

1966-01-01

Nucleoli of animal cells cultured in vitro are modified by a component of "nonimmune" animal serum. Modified nucleoli bind fluorescein-conjugated nonimmune serum proteins, as shown by calcium ion-dependent fluorescence. Analysis of serum indicates that the nucleolar-binding component is a globulin, with an electrophoretic mobility in the same region as the slow alpha-1 component in pH 8.6 Veronal buffer. The component has a low sedimentation constant (2.4S), and appears to contain glycoprotein with relatively high sialic acid content (8.5%); the latter moiety may be essential to reaction with nucleoli. The nucleolar component reacting with this alpha globulin fraction appears to be a histonelike basic protein. Primary cultures of animal cells have been supported for 1 wk through attachment, spreading, and outgrowth from colonies to confluent monolayers in medium containing a nucleolar-reactive serum fraction as the only protein supplement. PMID:4164214

Influence of physicochemical properties of laser-modified polystyrene on bovine serum albumin adsorption and rat C6 glioma cell behavior.

PubMed

Wang, Xuefeng; Ohlin, C André; Lu, Qinghua; Hu, Jun

2006-09-15

Biomaterial surface modification is an efficient way of improving cell-material interactions. In this study, sub-micrometer laser-induced periodic surface structures (LIPSS) were produced on polystyrene by laser irradiation. FT-IR analysis confirmed that this treatment also led to surface oxidation and anisotropic orientation of the produced carbonyl groups. As a consequence, the surface energy of the laser-treated polystyrene was 1.45 times that of the untreated polystyrene, as measured by contact-angle goniometry. Protein adsorption and rat C6 glioma cell behavior on the two substrates were investigated, showing that the changed physicochemical properties of laser-modified polystyrene surface led to an increase in the quantity of adsorbed bovine serum albumin and significantly affected the behavior of rat C6 glioma cells. In the early stages of cell spreading, cells explored their microenvironment using filopodium as the main sensor. Moreover, cells actively aligned themselves along the direction of LIPSS gradually and cell attachment and proliferation were significantly enhanced. 2006 Wiley Periodicals, Inc. J Biomed Mater Res, 2006.

Antibody Against Integrin Lymphocyte Function-Associated Antigen 1 Inhibits HIV Type 1 Infection in Primary Cells Through Caspase-8-Mediated Apoptosis

PubMed Central

Walker, Tiffany N.; Cimakasky, Lisa M.; Coleman, Ebony M.; Madison, M. Nia

2013-01-01

Abstract HIV-1 infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell-adhesion molecules such as lymphocyte function-associated antigen 1 (LFA-1) form localized domains on the cell surface. Studies show that LFA-1 on the surface of HIV-1 particles retains its adhesion function and enhances virus attachment to susceptible cells by binding its counterreceptor intercellular adhesion molecule 1 (ICAM-1). This virus–cell interaction augments virus infectivity by facilitating binding and entry events. In this study, we demonstrate that inhibition of the LFA-1/ICAM-1 interaction by a monoclonal antibody leads to decreased virus production and spread in association with increased apoptosis of HIV-infected primary T cells. The data indicate that the LFA-1/ICAM-1 interaction may limit apoptosis in HIV-1-infected T cells. This phenomenon appears similar to anoikis wherein epithelial cells are protected from apoptosis conferred by ligand-bound integrins. These results have implications for further understanding HIV pathogenesis and replication in peripheral compartments and lymphoid organs. PMID:22697794

Nanofabricated Collagen-Inspired Synthetic Elastomers for Primary Rat Hepatocyte Culture

PubMed Central

Bettinger, Christopher J.; Kulig, Katherine M.; Vacanti, Joseph P.

2009-01-01

Synthetic substrates that mimic the properties of extracellular matrix proteins hold significant promise for use in systems designed for tissue engineering applications. In this report, we designed a synthetic polymeric substrate that is intended to mimic chemical, mechanical, and topological characteristics of collagen. We found that elastomeric poly(ester amide) substrates modified with replica-molded nanotopographic features enhanced initial attachment, spreading, and adhesion of primary rat hepatocytes. Further, hepatocytes cultured on nanotopographic substrates also demonstrated reduced albumin secretion and urea synthesis, which is indicative of strongly adherent hepatocytes. These results suggest that these engineered substrates can function as synthetic collagen analogs for in vitro cell culture. PMID:18847357

Place Attachment, Place Identity and the Development of the Child's Self-Identity: Searching the Literature to Develop an Hypothesis

ERIC Educational Resources Information Center

Spencer, Christopher

2005-01-01

This is part of a campaign to encourage educational researchers, geographers in particular, to spread their literature searches beyond their immediate subject area. The question of place attachment and identity is reviewed through the psychologistal literature. The hypothesis is offered and supported, that place, in a geographical sense is also…

Structure, Function, and Assembly of Type 1 Fimbriae

NASA Astrophysics Data System (ADS)

Knight, Stefan D.; Bouckaert, Julie

Bacterial infections constitute a major global health problem, acutely accentuated by the rapid spread of antibiotic resistant bacterial strains. The widespread need for bacteria to attach - adhere - to target cells before they can initiate an infection may be used to advantage by targeting the bacterial adhesion tools such as pili and fimbriae for development of novel anti-bacterial vaccines and drugs. Type 1 fimbriae are widely expressed by Escherichia coli. and are used by uropathogenic strains to mediate attachment to specific niches in the urinary tract. These fimbriae belong to a class of fibrillar adhesion organelles assembled through the chaperone/usher pathway, one of the terminal branches of the general secretion pathway in Gram-negative bacteria. Our understanding of the assembly, structure and function of these structures has evolved significantly over the last decade. Here, we summarize current understanding of the function and biogenesis of fibrillar adhesion organelles, and provide some examples of recent progress towards interfering with bacterial adhesion as a means to prevent infection.

Treating cancer stem cells and cancer metastasis using glucose-coated gold nanoparticles

PubMed Central

Hu, Chenxia; Niestroj, Martin; Yuan, Daniel; Chang, Steven; Chen, Jie

2015-01-01

Cancer ranks among the leading causes of human mortality. Cancer becomes intractable when it spreads from the primary tumor site to various organs (such as bone, lung, liver, and then brain). Unlike solid tumor cells, cancer stem cells and metastatic cancer cells grow in a non-attached (suspension) form when moving from their source to other locations in the body. Due to the non-attached growth nature, metastasis is often first detected in the circulatory systems, for instance in a lymph node near the primary tumor. Cancer research over the past several decades has primarily focused on treating solid tumors, but targeted therapy to treat cancer stem cells and cancer metastasis has yet to be developed. Because cancers undergo faster metabolism and consume more glucose than normal cells, glucose was chosen in this study as a reagent to target cancer cells. In particular, by covalently binding gold nanoparticles (GNPs) with thio-PEG (polyethylene glycol) and thio-glucose, the resulting functionalized GNPs (Glu-GNPs) were created for targeted treatment of cancer metastasis and cancer stem cells. Suspension cancer cell THP-1 (human monocytic cell line derived from acute monocytic leukemia patients) was selected because it has properties similar to cancer stem cells and has been used as a metastatic cancer cell model for in vitro studies. To take advantage of cancer cells’ elevated glucose consumption over normal cells, different starvation periods were screened in order to achieve optimal treatment effects. Cancer cells were then fed using Glu-GNPs followed by X-ray irradiation treatment. For comparison, solid tumor MCF-7 cells (breast cancer cell line) were studied as well. Our irradiation experimental results show that Glu-GNPs are better irradiation sensitizers to treat THP-1 cells than MCF-7 cells, or Glu-GNPs enhance the cancer killing of THP-1 cells 20% more than X-ray irradiation alone and GNP treatment alone. This finding can help oncologists to design therapeutic strategies to target cancer stem cells and cancer metastasis. PMID:25844037

In Vitro Cytocompatibility of One- and Two-Dimensional Nanostructure-Reinforced Biodegradable Polymeric Nanocomposites

PubMed Central

Farshid, Behzad; Lalwani, Gaurav; Sitharaman, Balaji

2015-01-01

This study investigates the in vitro cytocompatibility of one- and two-dimensional (1-D and 2-D) carbon and inorganic nanomaterial reinforced polymeric nanocomposites fabricated using biodegradable polymer poly (propylene fumarate), crosslinking agent N-vinyl pyrrolidone (NVP) and following nanomaterials: single- and multi- walled carbon nanotubes, single- and multi- walled graphene oxide nanoribbons, graphene oxide nanoplatelets, molybdenum disulfide nanoplatelets, or tungsten disulfide nanotubes dispersed between 0.02–0.2 wt% concentrations in the polymer. The extraction media of unreacted components, crosslinked nanocomposites and their degradation products between 1X-100X dilutions were examined for effects on viability and attachment employing two cell lines: NIH3T3 fibroblasts and MC3T3 pre-osteoblasts. The extraction media of unreacted PPF/NVP elicited acute dose-dependent cytotoxicity attributed to leaching of unreacted components into cell culture media. However, extraction media of crosslinked nanocomposites showed no dose dependent adverse effects. Further, all crosslinked nanocomposites showed high viability (78–100%), high cellular attachment (40–55%), and spreading that was confirmed by confocal and scanning electron microscopy. Degradation products of nanocomposites showed a mild dose-dependent cytotoxicity possibly due to acidic degradation components of PPF. In general, compared to PPF control, none of the nanocomposites showed significant differences in cellular response to the unreacted components, crosslinked nanocomposites and their degradation products. The initial minor cytotoxic response and lower cell attachment numbers were observed only for a few nanocomposite groups; these effects were absent at later time points for all PPF nanocomposites. The favorable cytocompatibility results for all the nanocomposites opens avenues for in vivo safety and efficacy studies for bone tissue engineering applications. PMID:25367032

Tunable Volumetric Density and Porous Structure of Spherical Poly-ε-caprolactone Microcarriers, as Applied in Human Mesenchymal Stem Cell Expansion.

PubMed

Li, Jian; Lam, Alan Tin-Lun; Toh, Jessica Pei Wen; Reuveny, Shaul; Oh, Steve Kah-Weng; Birch, William R

2017-03-28

Polymeric microspheres may serve as microcarrier (MC) matrices, for the expansion of anchorage-dependent stem cells. They require surface properties that promote both initial cell adhesion and the subsequent spreading of cells, which is a prerequisite for successful expansion. When implemented in a three-dimensional culture environment, under agitation, their suspension under low shear rates depends on the MCs having a modest negative buoyancy, with a density of 1.02-1.05 g/cm 3 . Bioresorbable poly-ε-caprolactone (PCL), with a density of 1.14 g/cm 3 , requires a reduction in volumetric density, for the microspheres to achieve high cell viability and yields. Uniform-sized droplets, from solutions of PCL dissolved in dichloromethane (DCM), were generated by coaxial microfluidic geometry. Subsequent exposure to ethanol rapidly extracted the DCM solvent, solidifying the droplets and yielding monodisperse microspheres with a porous structure, which was demonstrated to have tunable porosity and a hollow inner core. The variation in process parameters, including the molecular weight of PCL, its concentration in DCM, and the ethanol concentration, served to effectively alter the diffusion flux between ethanol and DCM, resulting in a broad spectrum of volumetric densities of 1.04-1.11 g/cm 3 . The solidified microspheres are generally covered by a smooth thin skin, which provides a uniform cell culture surface and masks their internal porous structure. When coated with a cationic polyelectrolyte and extracellular matrix protein, monodisperse microspheres with a diameter of approximately 150 μm and densities ranging from 1.05-1.11 g/cm 3 are capable of supporting the expansion of human mesenchymal stem cells (hMSCs). Validation of hMSC expansion was carried out with a positive control of commercial Cytodex 3 MCs and a negative control of uncoated low-density PCL MCs. Static culture conditions generated more than 70% cell attachment and similar yields of sixfold cell expansion on all coated MCs, with poor cell attachment and growth on the negative control. Under agitation, coated porous microspheres, with a low density of 1.05 g/cm 3 , achieved robust cell attachment and resulted in high cell yields of ninefold cell expansion, comparable with those generated by commercial Cytodex 3 MCs.

Behaviour of human mesenchymal stem cells on a polyelectrolyte-modified HEMA hydrogel for silk-based ligament tissue engineering.

PubMed

Bosetti, M; Boccafoschi, F; Calarco, A; Leigheb, M; Gatti, S; Piffanelli, V; Peluso, G; Cannas, M

2008-01-01

The aim of this study was to design a functional bio-engineered material to be used as scaffold for autologous mesenchymal stem cells in ligament tissue engineering. Polyelectrolyte modified HEMA hydrogel (HEMA-co-METAC), applied as coating on silk fibroin fibres, has been formulated in order to take advantage of the biocompatibility of the polyelectrolyte by increasing its mechanical properties with silk fibres. Human bone marrow mesenchymal stem cells behaviour on such reinforced polyelectrolyte has been studied by evaluating cell morphology, cell number, attachment, spreading and proliferation together with collagen matrix production and its mRNA expression. Silk fibroin fibres matrices with HEMA-co-METAC coating exhibited acceptable mechanical behaviour compared to the natural ligament, good human mesenchymal stem cell adhesion and with mRNA expression studies higher levels of collagen types I and III expression when compared to control cells on polystyrene. These data indicate high expression of mRNA for proteins responsible for the functional characteristics of the ligaments and suggest a potential for use of this biomaterial in ligament tissue-engineering applications.

Integration of micro nano and bio technologies with layer-by-layer self-assembly

NASA Astrophysics Data System (ADS)

Kommireddy, Dinesh Shankar

In the past decade, layer-by-layer (LbL) nanoassembly has been used as a tool for immobilization and surface modification of materials with applications in biology and physical sciences. Often, in such applications, LbL assembly is integrated with various techniques to form functional surface coatings and immobilized matrices. In this work, integration of LbL with microfabrication and microfluidics, and tissue engineering are explored. In an effort to integrate microfabrication with LbL nanoassembly, microchannels were fabricated using soft-lithography and the surface of these channels was used for the immobilization of materials using LbL and laminar flow patterning. Synthesis of poly(dimethyldiallyl ammonium chloride)/poly(styrene sulfonate) and poly(dimethyldiallyl ammonium chloride)/bovine serum albumin microstrips is demonstrated with the laminar flow microfluidic reactor. Resulting micropatterns are 8-10 mum wide, separated with few micron gaps. The width of these microstrips as well as their position in the microchannel is controlled by varying the flow rate, time of interaction and concentration of the individual components, which is verified by numerical simulation. Spatially resolved pH sensitivity was observed by modifying the surface of the channel with a pH sensitive dye. In order to investigate the integration of LbL assembly with tissue engineering, glass substrates were coated with nanoparticle/polyelectrolyte layers, and two different cell types were used to test the applicability of these coatings for the surface modification of medical implants. Titanium dioxide (TiO 2), silicon dioxide, halloysite and montmorillonite nanoparticles were assembled with oppositely charged polyelectrolytes. In-vitro cytotoxicity tests of the nanoparticle substrates on human dermal firbroblasts (HDFs) showed that the nanoparticle surfaces do not have toxic effects on the cells. HDFs retained their phenotype on the nanoparticle coatings, by synthesizing type-I collagen. These cells also showed active proliferation on the nanoparticle substrates. Cells attached on TiO2 substrates showed faster rate of spreading compared with the other types of nanoparticle coatings. Mesenchymal stem cells (MSCs) were used as a second cell type to support and elaborate on the results obtained with the HDFs. Increasing surface roughness was observed with increasing number of layers of TiO2. Tests with a higher number of layers of TiO2, showed an increased attachment, proliferation and faster spreading of the MSCs on a larger number of layers of TiO2.

The requirements for herpes simplex virus type 1 cell-cell spread via nectin-1 parallel those for virus entry.

PubMed

Even, Deborah L; Henley, Allison M; Geraghty, Robert J

2006-08-01

Herpes simplex virus type 1 (HSV-1) spreads from an infected cell to an uninfected cell by virus entry, virus-induced cell fusion, and cell-cell spread. The three forms of virus spread require the viral proteins gB, gD, and gH-gL, as well as a cellular gD receptor. The mutual requirement for the fusion glycoproteins and gD receptor suggests that virus entry, cell fusion, and cell-cell spread occur by a similar mechanism. The goals of this study were to examine the role of the nectin-1alpha transmembrane domain and cytoplasmic tail in cell-cell spread and to obtain a better understanding of the receptor-dependent events occurring at the plasma membrane during cell-cell spread. We determined that an intact nectin-1alpha V-like domain was required for cell-cell spread, while a membrane-spanning domain and cytoplasmic tail were not. Chimeric forms of nectin-1 that were non-functional for virus entry did not mediate cell-cell spread regardless of whether they could mediate cell fusion. Also, cell-cell spread of syncytial isolates was dependent upon nectin-1alpha expression and occurred through a nectin-1-dependent mechanism. Taken together, our results indicate that nectin-1-dependent events occurring at the plasma membrane during cell-cell spread were equivalent to those for virus entry.

Air conditioned suit

NASA Technical Reports Server (NTRS)

Carl, G. R. (Inventor)

1973-01-01

An environmentally controlled suit is described consisting of an airtight outergarment attached by an airtight bellows to the wall of a sterile chamber, an undergarment providing for circulation of air near the skin of the wearer, and a circulation system comprised of air supply and distribution to the extremities of the undegarment and central collection and exhaust of air from the midsection of the undergarment. A workman wearing the undergarment and attached circulation system enters the outer garment through a tunnel in the chamber wall and the attached bellows to work in the chamber without any danger of spreading bacteria.

Control of intracellular pH and growth by fibronectin in capillary endothelial cells

NASA Technical Reports Server (NTRS)

Ingber, D. E.; Prusty, D.; Frangioni, J. V.; Cragoe, E. J. Jr; Lechene, C.; Schwartz, M. A.

1990-01-01

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.

Hydrolyzable tannins (chebulagic acid and punicalagin) target viral glycoprotein-glycosaminoglycan interactions to inhibit herpes simplex virus 1 entry and cell-to-cell spread.

PubMed

Lin, Liang-Tzung; Chen, Ting-Ying; Chung, Chueh-Yao; Noyce, Ryan S; Grindley, T Bruce; McCormick, Craig; Lin, Ta-Chen; Wang, Guey-Horng; Lin, Chun-Ching; Richardson, Christopher D

2011-05-01

Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues.

Whole-Teflon microfluidic chips

PubMed Central

Ren, Kangning; Dai, Wen; Zhou, Jianhua; Su, Jing; Wu, Hongkai

2011-01-01

Although microfluidics has shown exciting potential, its broad applications are significantly limited by drawbacks of the materials used to make them. In this work, we present a convenient strategy for fabricating whole-Teflon microfluidic chips with integrated valves that show outstanding inertness to various chemicals and extreme resistance against all solvents. Compared with other microfluidic materials [e.g., poly(dimethylsiloxane) (PDMS)] the whole-Teflon chip has a few more advantages, such as no absorption of small molecules, little adsorption of biomolecules onto channel walls, and no leaching of residue molecules from the material bulk into the solution in the channel. Various biological cells have been cultured in the whole-Teflon channel. Adherent cells can attach to the channel bottom, spread, and proliferate well in the channels (with similar proliferation rate to the cells in PDMS channels with the same dimensions). The moderately good gas permeability of the Teflon materials makes it suitable to culture cells inside the microchannels for a long time. PMID:21536918

Effect of environmental stress on the ability of Listeria monocytogenes Scott A to attach to food contact surfaces.

PubMed

Smoot, L M; Pierson, M D

1998-10-01

Attachment of Listeria monocytogenes Scott A to Buna-N rubber and stainless steel under different temperature and pH conditions at the time of cell growth or at the time of attachment was investigated. All experiments were conducted using sterile phosphate buffer to avoid cell growth during exposure to the test surfaces. Numbers of attached cells increased with increasing attachment temperature (10 to 45 degrees C) and exposure time for both test surfaces. Maximum levels of attached cells were obtained when cell growth occurred at 30 degrees C. Downward, but not upward, shifts in the cell suspension holding temperature prior to attachment to Buna-N rubber resulted in reduced adhered cell populations. Maximum levels of adhered cells to Buna-N rubber were not affected by adjustments of the attachment medium pH between 4 and 9. However, after short contact times (i.e., less than 30 min), levels of attached cells were lower when attachment occurred under alkaline conditions. Growth pH was also found to affect the levels of adhered cell populations to Buna-N rubber. L. monocytogenes Scott A attached to stainless steel at higher levels for all temperature and pH parameters evaluated in this study.

Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

PubMed

Matthysse, A G; Wyman, P M; Holmes, K V

1978-11-01

Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains.

Long-term liver-specific functions of hepatocytes in electrospun chitosan nanofiber scaffolds coated with fibronectin.

PubMed

Rajendran, Divya; Hussain, Ali; Yip, Derek; Parekh, Amit; Shrirao, Anil; Cho, Cheul H

2017-08-01

In this study, a new 3D liver model was developed using biomimetic nanofiber scaffolds and co-culture system consisting of hepatocytes and fibroblasts for the maintenance of long-term liver functions. The chitosan nanofiber scaffolds were fabricated by the electrospinning technique. To enhance cellular adhesion and spreading, the surfaces of the chitosan scaffolds were coated with fibronectin (FN) by adsorption and evaluated for various cell types. Cellular phenotype, protein expression, and liver-specific functions were extensively characterized by immunofluorescent and histochemical stainings, albumin enzyme-linked immunosorbent assay and Cytochrome p450 detoxification assays, and scanning electron microscopy. The electrospun chitosan scaffolds exhibited a highly porous and randomly oriented nanofibrous structure. The FN coating on the surface of the chitosan nanofibers significantly enhanced cell attachment and spreading, as expected, as surface modification with this cell adhesion molecule on the chitosan surface is important for focal adhesion formation and integrin binding. Comparison of hepatocyte mono-cultures and co-cultures in 3D culture systems indicated that the hepatocytes in co-cultures formed colonies and maintained their morphologies and functions for prolonged periods of time. The 3D liver tissue model developed in this study will provide useful tools toward the development of engineered liver tissues for drug screening and tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2119-2128, 2017. © 2017 Wiley Periodicals, Inc.

Plasma treatment induces internal surface modifications of electrospun poly(L-lactic) acid scaffold to enhance protein coating

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

Jin Seo, Hyok; Hee Lee, Mi; Kwon, Byeong-Ju

2013-08-21

Advanced biomaterials should also be bioactive with regard to desirable cellular responses, such as selective protein adsorption and cell attachment, proliferation, and differentiation. To enhance cell-material interactions, surface modifications have commonly been performed. Among the various surface modification approaches, atmospheric pressure glow discharge plasma has been used to change a hydrophobic polymer surface to a hydrophilic surface. Poly(L-lactic acid) (PLLA)-derived scaffolds lack cell recognition signals and the hydrophobic nature of PLLA hinders cell seeding. To make PLLA surfaces more conducive to cell attachment and spreading, surface modifications may be used to create cell-biomaterial interfaces that elicit controlled cell adhesion andmore » maintain differentiated phenotypes. In this study, (He) gaseous atmospheric plasma glow discharge was used to change the characteristics of a 3D-type polymeric scaffold from hydrophobic to hydrophilic on both the outer and inner surfaces of the scaffold and the penetration efficiency with fibronectin was investigated. Field-emission scanning electron microscope images showed that some grooves were formed on the PLLA fibers after plasma treatment. X-ray photoelectron spectroscopy data also showed chemical changes in the PLLA structure. After plasma treatment, -CN (285.76 eV) was increased in C1s and -NH{sub 2} (399.70 eV) was increased significantly and –N=CH (400.80 eV) and –NH{sub 3}{sup +} (402.05 eV) were newly appeared in N1s. These changes allowed fibronectin to penetrate into the PLLA scaffold; this could be observed by confocal microscopy. In conclusion, helium atmospheric pressure plasma treatment was effective in modifying the polymeric scaffold, making it hydrophilic, and this treatment can also be used in tissue engineering research as needed to make polymers hydrophilic.« less

Banded Spherulitic Morphology in Blends of Poly(propylene fumarate) and Poly( -caprolactone) and Interaction with MC3T3-E1 Cells

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

Wang, Kan; Jesse, Stephen; Wang, Shanfeng

2012-01-01

The thermal properties, morphological development, crystallization behavior, and miscibility of semicrystalline PCL and its 25, 50, and 75 wt% blends with amorphous PPF in spin-coated thin films crystallized at various crystallization temperatures (T{sub c}) from 25 to 52 C are investigated. The surface roughness of PPF/PCL ({phi}{sub PCL} = 75%) films increases with increasing T{sub c} and consequently the adsorption of serum proteins is also increased. No significant variance is found in surface hydrophilicity or in mouse MC3T3-E1 cell attachment, spreading, and proliferation on PPF/PCL ({phi}{sub PCL} = 75%) films crystallized isothermally at 25, 37, and 45 C, because ofmore » low ridge height, nonuniformity in structures, and PPF surface segregation« less

Staphylococcus aureus biofilms: recent developments in biofilm dispersal.

PubMed

Lister, Jessica L; Horswill, Alexander R

2014-01-01

Staphylococcus aureus is a major cause of nosocomial and community-acquired infections and represents a significant burden on the healthcare system. S. aureus attachment to medical implants and host tissue, and the establishment of a mature biofilm, play an important role in the persistence of chronic infections. The formation of a biofilm, and encasement of cells in a polymer-based matrix, decreases the susceptibility to antimicrobials and immune defenses, making these infections difficult to eradicate. During infection, dispersal of cells from the biofilm can result in spread to secondary sites and worsening of the infection. In this review, we discuss the current understanding of the pathways behind biofilm dispersal in S. aureus, with a focus on enzymatic and newly described broad-spectrum dispersal mechanisms. Additionally, we explore potential applications of dispersal in the treatment of biofilm-mediated infections.

Silk-fibronectin protein alloy fibres support cell adhesion and viability as a high strength, matrix fibre analogue

NASA Astrophysics Data System (ADS)

Jacobsen, Matthew M.; Li, David; Gyune Rim, Nae; Backman, Daniel; Smith, Michael L.; Wong, Joyce Y.

2017-04-01

Silk is a natural polymer with broad utility in biomedical applications because it exhibits general biocompatibility and high tensile material properties. While mechanical integrity is important for most biomaterial applications, proper function and integration also requires biomaterial incorporation into complex surrounding tissues for many physiologically relevant processes such as wound healing. In this study, we spin silk fibroin into a protein alloy fibre with whole fibronectin using wet spinning approaches in order to synergize their respective strength and cell interaction capabilities. Results demonstrate that silk fibroin alone is a poor adhesive surface for fibroblasts, endothelial cells, and vascular smooth muscle cells in the absence of serum. However, significantly improved cell attachment is observed to silk-fibronectin alloy fibres without serum present while not compromising the fibres’ mechanical integrity. Additionally, cell viability is improved up to six fold on alloy fibres when serum is present while migration and spreading generally increase as well. These findings demonstrate the utility of composite protein alloys as inexpensive and effective means to create durable, biologically active biomaterials.

Determining the Involvement and Therapeutic Implications of Host Cellular Factors in Hepatitis C Virus Cell-to-Cell Spread

PubMed Central

Barretto, Naina; Sainz, Bruno; Hussain, Snawar

2014-01-01

ABSTRACT Hepatitis C virus (HCV) infects 180 million people worldwide and is a leading cause of liver diseases such as fibrosis, cirrhosis, and hepatocellular carcinoma. It has been shown that HCV can spread to naive cells using two distinct entry mechanisms, “cell-free” entry of infectious extracellular virions that have been released by infected cells and direct “cell-to-cell” transmission. Here, we examined host cell requirements for HCV spread and found that the cholesterol uptake receptor NPC1L1, which we recently identified as being an antiviral target involved in HCV cell-free entry/spread, is also required for the cell-to-cell spread. In contrast, the very low density lipoprotein (VLDL) pathway, which is required for the secretion of cell-free infectious virus and thus has been identified as an antiviral target for blocking cell-free virus secretion/spread, is not required for cell-to-cell spread. Noting that HCV cell-free and cell-to-cell spread share some common factors but not others, we tested the therapeutic implications of these observations and demonstrate that inhibitors that target cell factors required for both forms of HCV spread exhibit synergy when used in combination with interferon (a representative inhibitor of intracellular HCV production), while inhibitors that block only cell-free spread do not. This provides insight into the mechanistic basis of synergy between interferon and HCV entry inhibitors and highlights the broader, previously unappreciated impact blocking HCV cell-to-cell spread can have on the efficacy of HCV combination therapies. IMPORTANCE HCV can spread to naive cells using distinct mechanisms: “cell-free” entry of extracellular virus and direct “cell-to-cell” transmission. Herein, we identify the host cell HCV entry factor NPC1L1 as also being required for HCV cell-to-cell spread, while showing that the VLDL pathway, which is required for the secretion of cell-free infectious virus, is not required for cell-to-cell spread. While both these host factors are considered viable antiviral targets, we demonstrate that only inhibitors that block factors required for both forms of HCV entry/spread (i.e., NPC1L1) exhibit synergy when used in combination with interferon, while inhibitors that block factors required only for cell-free spread (i.e., VLDL pathway components) do not. Thus, this study advances our understanding of HCV cell-to-cell spread, provides mechanistic insight into the basis of drug synergy, and highlights inhibition of HCV spread as a previously unappreciated consideration in HCV therapy design. PMID:24554660

Effect of TiO2 Nanofiller Concentration on the Mechanical, Thermal and Biological Properties of HDPE/TiO2 Nanocomposites

NASA Astrophysics Data System (ADS)

Mozumder, Mohammad Sayem; Mourad, Abdel-Hamid I.; Mairpady, Anusha; Pervez, Hifsa; Haque, Md Emdadul

2018-03-01

The necessity for advanced and effective biomimetic tissue engineering materials has increased massively as bone diseases such as osteoporosis and bone cancer have become a major public health problem. Therefore, the objective of this study is to develop titanium dioxide (TiO2) nanoparticles-enriched high-density polyethylene (HDPE) nanocomposites that could serve as potential biomaterials. HDPE/TiO2 nanocomposites with varying TiO2 nanoparticles content were fabricated by using injection molding technique and were subjected to mechanical, thermal and biological characterization. SEM-EDS analysis confirmed even dispersion of TiO2 nanoparticles into the HDPE matrix. It was observed from the mechanical testing that the addition of TiO2 nanoparticles to HDPE noticeably improved the stiffness (from 345 to 378 MPa) while maintaining almost similar yield strength of pure HDPE. The thermal analyses revealed that TiO2 nanoparticles inclusion to HDPE matrix enhanced the thermal stability of nanocomposites, as the overall rate of crystallization increased by almost 4%. Furthermore, biocompatibility of nanocomposites was also studied by means of various cell culture experiments; human osteoblasts (hFOB) were seeded on the HDPE/TiO2 nanocomposites and were visualized through SEM after 72 h of incubation; surface morphology revealed normal cell growth and spreading with more attachment on PNC-10 that contains 10 wt.% of TiO2. Moreover, cell viability assays (i.e., MTT and cell attachment) revealed consistent increase in cell count and metabolic activity when triplicate cultures were incubated for 1, 3 and 7 days.

Effect of TiO2 Nanofiller Concentration on the Mechanical, Thermal and Biological Properties of HDPE/TiO2 Nanocomposites

NASA Astrophysics Data System (ADS)

Mozumder, Mohammad Sayem; Mourad, Abdel-Hamid I.; Mairpady, Anusha; Pervez, Hifsa; Haque, Md Emdadul

2018-05-01

The necessity for advanced and effective biomimetic tissue engineering materials has increased massively as bone diseases such as osteoporosis and bone cancer have become a major public health problem. Therefore, the objective of this study is to develop titanium dioxide (TiO2) nanoparticles-enriched high-density polyethylene (HDPE) nanocomposites that could serve as potential biomaterials. HDPE/TiO2 nanocomposites with varying TiO2 nanoparticles content were fabricated by using injection molding technique and were subjected to mechanical, thermal and biological characterization. SEM-EDS analysis confirmed even dispersion of TiO2 nanoparticles into the HDPE matrix. It was observed from the mechanical testing that the addition of TiO2 nanoparticles to HDPE noticeably improved the stiffness (from 345 to 378 MPa) while maintaining almost similar yield strength of pure HDPE. The thermal analyses revealed that TiO2 nanoparticles inclusion to HDPE matrix enhanced the thermal stability of nanocomposites, as the overall rate of crystallization increased by almost 4%. Furthermore, biocompatibility of nanocomposites was also studied by means of various cell culture experiments; human osteoblasts (hFOB) were seeded on the HDPE/TiO2 nanocomposites and were visualized through SEM after 72 h of incubation; surface morphology revealed normal cell growth and spreading with more attachment on PNC-10 that contains 10 wt.% of TiO2. Moreover, cell viability assays (i.e., MTT and cell attachment) revealed consistent increase in cell count and metabolic activity when triplicate cultures were incubated for 1, 3 and 7 days.

Osteogenic differentiation on DLC-PDMS-h surface.

PubMed

Soininen, Antti; Kaivosoja, Emilia; Sillat, Tarvo; Virtanen, Sannakaisa; Konttinen, Yrjö T; Tiainen, Veli-Matti

2014-10-01

The hypothesis was that anti-fouling diamond-like carbon polydimethylsiloxane hybrid (DLC-PDMS-h) surface impairs early and late cellular adhesion and matrix-cell interactions. The effect of hybrid surface on cellular adhesion and cytoskeletal organization, important for osteogenesis of human mesenchymal stromal cells (hMSC), where therefore compared with plain DLC and titanium (Ti). hMSCs were induced to osteogenesis and followed over time using scanning electron microscopy (SEM), time-of-flight secondary ion mass spectrometry (ToF-SIMS), immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR), and hydroxyapatite (HA) staining. SEM at 7.5 hours showed that initial adherence and spreading of hMSC was poor on DLC-PDMS-h. At 5 days some hMSC were undergoing condensation and apoptotic fragmentation, whereas cells on DLC and Ti grew well. DAPI-actin-vinculin triple staining disclosed dwarfed cells with poorly organized actin cytoskeleton-focal complex/adhesion-growth substrate attachments on hybrid coating, whereas spread cells, organized microfilament bundles, and focal adhesions were seen on DLC and in particular on Ti. Accordingly, at day one ToF-SIMS mass peaks showed poor protein adhesion to DLC-PDMS-h compared with DLC and Ti. COL1A1, ALP, OP mRNA levels at days 0, 7, 14, 21, and/or 28 and lack of HA deposition at day 28 demonstrated delayed or failed osteogenesis on DLC-PDMS-h. Anti-fouling DLC-PDMS-h is a poor cell adhesion substrate during the early protein adsorption-dependent phase and extracellular matrix-dependent late phase. Accordingly, some hMSCs underwent anoikis-type apoptosis and failed to complete osteogenesis, due to few focal adhesions and poor cell-to-ECM contacts. DLC-PDMS-h seems to be a suitable coating for non-integrating implants/devices designed for temporary use. © 2014 Wiley Periodicals, Inc.

Geraniin extracted from the rind of Nephelium lappaceum binds to dengue virus type-2 envelope protein and inhibits early stage of virus replication.

PubMed

Abdul Ahmad, Siti Aisyah; Palanisamy, Uma D; Tejo, Bimo A; Chew, Miaw Fang; Tham, Hong Wai; Syed Hassan, Sharifah

2017-11-21

The rapid rise and spread in dengue cases, together with the unavailability of safe vaccines and effective antiviral drugs, warrant the need to discover and develop novel anti-dengue treatments. In this study the antiviral activity of geraniin, extracted from the rind of Nephelium lappaceum, against dengue virus type-2 (DENV-2) was investigated. Geraniin was prepared from Nephelium lappaceum rind by reverse phase C-18 column chromatography. Cytotoxicity of geraniin towards Vero cells was evaluated using MTT assay while IC 50 value was determined by plaque reduction assay. The mode-of-action of geraniin was characterized using the virucidal, attachment, penetration and the time-of-addition assays'. Docking experiments with geraniin molecule and the DENV envelope (E) protein was also performed. Finally, recombinant E Domain III (rE-DIII) protein was produced to physiologically test the binding of geraniin to DENV-2 E-DIII protein, through ELISA competitive binding assay. Cytotoxicity assay confirmed that geraniin was not toxic to Vero cells, even at the highest concentration tested. The compound exhibited DENV-2 plaque formation inhibition, with an IC 50 of 1.75 μM. We further revealed that geraniin reduced viral infectivity and inhibited DENV-2 from attaching to the cells but had little effect on its penetration. Geraniin was observed to be most effective when added at the early stage of DENV-2 infection. Docking experiments showed that geraniin binds to DENV E protein, specifically at the DIII region, while the ELISA competitive binding assay confirmed geraniin's interaction with rE-DIII with high affinity. Geraniin from the rind of Nephelium lappaceum has antiviral activity against DENV-2. It is postulated that the compound inhibits viral attachment by binding to the E-DIII protein and interferes with the initial cell-virus interaction. Our results demonstrate that geraniin has the potential to be developed into an effective antiviral treatment, particularly for early phase dengue viral infection.

Comparison of candidate materials for a synthetic osteo-odonto keratoprosthesis device.

PubMed

Tan, Xiao Wei; Perera, A Promoda P; Tan, Anna; Tan, Donald; Khor, K A; Beuerman, Roger W; Mehta, Jodhbir S

2011-01-05

Osteo-odonto keratoprosthesis is one of the most successful forms of keratoprosthesis surgery for end-stage corneal and ocular surface disease. There is a lack of detailed comparison studies on the biocompatibilities of different materials used in keratoprosthesis. The aim of this investigation was to compare synthetic bioinert materials used for keratoprosthesis surgery with hydroxyapatite (HA) as a reference. Test materials were sintered titanium oxide (TiO(2)), aluminum oxide (Al(2)O(3)), and yttria-stabilized zirconia (YSZ) with density >95%. Bacterial adhesion on the substrates was evaluated using scanning electron microscopy and the spread plate method. Surface properties of the implant discs were scanned using optical microscopy. Human keratocyte attachment and proliferation rates were assessed by cell counting and MTT assay at different time points. Morphologic analysis and immunoblotting were used to evaluate focal adhesion formation, whereas cell adhesion force was measured with a multimode atomic force microscope. The authors found that bacterial adhesion on the TiO(2), Al(2)O(3), and YSZ surfaces were lower than that on HA substrates. TiO(2) significantly promoted keratocyte proliferation and viability compared with HA, Al(2)O(3,) and YSZ. Immunofluorescent imaging analyses, immunoblotting, and atomic force microscope measurement revealed that TiO(2) surfaces enhanced cell spreading and cell adhesion compared with HA and Al(2)O(3). TiO(2) is the most suitable replacement candidate for use as skirt material because it enhanced cell functions and reduced bacterial adhesion. This would, in turn, enhance tissue integration and reduce device failure rates during keratoprosthesis surgery.

Plasmid-dependent attachment of Agrobacterium tumefaciens to plant tissue culture cells.

PubMed Central

Matthysse, A G; Wyman, P M; Holmes, K V

1978-01-01

Kinetic, microscopic, and biochemical studies show that virulent Ti (tumor inducing)-plasmid-containing strains of Agrobacterium attach to normal tobacco and carrot tissue culture cells. Kinetic studies showed that virulent strains of A. tumefaciens attach to the plant tissue culture cells in increasing numbers during the first 1 to 2 h of incubation of the bacteria with the plant cells. Five Ti-plasmid-containing virulent Agrobacterium strains showed greater attachment to tobacco cells than did five avirulent strains. Light and scanning electron microscopic observations confirmed that virulent strains showed little attachment. Bacterial attachment was blocked by prior incubation of the plant cells with lipopolysaccharide extracted from A. tumefaciens, but not from A. radiobacter, suggesting that bacterial lipopolysaccharide is one of the components involved in the attachment process. At least one other bacterial product may be required for attachment in tissue culture because the virulent A. tumefaciens NT1, which lacks the Ti plasmid, does not itself attach to tobacco cells, but its lipopolysaccharide does inhibit the attachment of virulent strains. Images PMID:730370

Qualitative flow visualization of flame attachment on slopes

Treesearch

Torben P. Grumstrup; Sara S. McAllister; Mark A. Finney

2017-01-01

Heating of unburned fuel by attached flames and plume of a wildfire can produce high spread rates that have resulted in firefighter fatalities worldwide. Qualitative flow fields of the plume of a gas burner embedded in a table tilted to 0°, 10°, 20°, and 30° above horizontal were imaged using the retroreflective shadowgraph technique as a means to understand plume...

Attachment of Salmonella strains to a plant cell wall model is modulated by surface characteristics and not by specific carbohydrate interactions.

PubMed

Tan, Michelle Sze-Fan; Moore, Sean C; Tabor, Rico F; Fegan, Narelle; Rahman, Sadequr; Dykes, Gary A

2016-09-15

Processing of fresh produce exposes cut surfaces of plant cell walls that then become vulnerable to human foodborne pathogen attachment and contamination, particularly by Salmonella enterica. Plant cell walls are mainly composed of the polysaccharides cellulose, pectin and hemicelluloses (predominantly xyloglucan). Our previous work used bacterial cellulose-based plant cell wall models to study the interaction between Salmonella and the various plant cell wall components. We demonstrated that Salmonella attachment was favoured in the presence of pectin while xyloglucan had no effect on its attachment. Xyloglucan significantly increased the attachment of Salmonella cells to the plant cell wall model only when it was in association with pectin. In this study, we investigate whether the plant cell wall polysaccharides mediate Salmonella attachment to the bacterial cellulose-based plant cell wall models through specific carbohydrate interactions or through the effects of carbohydrates on the physical characteristics of the attachment surface. We found that none of the monosaccharides that make up the plant cell wall polysaccharides specifically inhibit Salmonella attachment to the bacterial cellulose-based plant cell wall models. Confocal laser scanning microscopy showed that Salmonella cells can penetrate and attach within the tightly arranged bacterial cellulose network. Analysis of images obtained from atomic force microscopy revealed that the bacterial cellulose-pectin-xyloglucan composite with 0.3 % (w/v) xyloglucan, previously shown to have the highest number of Salmonella cells attached to it, had significantly thicker cellulose fibrils compared to other composites. Scanning electron microscopy images also showed that the bacterial cellulose and bacterial cellulose-xyloglucan composites were more porous when compared to the other composites containing pectin. Our study found that the attachment of Salmonella cells to cut plant cell walls was not mediated by specific carbohydrate interactions. This suggests that the attachment of Salmonella strains to the plant cell wall models were more dependent on the structural characteristics of the attachment surface. Pectin reduces the porosity and space between cellulose fibrils, which then forms a matrix that is able to retain Salmonella cells within the bacterial cellulose network. When present with pectin, xyloglucan provides a greater surface for Salmonella cells to attach through the thickening of cellulose fibrils.

Highly sensitive label-free dual sensor array for rapid detection of wound bacteria.

PubMed

Sheybani, Roya; Shukla, Anita

2017-06-15

Wound infections are a critical healthcare concern worldwide. Rapid and effective antibiotic treatments that can mitigate infection severity and prevent the spread of antibiotic resistance are contingent upon timely infection detection. In this work, dual electrochemical pH and cell-attachment sensor arrays were developed for the real-time spatial and temporal monitoring of potential wound infections. Biocompatible polymeric device coatings were integrated to stabilize the sensors and promote bacteria attachment while preventing non-specific cell and protein fouling. High sensitivity (bacteria concentration of 10 2 colony forming units (CFU)/mL and -88.1±6.3mV/pH over a pH range of 1-13) and stability over 14 days were achieved without the addition of biological recognition elements. The dual sensor array was demonstrated to successfully monitor the growth of both gram-positive (Staphylococcus aureus and Streptococcus pyogenes) and gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) over time through lag and log growth phases and following antibiotic administration and in simulated shallow wounds conditions. The versatile fabrication methods utilized in sensor development, superior sensitivity, prolonged stability, and lack of non-specific sensor fouling may enable long-term in situ sensor array operation in low resource settings. Copyright © 2016 Elsevier B.V. All rights reserved.

Differentiation and characteristics of the enhanced green fluorescent protein gene transgenic goat neural stem cells cultured in attached and non-attached plates.

PubMed

Zheng, Yue-Mao; Dang, Yong-Hui; Qiu, Shuang; Qi, Ying-Pei; Xu, Yong-Ping; Sai, Wu-Jia-Fu

2011-08-01

The aims of this study were (i) to determine whether NSCs (neural stem cells) could be isolated from the brain of embryonic day 98 fetal goat, (ii) to determine if these stem cells have the capability of multipotent differentiation following transfection with a reporter gene, EGFP (enhanced green fluorescent protein) and (iii) to study the characteristics of the stem cells cultured in attached and non-attached plates. NSCs were isolated from embryonic day 98 fetal goat brain, transfected with EGFP gene using lipofection, and subcultured in attached and non-attached plates respectively. The transgenic stem cells were induced to differentiate into osteogenic and endothelial cells in vitro respectively. Markers associated with undifferentiated NSCs and their differentiated cells were tested by RT-PCR (reverse transcription-PCR). The results demonstrated that stem cells could be isolated from embryonic day 98 fetal goat brain, and EGFP gene could be transfected into the cells. The transgenic NSCs were capable of self-renewal, a defining property of stem cells, and were grown as free-floating neurospheres in non-attached plates. When the neurospheres were transferred and cultured in attached plates, cells migrate from the neurospheres and are grown as spindle cells. The stem cells were grown as quasi-circular cells when the single stem cells were cultured in attached plates. Both the NSCs cultured in non-attached and attached plates could express Hes1 (hairy and enhancer of split 1), Oct4 (octamer-binding protein 4), Nanog, Sox2 [SRY (sex-determining region Y)-box 2] and Nestin, while following differentiation cells expressed markers for osteogenic cells (Osteocalcin+ and Osteonectin+) and endothelium (CD34+ and eNOS+). The results demonstrated that the goat EGFP gene transgenic NSCs have the capability of multipotent differentiation, which means that the transgenic NSCs may be useful in cell transplantation studies in future.

Fossil slabs attached to unsubducted fragments of the Farallon plate.

PubMed

Wang, Yun; Forsyth, Donald W; Rau, Christina J; Carriero, Nina; Schmandt, Brandon; Gaherty, James B; Savage, Brian

2013-04-02

As the Pacific-Farallon spreading center approached North America, the Farallon plate fragmented into a number of small plates. Some of the microplate fragments ceased subducting before the spreading center reached the trench. Most tectonic models have assumed that the subducting oceanic slab detached from these microplates close to the trench, but recent seismic tomography studies have revealed a high-velocity anomaly beneath Baja California that appears to be a fossil slab still attached to the Guadalupe and Magdalena microplates. Here, using surface wave tomography, we establish the lateral extent of this fossil slab and show that it is correlated with the distribution of high-Mg andesites thought to derive from partial melting of the subducted oceanic crust. We also reinterpret the high seismic velocity anomaly beneath the southern central valley of California as another fossil slab extending to a depth of 200 km or more that is attached to the former Monterey microplate. The existence of these fossil slabs may force a reexamination of models of the tectonic evolution of western North America over the last 30 My.

Study on performance of magnetic fluorescent nanoparticles as gene carrier and location in pig kidney cells

NASA Astrophysics Data System (ADS)

Wang, Yan; Cui, Haixin; Sun, Changjiao; Du, Wei; Cui, Jinhui; Zhao, Xiang

2013-03-01

We evaluated the performance of green fluorescent magnetic Fe3O4 nanoparticles (NPs) as gene carrier and location in pig kidney cells. When the mass ratio of NPs to green fluorescent protein plasmid DNA reached 1:16 or above, DNA molecules can be combined completely with NPs, which indicates that the NPs have good ability to bind negative DNA. Atomic force microscopy (AFM) experiments were carried out to investigate the binding mechanism between NPs and DNA. AFM images show that individual DNA strands come off of larger pieces of netlike agglomerations and several spherical nanoparticles are attached to each individual DNA strand and interact with each other. The pig kidney cells were labelled with membrane-specific red fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and nucleus-specific blue fluorescent dye 4',6-diamidino-2-phenylindole dihydrochloride. We found that green fluorescent nanoparticles can past the cell membrane and spread throughout the interior of the cell. The NPs seem to locate more frequently in the cytoplasm than in the nucleus.

RIC8A is essential for the organisation of actin cytoskeleton and cell-matrix interaction.

PubMed

Ruisu, Katrin; Meier, Riho; Kask, Keiu; Tõnissoo, Tambet; Velling, Teet; Pooga, Margus

2017-08-15

RIC8A functions as a chaperone and guanine nucleotide exchange factor for a subset of G protein α subunits. Multiple G protein subunits mediate various signalling events that regulate cell adhesion and migration and the involvement of RIC8A in some of these processes has been demonstrated. We have previously shown that the deficiency of RIC8A causes a failure in mouse gastrulation and neurogenesis - major events in embryogenesis that rely on proper association of cells with the extracellular matrix (ECM) and involve active cell migration. To elaborate on these findings, we used Ric8a -/- mouse embryonic stem cells and Ric8a-deficient mouse embryonic fibroblasts, and found that RIC8A plays an important role in the organisation and remodelling of actin cytoskeleton and cell-ECM association. Ric8a-deficient cells were able to attach to different ECM components, but were unable to spread correctly, and did not form stress fibres or focal adhesion complexes. We also found that the presence of RIC8A is necessary for the activation of β1 integrins and integrin-mediated cell migration. Copyright © 2017 Elsevier Inc. All rights reserved.

The lid wiper and muco-cutaneous junction anatomy of the human eyelid margins: an in vivo confocal and histological study

PubMed Central

Knop, Erich; Knop, Nadja; Zhivov, Andrey; Kraak, Robert; Korb, Donald R; Blackie, Caroline; Greiner, Jack V; Guthoff, Rudolf

2011-01-01

The inner border of the eyelid margin is critically important for ocular surface integrity because it guarantees the thin spread of the tear film. Its exact morphology in the human is still insufficiently known. The histology in serial sections of upper and lower lid margins in whole-mount specimens from 10 human body donors was compared to in vivo confocal microscopy of eight eyes with a Heidelberg retina-tomograph (HRT II) and attached Rostock cornea module. Behind the posterior margin of the Meibomian orifices, the cornified epidermis stopped abruptly and was replaced by a continuous layer of para-keratinized (pk) cells followed by discontinuous pk cells. The pk cells covered the muco-cutaneous junction (MCJ), the surface of which corresponded to the line of Marx (0.2–0.3 mm wide). Then a stratified epithelium with a conjunctival structure of cuboidal cells, some pk cells, and goblet cells formed an epithelial elevation of typically about 100 μm initial thickness (lid wiper). This continued for 0.3–1.5 mm and formed a slope. The MCJ and lid wiper extended all along the lid margin from nasal to temporal positions in the upper and lower lids. Details of the epithelium and connective tissue were also detectable using the Rostock cornea module. The human inner lid border has distinct zones. Due to its location and morphology, the epithelial lip of the lid wiper appears a suitable structure to spread the tear film and is distinct from the MCJ/line of Marx. Better knowledge of the lid margin appears important for understanding dry eye disease and its morphology can be analysed clinically by in vivo confocal microscopy. PMID:21413985

Interaction of Chlamydia trachomatis organisms and HeLa 229 cells.

PubMed Central

Kuo, C C; Grayston, T

1976-01-01

The infection of HeLa 229 cells in monolayer culture with trachoma (B/TW-5/OT) and lymphogranuloma venereum (LGV) (L2/434/Bu) organism was studied in terms of two parameters: radioactivity counts of cell-associated tritium labeled organisms at the initial stage of inoculation for measurement of attachment, and inclusion counts of infection cells after incubation for measurement of growth. Factors affecting attachment and inclusion formation and correlation of the two are presented. It was shown that attachment is an important initial step in infection by Chlamydia trachomatis. The rate of attachment was temperature dependent. The attachment of LGV organisms was affected more profoundly by temperature than was that of trachoma organisms. Attachment and inclusion formation of trachoma and LGV organisms were inhibited by heparin. Diethylaminoethyl-dextran was again shown to enhance attachment and inclusion formation of trachoma but not LGV organisms. NaF had no effect on attachment, but inhibited inclusion formation of both trachoma and LGV organisms. Both attachment and inclusion formation of trachoma organisms were strongly enhanced by centrifugation of the inoculum onto the cell monolayer. Although inclusion formation of trachoma organism was much greater in susceptible cells (HeLa 229) than relatively insusceptible cells (fetal tonsil), attachment was only slightly greater. The results based on the test of two cell lines suggested that attachment prpbably is not a critical factor in determing a cell line's susceptibility to infection with trachoma organisms. PMID:179950

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading.

PubMed

Li, Yuan; Lovett, David; Zhang, Qiao; Neelam, Srujana; Kuchibhotla, Ram Anirudh; Zhu, Ruijun; Gundersen, Gregg G; Lele, Tanmay P; Dickinson, Richard B

2015-08-18

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Moving Cell Boundaries Drive Nuclear Shaping during Cell Spreading

PubMed Central

Li, Yuan; Lovett, David; Zhang, Qiao; Neelam, Srujana; Kuchibhotla, Ram Anirudh; Zhu, Ruijun; Gundersen, Gregg G.; Lele, Tanmay P.; Dickinson, Richard B.

2015-01-01

The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes. PMID:26287620

Hydrocarbon Deposition Attenuates Osteoblast Activity on Titanium

PubMed Central

Hayashi, R.; Ueno, T.; Migita, S.; Tsutsumi, Y.; Doi, H.; Ogawa, T.; Hanawa, T.; Wakabayashi, N.

2014-01-01

Although the reported percentage of bone-implant contact is far lower than 100%, the cause of such low levels of bone formation has rarely been investigated. This study tested the negative biological effect of hydrocarbon deposition onto titanium surfaces, which has been reported to be inevitable. Osteogenic MC3T3-E1 cells were cultured on titanium disks on which the carbon concentration was experimentally regulated to achieve carbon/titanium (C/Ti) ratios of 0.3, 0.7, and 1.0. Initial cellular activities such as cell attachment and cell spreading were concentration-dependently suppressed by the amount of carbon on the titanium surface. The osteoblastic functions of alkaline phosphatase activity and calcium mineralization were also reduced by more than 40% on the C/Ti (1.0) surface. These results indicate that osteoblast activity is influenced by the degree of hydrocarbon contamination on titanium implants and suggest that hydrocarbon decomposition before implant placement may increase the biocompatibility of titanium. PMID:24868012

A miniaturized multipurpose platform for rapid, label-free, and simultaneous separation, patterning, and in vitro culture of primary and rare cells.

PubMed

Didar, Tohid Fatanat; Bowey, Kristen; Almazan, Guillermina; Tabrizian, Maryam

2014-02-01

Given that current cell isolation techniques are expensive, time consuming, yield low isolation purities, and/or alter target cell properties, a versatile, cost effective, and easy-to-operate microchip with the capability to simultaneously separate, capture, pattern, and culture rare and primary cells in vitro is developed. The platform is based on target cell adhesion onto the micro-fabricated interfaces produced by microcontact printing of cell-specific antibodies. Results show over 95% separation efficiency in less than 10 min for the separation of oligodendrocyte progenitor cells (OPCs) and cardiomyocytes from rat brain and heart mixtures, respectively. Target cell attachment and single cell spreading can be precisely controlled on the basis of the designed patterns. Both cell types can maintain their biofunctionality. Indeed, isolated OPCs can proliferate and differentiate into mature oligodendrocytes, while isolated cardiomyocytes retain their contractile properties on the separation platform. Successful separation of two dissimilar cell types present in varying concentrations in their respective cell mixtures and the demonstration of their integrity after separation open new avenues for time and cost-effective sorting of various cell types using the developed miniaturized platform. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Activation of Rho GTPase Cdc42 promotes adhesion and invasion in colorectal cancer cells.

PubMed

Gao, Lei; Bai, Lan; Nan, Qing zhen

2013-07-25

The purpose of this study was to investigate the role of activated Rho GTPase cell division control protein 42 homolog (Cdc42) in colorectal cancer cell adhesion, migration, and invasion. The constitutively active form of Cdc42 (GFP-Cdc42L61) or control vector was overexpressed in the colorectal cancer cell line SW480. The localization of active Cdc42 was monitored by immunofluorescence staining, and the effects of active Cdc42 on cell migration and invasion were examined using an attachment assay, a wound healing assay, and a Matrigel migration assay in vitro. Immunofluorescence staining revealed that constitutively active Cdc42 predominately localized to the plasma membrane. Compared to SW480 cells transfected with the control vector, overexpression of constitutively active Cdc42 in SW480 cells promoted filopodia formation and cell stretch and dramatically enhanced cell adhesion to the coated plates. The wound healing assay revealed a significant increase of migration capability in SW480 cells expressing active Cdc42 compared to the control cells. Additionally, the Matrigel invasion assay demonstrated that active Cdc42 significantly promoted SW480 cell migration through the chamber. Our results suggest that active Rho GTPase Cdc42 can greatly enhance colorectal cancer cell SW480 to spread, migrate, and invade, which may contribute to colorectal cancer metastasis.

Adhesion of Blood Plasma Proteins and Platelet-rich Plasma on l-Valine-Based Poly(ester urea).

PubMed

Childers, Erin P; Peterson, Gregory I; Ellenberger, Alex B; Domino, Karen; Seifert, Gabrielle V; Becker, Matthew L

2016-10-10

The competitive absorption of blood plasma components including fibrinogen (FG), bovine serum albumin (BSA), and platelet-rich plasma (PRP) on l-valine-based poly(ester urea) (PEU) surfaces were investigated. Using four different PEU polymers, possessing compositionally dependent trends in thermal, mechanical, and critical surface tension measurements, water uptake studies were carried out to determine in vitro behavior of the materials. Quartz crystal microbalance (QCM) measurements were used to quantify the adsorption characteristics of PRP onto PEU thin films by coating the surfaces initially with FG or BSA. Pretreatment of the PEU surfaces with FG inhibited the adsorption of PRP and BSA decreased the absorption 4-fold. In vitro studies demonstrated that cells cultured on l-valine-based PEU thin films allowed attachment and spreading of rat aortic cells. These measurements will be critical toward efforts to use this new class of materials in blood-contacting biomaterials applications.

Maintenance of in vivo induced cytochrome P-450s in hepatocyte monolayers at non freezing temperatures.

PubMed

Evans, Peter J

2015-04-01

Cytochrome P450s (CYPs) induced in rats by 3-methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (Dex) were investigated. The inducers had no effect on hepatocyte yield, viability, attachment or spreading on collagen. 3-MC induced ethoxyresorufin deethylase (EROD). Under normothermic conditions the activity fell in culture. However, it was maintained when cells were preserved at 10°C under a gelatin gel. Upon reactivation the activity mirrored that of freshly isolated cells at 37°C. Induced levels were stable for at least 6h , the time to form a confluent monolayer. The investigation was extended to other CYPs by looking at patterns of testosterone metabolism. Phenobarbital had the greatest influence in terms of the quantity and number of metabolites. Culture at 37°C decreased the peaks dramatically within 24 h. All 7 peaks were maintained in the preservation system. Copyright © 2015 Elsevier Inc. All rights reserved.

[Cytocompatibility of nanophase hydroxyapatite ceramics].

PubMed

Wen, Bo; Chen, Zhi-qing; Jiang, Yin-shan; Yang, Zheng-wen; Xu, Yong-zhong

2004-12-01

To evaluate the cytocompatibility of nanophase hydroxyapatite ceramics in vitro. Hydroxyapatite (HA) was prepared via wet method. The grain size of the hydroxyapatite in the study was determined by scanning electron microscope and atomic force microscope with image analysis software. Primary osteoblast culture was established from rat calvaria. Cell adherence and proliferation on nanophase hydroxyapatite ceramics and conventional hydroxyapatite ceramics were examined at 1, 3, 5, 7 days. Morphology of the cells was observed by microscope. The average grain size of the nanophase and conventional HA was 55 nm and 780 nm, respectively. Throughout 7 days period, osteoblast proliferation on the HA was similar to that on tissue culture borosilicate glass controls, osteoblasts could attach, spread and proliferate on HA. However, compared to conventional ceramics, osteoblast proliferation on nanophase HA was significantly better after 1, 3, 5 and 7 days. Cytocompatibility of nanophase HA was significantly better than conventional ceramics.

Development anmd testing of electrophoresis solutions. Task I.1: Development of optimal buffer system

NASA Technical Reports Server (NTRS)

1985-01-01

Two buffers were explored for testing: low ionic strength electrophoresis buffer with and without density gradient material. It was found that the electrophoresis routine was better tolerated when Ficoll was present. The results of a viability study of primary human fetal kidney (HFK-1) cells at the first passage are shown. Cell strain HFK-1 was used in several experiments at the first and second passage. The HFK consisted mainly of fibroblasts, and HFK-1 has a high epithelioid cell content. The chromosomes of HFK were examined and found to be euploid. The stock medium for cell electrophoresis is described. In this solution density gradient solutes such as sucrose and Ficoll are dissolved to bring the osmolarity to 0.30. Its ionic strength is less than 0.01M, and its conductivity is usually 0.0011 mho/cm. Methods for viability determination included direct microscopic counting of the percent cells attached and spread within 24 hr of plating test cultures or electrophoretically separated fractions. The Cytograf viability assay concept was tested, and shown that blue stained cells scatter less light into the 0.8 to 3.3 deg angular interval than do unstained cells.

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

PubMed

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

2016-12-01

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

Macroporous Hydrogel Scaffolds for Three-Dimensional Cell Culture and Tissue Engineering.

PubMed

Fan, Changjiang; Wang, Dong-An

2017-10-01

Hydrogels have been promising candidate scaffolds for cell delivery and tissue engineering due to their tissue-like physical properties and capability for homogeneous cell loading. However, the encapsulated cells are generally entrapped and constrained in the submicron- or nanosized gel networks, seriously limiting cell growth and tissue formation. Meanwhile, the spatially confined settlement inhibits attachment and spreading of anchorage-dependent cells, leading to their apoptosis. In recent years, macroporous hydrogels have attracted increasing attention in use as cell delivery vehicles and tissue engineering scaffolds. The introduction of macropores within gel scaffolds not only improves their permeability for better nutrient transport but also creates space/interface for cell adhesion, proliferation, and extracellular matrix deposition. Herein, we will first review the development of macroporous gel scaffolds and outline the impact of macropores on cell behaviors. In the first part, the advantages and challenges of hydrogels as three-dimensional (3D) cell culture scaffolds will be described. In the second part, the fabrication of various macroporous hydrogels will be presented. Third, the enhancement of cell activities within macroporous gel scaffolds will be discussed. Finally, several crucial factors that are envisaged to propel the improvement of macroporous gel scaffolds are proposed for 3D cell culture and tissue engineering.

Localized gene delivery using antibody tethered adenovirus from polyurethane heart valve cusps and intra-aortic implants.

PubMed

Stachelek, S J; Song, C; Alferiev, I; Defelice, S; Cui, X; Connolly, J M; Bianco, R W; Levy, R J

2004-01-01

The present study investigated a novel approach for gene therapy of heart valve disease and vascular disorders. We formulated and characterized implantable polyurethane films that could also function as gene delivery systems through the surface attachment of replication defective adenoviruses using an anti-adenovirus antibody tethering mechanism. Our hypothesis was that we could achieve site-specific gene delivery to cells interacting with these polyurethane implants, and thereby demonstrate the potential for intravascular devices that could also function as gene delivery platforms for therapeutic vectors. Previous research by our group has demonstrated that polyurethane elastomers can be derivatized post-polymerization through a series of chemical reactions activating the hard segment amide groups with alkyl bromine residues, which can enable a wide variety of subsequent chemical modifications. Furthermore, prior research by our group investigating gene delivery intravascular stents has shown that collagen-coated balloon expandable stents can be configured with anti-adenovirus antibodies via thiol-based chemistry, and can then tether adenoviral vectors at doses that lead to high levels of localized arterial neointima expression, but with virtually no distal spread of vector. Thus, we sought to create two-device configurations for our investigations building on this previous research. (1) Polyurethane films coated with Type I collagen were thiol activated to permit covalent attachment of anti-adenovirus antibodies to enable gene delivery via vector tethering. (2) We also formulated polyurethane films with direct covalent attachment of anti-adenovirus antibodies to polyurethane hard segments derivatized with alkyl-thiol groups, thereby also enabling tethering of replication-defective adenoviruses. Both formulations demonstrated highly localized and efficient transduction in cell culture studies with rat arterial smooth muscle cells. In vivo experiments with collagen-coated polyurethane films investigated an abdominal aorta implant model in pigs using a button configuration that simulated the blood contacting environment of a vascular graft. One week explants of the collagen-coated polyurethane films demonstrated 14.3+/-2.5% of neointimal cells on the surface of the implant transduced with green fluorescent protein - adenovirus (AdGFP) vector loadings of 1 x 10(8) PFU. PCR studies demonstrated no detectable vector DNA in blood or distal organs. Similarly, polyurethane films with direct attachment of antivector antibodies to the surface were used in sheep pulmonary valve leaflet replacement studies, simulating the blood contacting environment of a prosthetic heart valve cusp. Polyurethane films with antibody tethered AdGFP vector (10(8) PFU) demonstrated 25.1+/-5.7% of attached cells transduced in these 1 week studies, with no detectable vector DNA in blood or distal organs. In vivo GFP expression was confirmed with immunohistochemistry. It is concluded that site-specific intravascular delivery of adenoviral vectors for gene therapy can be achieved with polyurethane implants utilizing the antivector antibody tethering mechanism.

Effect of microbubble ligation to cells on ultrasound signal enhancement: implications for targeted imaging.

PubMed

Lankford, Miles; Behm, Carolyn Z; Yeh, James; Klibanov, Alexander L; Robinson, Peter; Lindner, Jonathan R

2006-10-01

Molecular imaging with contrast-enhanced ultrasound (CEU) relies on the detection of microbubbles retained in regions of disease. The aim of this study was to determine whether microbubble attachment to cells influences their acoustic signal generation and stability. Biotinylated microbubbles were attached to streptavidin-coated plates to derive density versus intensity relations during low- and high-power imaging. To assess damping from microbubble attachment to solid or cell surfaces, in vitro imaging was performed for microbubbles charge-coupled to methacrylate spheres and for vascular cell adhesion molecule-1-targeted microbubbles attached to endothelial cells. Signal enhancement on plates increased according to acoustic power and microbubble site density up to 300 mm. Microbubble signal was reduced by attachment to solid spheres during high- and low-power imaging but was minimally reduced by attachment to endothelial cells and only at low power. Attachment of targeted microbubbles to rigid surfaces results in damping and a reduction of their acoustic signal, which is not seen when microbubbles are attached to cells. A reliable concentration versus intensity relationship can be expected from microbubble attachment to 2-dimensional surfaces until a very high site density is reached.

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1

PubMed Central

Dios-Esponera, Ana; Isern de Val, Soledad; Sevilla-Movilla, Silvia; García-Verdugo, Rosa; García-Bernal, David; Arellano-Sánchez, Nohemí; Cabañas, Carlos; Teixidó, Joaquin

2015-01-01

Stimulation by chemokines of integrin α4β1–dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase–inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76–, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation. PMID:26202465

Pore size and LbL chitosan coating influence mesenchymal stem cell in vitro fibrosis and biomineralization in 3D porous poly(epsilon-caprolactone) scaffolds.

PubMed

Mehr, Nima Ghavidel; Li, Xian; Chen, Gaoping; Favis, Basil D; Hoemann, Caroline D

2015-07-01

Poly(epsilon-caprolactone) (PCL) is a hydrophobic bioplastic under development for bone tissue engineering applications. Limited information is available on the role of internal geometry and cell-surface attachment on osseous integration potential. We tested the hypothesis that human bone marrow mesenchymal stem cells (MSCs) deposit more mineral inside porous 3D PCL scaffolds with fully interconnected 84 or 141 µm pores, when the surfaces are coated with chitosan via Layer-by-Layer (LbL)-deposited polyelectrolytes. Freshly trypsinized MSCs were seeded on PCL 3D cylinders using a novel static cold seeding method in 2% serum to optimally populate all depths of the scaffold discs, followed by 10 days of culture in proliferation medium and 21 additional days in osteogenic medium. MSCs were observed by SEM and histology to spread faster and to proliferate more on chitosan-coated pore surfaces. Most pores, with or without chitosan, became filled by collagen networks sparsely populated with fibroblast-like cells. After 21 days of culture in osteogenic medium, sporadic matrix mineralization was detected histologically and by micro-CT in highly cellular surface layers that enveloped all scaffolds and in cell aggregates in 141 µm pores near the edges. LbL-chitosan promoted punctate mineral deposition on the surfaces of 84 µm pores (p < 0.05 vs. PCL-only) but not the 141 µm pores. This study revealed that LbL-chitosan coatings are sufficient to promote MSC attachment to PCL but only enhance mineral formation in 84 µm pores, suggesting a potential inhibitory role for MSC-derived fibroblasts in osteoblast terminal differentiation. © 2014 Wiley Periodicals, Inc.

Single molecular force across single integrins dictates cell spreading.

PubMed

Chowdhury, Farhan; Li, Isaac T S; Leslie, Benjamin J; Doğanay, Sultan; Singh, Rishi; Wang, Xuefeng; Seong, Jihye; Lee, Sang-Hak; Park, Seongjin; Wang, Ning; Ha, Taekjip

2015-10-01

Cells' ability to sense and interpret mechanical signals from the extracellular milieu modulates the degree of cell spreading. Yet how cells detect such signals and activate downstream signaling at the molecular level remain elusive. Herein, we utilize tension gauge tether (TGT) platform to investigate the underlying molecular mechanism of cell spreading. Our data from both differentiated cells of cancerous and non-cancerous origin show that for the same stiff underlying glass substrates and for same ligand density it is the molecular forces across single integrins that ultimately determine cell spreading responses. Furthermore, by decoupling molecular stiffness and molecular tension we demonstrate that molecular stiffness has little influence on cell spreading. Our data provide strong evidence that links molecular forces at the cell-substrate interface to the degree of cell spreading.

Influence of TiN coating on the biocompatibility of medical NiTi alloy.

PubMed

Jin, Shi; Zhang, Yang; Wang, Qiang; Zhang, Dan; Zhang, Song

2013-01-01

The biocompatibility of TiN coated nickel-titanium shape memory alloy (NiTi-SMA) was evaluated to compare with that of the uncoated NiTi-SMA. Based on the orthodontic clinical application, the surface properties and biocompatibility were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), wettability test, mechanical test and in vitro tests including MTT, cell apoptosis and cell adhesion tests. It was observed that the bonding between the substrate and TiN coating is excellent. The roughness and wettability increased as for the TiN coating compared with the uncoated NiTi-SMA. MTT test showed no significant difference between the coated and uncoated NiTi-SMA, however the percentage of early cell apoptosis was significantly higher as for the uncoated NiTi alloy. SEM results showed that TiN coating could enhance the cell attachment, spreading and proliferation on NiTi-SMA. The results indicated that TiN coating bonded with the substrate well and could lead to a better biocompatibility. Copyright © 2012 Elsevier B.V. All rights reserved.

Hemocyte-lineage marker proteins in a crustacean, the freshwater crayfish, Pacifastacus leniusculus.

PubMed

Wu, Chenglin; Söderhäll, Irene; Kim, Young-A; Liu, Haipeng; Söderhäll, Kenneth

2008-10-01

To identify proteins associated with development of different hemocyte types in the freshwater crayfish Pacifastacus leniusculus, 2-DE followed by MS analysis was carried out with hematopoietic tissue (Hpt) cells, semigranular cells (SGC) and granular cells (GC). Within the hemocyte lineages one two-domain Kazal proteinase inhibitor (KPI) was found to be specific for SGC, while a superoxide dismutase (SOD) was specific for GC at protein as well as at mRNA level. The proliferation cell nuclear antigen (PCNA) was detected at the mRNA level in Hpt cells only. We also provide evidence that SGC and GC most likely differentiate to maturation as separate lineages. We found that after laminarin or lipopolysaccharide (LPS) injection into crayfish, the transcript levels of PCNA and SOD increased in the Hpt cells, whereas the KPI transcript never was present in Hpt regardless of any challenge. RNA interference of PCNA in the Hpt cells led to that most of the cells did not spread or attach to the tissue culture dish. These results suggest that PCNA, KPI and SOD can be used as markers for Hpt cells, SGC and GC, respectively, and in conjunction with these results, a model is proposed how the Hpt responds to a microbial challenge by proliferation and release of Hpt cells.

Fibrin gel as a scaffold for skin substitute – production and clinical experience.

PubMed

Kljenak, Antun; Tominac Trcin, Mirna; Bujić, Marina; Dolenec, Tamara; Jevak, Martina; Mršić, Gordan; Zmiš, Gordana; Barčot, Zoran; Muljačić, Ante; Popović, Maja

2016-06-01

The purpose of this study was to create a fibrin-based human skin substitute in vitro with epidermal and dermal component and to assess its healing potential in deep partial and full thickness burns. Fibrin scaffolds were prepared from commercial fibrin glue kits. Human fibroblasts were cultured in fibrin gel. Human keratinocytes were seeded on the top of the gel. Viability of cells was determined fluorimetrically. Scanning electron microscope and immunocytochemistry analysis of cultured cells were performed. After hydrosurgical preparation of deep burn necrotic tissue, wound bed was prepared for skin substitutes. Progress of healing was documented using visual estimation and photos. Scanning electron microscope images showed good cell attachment and colony spreading of keratinocytes and fibroblasts on fibrin scaff old. Immunofluorescent staining of cell cultures on fibrin scaffold showed expression of vimentin, a marker of fibroblast cells, cytokeratin 19, a marker of epithelial stem cells, as well as involucrin, a marker of differentiated keratinocytes. Clinical results clearly showed that appearance of the skin did not differ significantly from the areas of transplanted skin using split-thickness skin graft techniques. In conclusion, using these fibrin-cultured autografts on massive full-thickness burn resulted in good healing.

Effect of bovine manure on fecal coliform attachment to soil and soil particles of different sizes.

PubMed

Guber, Andrey K; Pachepsky, Yakov A; Shelton, Daniel R; Yu, Olivia

2007-05-01

Manure-borne bacteria can be transported in runoff as free cells, cells attached to soil particles, and cells attached to manure particles. The objectives of this work were to compare the attachment of fecal coliforms (FC) to different soils and soil fractions and to assess the effect of bovine manure on FC attachment to soil and soil fractions. Three sand fractions of different sizes, the silt fraction, and the clay fraction of loam and sandy clay loam soils were separated and used along with soil samples in batch attachment experiments with water-FC suspensions and water-manure-FC suspensions. In the absence of manure colloids, bacterial attachment to soil, silt, and clay particles was much higher than the attachment to sand particles having no organic coating. The attachment to the coated sand particles was similar to the attachment to silt and clay. Manure colloids in suspensions decreased bacterial attachment to soils, clay and silt fractions, and coated sand fractions, but did not decrease the attachment to sand fractions without the coating. The low attachment of bacteria to silt and clay particles in the presence of manure colloids may cause predominantly free-cell transport of manure-borne FC in runoff.

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein.

PubMed

Gill, Kamal S; Beier, Frank; Goldberg, Harvey A

2008-07-01

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16x molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading.

Rho-ROCK signaling differentially regulates chondrocyte spreading on fibronectin and bone sialoprotein

PubMed Central

Gill, Kamal S.; Beier, Frank; Goldberg, Harvey A.

2008-01-01

The mammalian growth plate is a dynamic structure rich in extracellular matrix (ECM). Interactions of growth plate chondrocytes with ECM proteins regulate cell behavior. In this study, we compared chondrocyte adhesion and spreading dynamics on fibronectin (FN) and bone sialoprotein (BSP). Chondrocyte adhesion and spreading were also compared with fibroblasts to analyze potential cell-type-specific effects. Chondrocyte adhesion to BSP is independent of posttranslational modifications but is dependent on the RGD sequence in BSP. Whereas chondrocytes and fibroblasts adhered at similar levels on FN and BSP, cells displayed more actin-dependent spread on FN despite a 16× molar excess of BSP adsorbed to plastic. To identify intracellular mediators responsible for this difference in spreading, we investigated focal adhesion kinase (FAK)-Src and Rho-Rho kinase (ROCK) signaling. Although activated FAK localized to the vertices of adhered chondrocytes, levels of FAK activation did not correlate with the extent of spreading. Furthermore, Src inhibition reduced chondrocyte spreading on both FN and BSP, suggesting that FAK-Src signaling is not responsible for less cell spreading on BSP. In contrast, inhibition of Rho and ROCK in chondrocytes increased cell spreading on BSP and membrane protrusiveness on FN but did not affect cell adhesion. In fibroblasts, Rho inhibition increased fibroblast spreading on BSP while ROCK inhibition changed membrane protrusiveness of FN and BSP. In summary, we identify a novel role for Rho-ROCK signaling in regulating chondrocyte spreading and demonstrate both cell- and matrix molecule-specific mechanisms controlling cell spreading. PMID:18463228

Initial association of fresh microbial products to soil particles: a joint density fractionation and NanoSIMS study

NASA Astrophysics Data System (ADS)

Hatton, Pierre-Joseph; Remusat, Laurent; Brewer, Elizabeth; Derrien, Delphine

2014-05-01

While soil microorganisms are increasingly seen as shaping stable soil organic matter (OM) formation, the mechanisms controlling the attachment of microbial metabolites to soil particles are not fully understood yet. We investigate the spatial distribution of freshly produced microbial products among density-isolated fractions of soil using stable C and N isotopes and Nano-scale secondary ion mass spectrometry (NanoSIMS). A surface forest soil was amended with uniformly 13C/15N labeled glycine and incubated for 8 hours in gamma-irradiated and non-sterile soils. Sequential density fractionation was then performed to isolate various classes of aggregates and of single mineral particles. Eight hours after the labeled glycine addition, 7 % of the 13C and 15N was tightly bound to soil assemblages. Comparison of sterile and non-sterile treatments revealed that microbial activity was almost completely responsible for this rapid association (>85 %). The distributions of glycine-derived 13C and 15N, considered as markers of new microbial products, were mapped on particles of the non-sterile treatment using NanoSIMS. New microbial products were heterogeneously distributed and spatially decoupled at the surface of on soil particles. 13C microbial products were scarce and presumably within or in the vicinity of microbial cells. In contrast, 15N microbial products seemed evenly spread at the surface of soil particles, likely as soluble exoenzymes diffusing away from their parent cell. Macroscopic measurements among density fractions suggested that the diffusion of such 15N microbial products was spatially limited yet, because of pore space architecture. NanoSIMS images further allowed gaining insight into the attachment of the new microbial products on particle surfaces already covered by OM, in a multilayer fashion. Using an internal calibration method to determine C/N ratios of NanoSIMS images, we showed the preferential attachment of soluble microbial N-metabolites to N-rich mineral-attached OM (C/N ratios mostly < 16). Exceptions were found in dense particles, supposed to contained aluminium and iron (hydr)oxides, with the microbial N-metabolites apparently preferentially attached to C-rich mineral-attached OM (C/N ratios > 80). This work provided visual evidences that the attachment of new microbial products to the soil matrix is mediated by distinct processes for N-rich and C-rich metabolites. It also demonstrated that the pore space architecture has impact on the formation of stable OM by limiting the diffusion of soluble microbial metabolites and their access to reactive and stabilising surfaces.

Evidence of femtosecond-laser pulse induced cell membrane nanosurgery

NASA Astrophysics Data System (ADS)

Katchinskiy, Nir; Godbout, Roseline; Elezzabi, Abdulhakem Y.

2017-02-01

The mechanism of femtosecond laser nanosurgical attachment is investigated in the following article. Using sub-10 femtosecond laser pulses with 800 nm central wavelength were used to attach retinoblastoma cells. During the attachment process the cell membrane phospholipid bilayers hemifuse into one shared phospholipid bilayer, at the location of attachment. Transmission electron microscopy was used in order to verify the above hypothesis. Based on the imaging results, it was concluded that the two cell membrane coalesce to form one single shared membrane. The technique of cell-cell attachment via femtosecond laser pulses could potentially serve as a platform for precise cell membrane manipulation. Manipulation of the cellular membrane is valuable for studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

Biofunctionalized 3-D Carbon Nano-Network Platform for Enhanced Fibroblast Cell Adhesion

NASA Astrophysics Data System (ADS)

Chowdhury, A. K. M. Rezaul Haque; Tavangar, Amirhossein; Tan, Bo; Venkatakrishnan, Krishnan

2017-03-01

Carbon nanomaterials have been investigated for various biomedical applications. In most cases, however, these nanomaterials must be functionalized biologically or chemically due to their biological inertness or possible cytotoxicity. Here, we report the development of a new carbon nanomaterial with a bioactive phase that significantly promotes cell adhesion. We synthesize the bioactive phase by introducing self-assembled nanotopography and altered nano-chemistry to graphite substrates using ultrafast laser. To the best of our knowledge, this is the first time that such a cytophilic bio-carbon is developed in a single step without requiring subsequent biological/chemical treatments. By controlling the nano-network concentration and chemistry, we develop platforms with different degrees of cell cytophilicity. We study quantitatively and qualitatively the cell response to nano-network platforms with NIH-3T3 fibroblasts. The findings from the in vitro study indicate that the platforms possess excellent biocompatibility and promote cell adhesion considerably. The study of the cell morphology shows a healthy attachment of cells with a well-spread shape, overextended actin filaments, and morphological symmetry, which is indicative of a high cellular interaction with the nano-network. The developed nanomaterial possesses great biocompatibility and considerably stimulates cell adhesion and subsequent cell proliferation, thus offering a promising path toward engineering various biomedical devices.

High Ringxiety: Attachment Anxiety Predicts Experiences of Phantom Cell Phone Ringing.

PubMed

Kruger, Daniel J; Djerf, Jaikob M

2016-01-01

Mobile cell phone users have reported experiencing ringing and/or vibrations associated with incoming calls and messages, only to find that no call or message had actually registered. We believe this phenomenon can be understood as a human signal detection issue, with potentially important influences from psychological attributes. We hypothesized that individuals higher in attachment anxiety would report more frequent phantom cell phone experiences, whereas individuals higher in attachment avoidance would report less frequent experiences. If these experiences are primarily psychologically related to attributes of interpersonal relationships, associations with attachment style should be stronger than for general sensation seeking. We also predicted that certain contexts would interact with attachment style to increase or decrease the likelihood of experiencing phantom cell phone calls and messages. Attachment anxiety directly predicted the frequency of phantom ringing and notification experiences, whereas attachment avoidance and sensation seeking did not directly predict frequency. Attachment anxiety and attachment avoidance interacted with contextual factors (expectations for a call or message and concerned about an issue that one may be contacted about) in the expected directions for predicting phantom cell phone experiences.

Gonococcal attachment to eukaryotic cells

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

James, J.F.; Lammel, C.J.; Draper, D.L.

The attachment of Neisseria gonorrhoeae to eukaryotic cells grown in tissue culture was analyzed by use of light and electron microscopy and by labeling of the bacteria with (/sup 3/H)- and (/sup 14/C)adenine. Isogenic piliated and nonpiliated N. gonorrhoeae from opaque and transparent colonies were studied. The results of light microscopy studies showed that the gonococci attached to cells of human origin, including Flow 2000, HeLa 229, and HEp 2. Studies using radiolabeled gonococci gave comparable results. Piliated N. gonorrhoeae usually attached in larger numbers than nonpiliated organisms, and those from opaque colonies attached more often than isogenic variants frommore » transparent colonies. Day-to-day variation in rate of attachment was observed. Scanning electron microscopy studies showed the gonococcal attachment to be specific for microvilli of the host cells. It is concluded that more N. gonorrhoeae from opaque colonies, as compared with isogenic variants from transparent colonies, attach to eukaryotic cells grown in tissue culture.« less

Analysis of Initial Cell Spreading Using Mechanistic Contact Formulations for a Deformable Cell Model

PubMed Central

Odenthal, Tim; Smeets, Bart; Van Liedekerke, Paul; Tijskens, Engelbert; Van Oosterwyck, Hans; Ramon, Herman

2013-01-01

Adhesion governs to a large extent the mechanical interaction between a cell and its microenvironment. As initial cell spreading is purely adhesion driven, understanding this phenomenon leads to profound insight in both cell adhesion and cell-substrate interaction. It has been found that across a wide variety of cell types, initial spreading behavior universally follows the same power laws. The simplest cell type providing this scaling of the radius of the spreading area with time are modified red blood cells (RBCs), whose elastic responses are well characterized. Using a mechanistic description of the contact interaction between a cell and its substrate in combination with a deformable RBC model, we are now able to investigate in detail the mechanisms behind this universal power law. The presented model suggests that the initial slope of the spreading curve with time results from a purely geometrical effect facilitated mainly by dissipation upon contact. Later on, the spreading rate decreases due to increasing tension and dissipation in the cell's cortex as the cell spreads more and more. To reproduce this observed initial spreading, no irreversible deformations are required. Since the model created in this effort is extensible to more complex cell types and can cope with arbitrarily shaped, smooth mechanical microenvironments of the cells, it can be useful for a wide range of investigations where forces at the cell boundary play a decisive role. PMID:24146605

Caffeic acid, a coffee-related organic acid, inhibits infection by severe fever with thrombocytopenia syndrome virus in vitro.

PubMed

Ogawa, Motohiko; Shirasago, Yoshitaka; Ando, Shuji; Shimojima, Masayuki; Saijo, Masayuki; Fukasawa, Masayoshi

2018-04-05

Severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) causes tick-borne hemorrhagic fever in East Asia. The disease is characterized by high morbidity and mortality. Here, we evaluated the effects of caffeic acid (CA), a coffee-related organic acid with antiviral effects, against SFTSV infection. CA dose-dependently inhibited SFTSV infection in permissive human hepatoma Huh7.5.1-8 cells when SFTSV was added into the culture medium with CA. However, quinic acid (QA), another coffee-related organic acid, did not inhibit SFTSV infection. The 50% inhibitory concentration (IC 50 ) of CA against SFTSV was 0.048 mM, whereas its 50% cytotoxic concentration was 7.6 mM. The selectivity index (SI) was 158. Pre-incubation of SFTSV with CA for 4 h resulted in a greater inhibition of SFTSV infection (IC 50  = 0.019 mM; SI = 400). The pre-incubation substantially decreased viral attachment to the cells. CA treatment of the SFTSV-infected cells also inhibited the infection, albeit less effectively. CA activity after cell infection with SFTSV was more pronounced at a low multiplicity of infection (MOI) of 0.01 per cell (IC 50  = 0.18 mM) than at a high MOI of 1 per cell (IC 50  > 1 mM). Thus, CA inhibited virus spread by acting directly on the virus rather than on the infected cells. In conclusion, CA acted on SFTSV and inhibited viral infection and spread, mainly by inhibiting the binding of SFTSV to the cells. We therefore demonstrated CA to be a potential anti-SFTSV drug for preventing and treating SFTS. Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Effect of D-valine and cytosine arabinoside on (/sup 3/H)thymidine incorporation in rat and rabbit epididymal epithelial cell cultures

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

Orgebin-Crist, M.C.; Jonas-Davies, J.; Storey, P.

1984-01-01

Epithelial cell enriched primary cultures were established from the rat and the rabbit epididymis. Epithelial cell aggregates, obtained after pronase digestion of minced epididymis, attached to the culture dish and after 72 h in vitro spread out to form discrete patches of cells. These cells have an epithelioid morphology and form a monolayer of closely apposed polygonal cells where DNA synthesis, as judged by (/sup 3/H)thymidine uptake, is very low. In L-valine medium the nonepithelial cell contamination was no more than 10% in rat and rabbit epididymal primary cultures. The labeling index of rat epididymal cells cultured in D-valine mediummore » was significantly lower than that of cells cultured in L-valine medium. In contrast, the labeling index of rabbit epididymal cells cultured in D-valine medium was significantly higher than that of cells cultured in L-valine medium. Cytosine arabinoside decreased the number of labeled cells in both L-valine and D-valine cultures. From these results, it appears that D-valine is a selective agent for rat epididymal epithelial cells, but not for rabbit epithelial cells, and that cytosine arabinoside is a simple and effective means to control the proliferation of fibroblast-like cells in both rat and rabbit epididymal cell cultures.« less

Substrate effects on endothelial cell adherence rates.

PubMed

Scott, W J; Mann, P

1990-01-01

Endothelial cell attachment to a synthetic substrate was studied using an in vitro model system. Attachment rate was defined as the number of tritium-labeled endothelial cells attached to a synthetic substrate after 30 minutes. The surface of the synthetic substrate was chemically modified with either laminin or fibronectin. Labeled endothelial cells attached more rapidly to synthetic substrate, chemically modified with biomolecules, as compared with the untreated substrate controls. Unlabeled endothelial cells were grown to confluency on a second set of modified and untreated substrates. The cells were removed with 1% Triton, and the rate of re-endothelialization with tritium-labeled endothelial cells was determined. The rate was 11-13 times that of the same cells on untreated substrate. These data confirm that biomolecules increase the attachment rate of endothelial cells to synthetic substrate, and also suggest that endothelial cells may secrete a Triton-insoluble product (Sigma, St. Louis, MO) into subendothelial matrix that increases re-endothelialization.

Bone-repair properties of biodegradable hydroxyapatite nano-rod superstructures

NASA Astrophysics Data System (ADS)

D'Elía, Noelia L.; Mathieu, Colleen; Hoemann, Caroline D.; Laiuppa, Juan A.; Santillán, Graciela E.; Messina, Paula V.

2015-11-01

Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants.Nano-hydroxyapatite (nano-HAp) materials show an analogous chemical composition to the biogenic mineral components of calcified tissues and depending on their topography they may mimic the specific arrangement of the crystals in bone. In this work, we have evaluated the potential of four synthesized nano-HAp superstructures for the in vitro conditions of bone-repair. Experiments are underway to investigate the effects of the material microstructure, surface roughness and hydrophilicity on their osseo-integration, osteo-conduction and osteo-induction abilities. Materials were tested in the presence of both, rat primary osteoblasts and rabbit mesenchymal stem cells. The following aspects are discussed: (i) cytotoxicity and material degradation; (ii) rat osteoblast spreading, proliferation and differentiation; and (iii) rabbit mesenchymal stem cell adhesion on nano-HAp and nano-HAp/collagen type I coatings. We effectively prepared a material based on biomimetic HAp nano-rods displaying the appropriate surface topography, hydrophilicity and degradation properties to induce the in vitro desired cellular responses for bone bonding and healing. Cells seeded on the selected material readily attached, proliferated and differentiated, as confirmed by cell viability, mitochondrial metabolic activity, alkaline phosphatase (ALP) activity and cytoskeletal integrity analysis by immunofluorescence localization of alpha-smooth muscle actin (α-SMA) protein. These results highlight the influence of material's surface characteristics to determine their tissue regeneration potential and their future use in engineering osteogenic scaffolds for orthopedic implants. Electronic supplementary information (ESI) available: Calculation of roughness parameters Rz, Rz,max, and Rz, prom. Nano-HAp powder degradation after immersion in phosphate buffer (pH = 7.4). Optical phase contrast microphotographs of MSC adhesion on nano-HAp and nano-HAp/Co I coatings at different concentrations. Laser scanning confocal microphotographs of MSCs' α-SMA expression spreading on large amounts of nano-HAp (MI) coatings. Immunofluorescence microphotograph analysis by image software. See DOI: 10.1039/c5nr04850h

Structural characterization of nanocrystalline hydroxyapatite and adhesion of pre-osteoblast cells

NASA Astrophysics Data System (ADS)

Zhu, Xiaolong; Eibl, Oliver; Berthold, Christoph; Scheideler, Lutz; Geis-Gerstorfer, Jürgen

2006-06-01

Nanocrystalline hydroxyapatite (Nano HA), a prototype of minerals of bones and teeth, attracts increasing interest in medicine and dentistry. Different parameters for synthesis and post-treatment were investigated to determine their effects on crystallinity of nano HA, and in vitro cell responses to nano HA were studied. XRD and TEM analyses indicate that the crystallinity of nano HA synthesized by a chemical method was within the range of 15-50 nm, which is adapted to natural minerals of hard tissues. Increasing the ageing temperature significantly increased the crystallinity of nano HA, while lengthening the ageing time or varying the post-ageing drying process did not have any influence on its crystallinity. Nano HA annealed between 300 and 900 °C showed a small increase in crystallinity with increasing annealing temperature due to the long-range ordering effect. Cell attachment and spreading on nano HA were lower than those on pure titanium, and decreased as the crystallinity of nano HA increased. However, cells on nano HA demonstrated well-developed filopodia and lamelliopodia, which facilitate migration of the cells on it. This may benefit osteogenesis at the interface between bone and nano HA in vivo.

Nanolayer formation on titanium by phosphonated gelatin for cell adhesion and growth enhancement

PubMed Central

Zhou, Xiaoyue; Park, Shin-Hye; Mao, Hongli; Isoshima, Takashi; Wang, Yi; Ito, Yoshihiro

2015-01-01

Phosphonated gelatin was prepared for surface modification of titanium to stimulate cell functions. The modified gelatin was synthesized by coupling with 3-aminopropylphosphonic acid using water-soluble carbodiimide and characterized by 31P nuclear magnetic resonance and gel permeation chromatography. Circular dichroism revealed no differences in the conformations of unmodified and phosphonated gelatin. However, the gelation temperature was changed by the modification. Even a high concentration of modified gelatin did not form a gel at room temperature. Time-of-flight secondary ion mass spectrometry showed direct bonding between the phosphonated gelatin and the titanium surface after binding. The binding behavior of phosphonated gelatin on the titanium surface was quantitatively analyzed by a quartz crystal microbalance. Ellipsometry showed the formation of a several nanometer layer of gelatin on the surface. Contact angle measurement indicated that the modified titanium surface was hydrophobic. Enhancement of the attachment and spreading of MC-3T3L1 osteoblastic cells was observed on the phosphonated gelatin-modified titanium. These effects on cell adhesion also led to growth enhancement. Phosphonation of gelatin was effective for preparation of a cell-stimulating titanium surface. PMID:26366080

In vitro evaluation of crosslinked electrospun fish gelatin scaffolds.

PubMed

Gomes, S R; Rodrigues, G; Martins, G G; Henriques, C M R; Silva, J C

2013-04-01

Gelatin from cold water fish skin was electrospun, crosslinked and investigated as a substrate for the adhesion and proliferation of cells. Gelatin was first dissolved in either water or concentrated acetic acid and both solutions were successfully electrospun. Cross-linking was achieved via three different routes: glutaraldehyde vapor, genipin and dehydrothermal treatment. Solution's properties (surface tension, electrical conductivity and viscosity) and scaffold's properties (chemical bonds, weight loss and fiber diameters) were measured. Cellular viability was analyzed culturing 3T3 fibroblasts plated on the scaffolds and grown up to 7 days. The cells were fixed and observed with SEM or stained for DNA and F-actin and observed with confocal microscopy. In all scaffolds, the cells attached and spread with varying degrees. The evaluation of cell viability showed proliferation of cells until confluence in scaffolds crosslinked by glutaraldehyde and genipin; however the rate of growth in genipin crosslinked scaffolds was slow, recovering only by day five. The results using the dehydrothermal treatment were the less satisfactory. Our results show that glutaraldehyde treated fish gelatin is the most suitable substrate, of the three studied, for fibroblast adhesion and proliferation. Copyright © 2012 Elsevier B.V. All rights reserved.

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover

NASA Technical Reports Server (NTRS)

Mooney, D. J.; Hansen, L. K.; Langer, R.; Vacanti, J. P.; Ingber, D. E.

1994-01-01

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

Effects of nerve cells and adhesion molecules on nerve conduit for peripheral nerve regeneration

PubMed Central

Fiorellini, Joseph P.

2017-01-01

Background For peripheral nerve regeneration, recent attentions have been paid to the nerve conduits made by tissue-engineering technique. Three major elements of tissue-engineering are cells, molecules, and scaffolds. Methods In this study, the attachments of nerve cells, including Schwann cells, on the nerve conduit and the effects of both growth factor and adhesion molecule on these attachments were investigated. Results The attachment of rapidly-proliferating cells, C6 cells and HS683 cells, on nerve conduit was better than that of slowly-proliferating cells, PC12 cells and Schwann cells, however, the treatment of nerve growth factor improved the attachment of slowly-proliferating cells. In addition, the attachment of Schwann cells on nerve conduit coated with fibronectin was as good as that of Schwann cells treated with glial cell line-derived neurotrophic factor (GDNF). Conclusions Growth factor changes nerve cell morphology and affects cell cycle time. And nerve growth factor or fibronectin treatment is indispensable for Schwann cell to be used for implantation in artificial nerve conduits. PMID:29090249

Community dynamics of attached and free cells and the effects of attached cells on chalcopyrite bioleaching by Acidithiobacillus sp.

PubMed

Yang, Hailin; Feng, Shoushuai; Xin, Yu; Wang, Wu

2014-02-01

The community dynamics of attached and free cells of Acidithiobacillus sp. were investigated and compared during chalcopyrite bioleaching process. In the mixed strains system, Acidithiobacillus ferrooxidans was the dominant species at the early stage while Acidithiobacillus thiooxidans owned competitive advantage from the middle stage to the end of bioprocess. Meanwhile, compared to A. ferrooxidans, more significant effects of attached cells on free biomass with A. thiooxidans were shown in either the pure or mixed strains systems. Moreover, the effects of attached cells on key chemical parameters were also studied in different adsorption-deficient systems. Consistently, the greatest reduction of key chemical ion was shown with A. thiooxidans and the loss of bioleaching efficiency was high to 50.5%. These results all demonstrated the bioleaching function of attached cells was more efficient than the free cells, especially with A. thiooxidans. These notable results would help us to further understand the chalcopyrite bioleaching. Copyright © 2013 Elsevier Ltd. All rights reserved.

Pseudomonas aeruginosa cells attached to a surface display a typical proteome early as 20 minutes of incubation

PubMed Central

Crouzet, Marc; Claverol, Stéphane; Lomenech, Anne-Marie; Le Sénéchal, Caroline; Costaglioli, Patricia; Barthe, Christophe; Garbay, Bertrand; Bonneu, Marc

2017-01-01

Biofilms are present in all environments and often result in negative effects due to properties of the biofilm lifestyle and especially antibiotics resistance. Biofilms are associated with chronic infections. Controlling bacterial attachment, the first step of biofilm formation, is crucial for fighting against biofilm and subsequently preventing the persistence of infection. Thus deciphering the underlying molecular mechanisms involved in attachment could allow discovering molecular targets from it would be possible to develop inhibitors against bacterial colonization and potentiate antibiotherapy. To identify the key components and pathways that aid the opportunistic pathogen Pseudomonas aeruginosa in attachment we performed for the first time a proteomic analysis as early as after 20 minutes of incubation using glass wool fibers as a surface. We compared the protein contents of the attached and unattached bacteria. Using mass spectrometry, 3043 proteins were identified. Our results showed that, as of 20 minutes of incubation, using stringent quantification criteria 616 proteins presented a modification of their abundance in the attached cells compared to their unattached counterparts. The attached cells presented an overall reduced gene expression and characteristics of slow-growing cells. The over-accumulation of outer membrane proteins, periplasmic folding proteins and O-antigen chain length regulators was also observed, indicating a profound modification of the cell envelope. Consistently the sigma factor AlgU required for cell envelope homeostasis was highly over-accumulated in attached cells. In addition our data suggested a role of alarmone (p)ppGpp and polyphosphate during the early attachment phase. Furthermore, almost 150 proteins of unknown function were differentially accumulated in the attached cells. Our proteomic analysis revealed the existence of distinctive biological features in attached cells as early as 20 minutes of incubation. Analysis of some mutants demonstrated the interest of this proteomic approach in identifying genes involved in the early phase of adhesion to a surface. PMID:28678862

Evidence of recent volcanic activity on the ultraslow-spreading Gakkel ridge.

PubMed

Edwards, M H; Kurras, G J; Tolstoy, M; Bohnenstiehl, D R; Coakley, B J; Cochran, J R

2001-02-15

Seafloor spreading is accommodated by volcanic and tectonic processes along the global mid-ocean ridge system. As spreading rate decreases the influence of volcanism also decreases, and it is unknown whether significant volcanism occurs at all at ultraslow spreading rates (<1.5 cm yr(-1)). Here we present three-dimensional sonar maps of the Gakkel ridge, Earth's slowest-spreading mid-ocean ridge, located in the Arctic basin under the Arctic Ocean ice canopy. We acquired this data using hull-mounted sonars attached to a nuclear-powered submarine, the USS Hawkbill. Sidescan data for the ultraslow-spreading (approximately 1.0 cm yr(-1)) eastern Gakkel ridge depict two young volcanoes covering approximately 720 km2 of an otherwise heavily sedimented axial valley. The western volcano coincides with the average location of epicentres for more than 250 teleseismic events detected in 1999, suggesting that an axial eruption was imaged shortly after its occurrence. These findings demonstrate that eruptions along the ultraslow-spreading Gakkel ridge are focused at discrete locations and appear to be more voluminous and occur more frequently than was previously thought.

Characterization of femtosecond-laser pulse induced cell membrane nanosurgical attachment.

PubMed

Katchinskiy, Nir; Godbout, Roseline; Elezzabi, Abdulhakem Y

2016-07-01

This article provides insight into the mechanism of femtosecond laser nanosurgical attachment of cells. We have demonstrated that during the attachment of two retinoblastoma cells using sub-10 femtosecond laser pulses, with 800 nm central wavelength, the phospholipid molecules of both cells hemifuse and form one shared phospholipid bilayer, at the attachment location. In order to verify the hypothesis that hemifusion takes place, transmission electron microscope images of the cell membranes of retinoblastoma cells were taken. It is shown that at the attachment interface, the two cell membranes coalesce and form one single membrane shared by both cells. Thus, further evidence is provided to support the hypothesis that laser-induced ionization process led to an ultrafast reversible destabilization of the phospholipid layer of the cellular membrane, which resulted in cross-linking of the phospholipid molecules in each membrane. This process of hemifusion occurs throughout the entire penetration depth of the femtosecond laser pulse train. Thus, the attachment between the cells takes place across a large surface area, which affirms our findings of strong physical attachment between the cells. The femtosecond laser pulse hemifusion technique can potentially provide a platform for precise molecular manipulation of cellular membranes. Manipulation of the cellular membrane is an important procedure that could aid in studying diseases such as cancer; where the expression level of plasma proteins on the cell membrane is altered.

Epidemic of cell phone virus

NASA Astrophysics Data System (ADS)

Wang, Pu; González, Marta; Barabási, Albert-László.

2008-03-01

Standard operating systems and Bluetooth technology will be a trend for future cell phone features. These will enable cell phone viruses to spread either through SMS or by sending Bluetooth requests when cell phones are physically close enough. The difference in spreading methods gives these two types of viruses' different epidemiological characteristics. SMS viruses' spread is mainly based on people's social connections, whereas the spreading of Bluetooth viruses is affected by people's mobility patterns and population distribution. Using cell phone data recording calls, SMS and locations of more than 6 million users, we study the spread of SMS and Bluetooth viruses and characterize how the social network and the mobility of mobile phone users affect such spreading processes.

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity.

PubMed

Linnemann, Amelia K; Krawetz, Stephen A

2009-05-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14-18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment.

Chlorine stress mediates microbial surface attachment in drinking water systems.

PubMed

Liu, Li; Le, Yang; Jin, Juliang; Zhou, Yuliang; Chen, Guowei

2015-03-01

Microbial attachment to drinking water pipe surfaces facilitates pathogen survival and deteriorates disinfection performance, directly threatening the safety of drinking water. Notwithstanding that the formation of biofilm has been studied for decades, the underlying mechanisms for the origins of microbial surface attachment in biofilm development in drinking water pipelines remain largely elusive. We combined experimental and mathematical methods to investigate the role of environmental stress-mediated cell motility on microbial surface attachment in chlorination-stressed drinking water distribution systems. Results show that at low levels of disinfectant (0.0-1.0 mg/L), the presence of chlorine promotes initiation of microbial surface attachment, while higher amounts of disinfectant (>1.0 mg/L) inhibit microbial attachment. The proposed mathematical model further demonstrates that chlorination stress (0.0-5.0 mg/L)-mediated microbial cell motility regulates the frequency of cell-wall collision and thereby controls initial microbial surface attachment. The results reveal that transport processes and decay patterns of chlorine in drinking water pipelines regulate microbial cell motility and, thus, control initial surface cell attachment. It provides a mechanistic understanding of microbial attachment shaped by environmental disinfection stress and leads to new insights into microbial safety protocols in water distribution systems.

Insights to the effects of free cells on community structure of attached cells and chalcopyrite bioleaching during different stages.

PubMed

Feng, Shoushuai; Yang, Hailin; Wang, Wu

2016-01-01

The effects of free cells on community structure of attached cells and chalcopyrite bioleaching by Acidithiobacillus sp. during different stages were investigated. The attached cells of Acidithiobacillus thiooxidans owned the community advantage from 14thd to the end of bioprocess in the normal system. The community structure of attached cells was greatly influenced in the free cells-deficient systems. Compared to A. thiooxidans, the attached cells community of Acidithiobacillus ferrooxidans had a higher dependence on its free cells. Meanwhile, the analysis of key biochemical parameters revealed that the effects of free cells on chalcopyrite bioleaching in different stages were diverse, ranging from 32.8% to 64.3%. The bioleaching contribution of free cells of A. ferrooxidans in the stationary stage (8-14thd) was higher than those of A. thiooxidans, while the situation was gradually reversed in the jarosite passivation inhibited stage (26-40thd). These results may be useful in guiding chalcopyrite bioleaching. Copyright © 2015 Elsevier Ltd. All rights reserved.

Influence of substrate diffusion on degradation of dibenzofuran and 3-chlorodibenzofuran by attached and suspended bacteria.

PubMed Central

Harms, H; Zehnder, A J

1994-01-01

Dibenzofuran uptake-associated kinetic parameters of suspended and attached Sphingomonas sp. strain HH19k cells were compared. The suspended cells were studied in a batch system, whereas glass beads in percolated columns were used as the solid support for attached cells. The maximum specific activities of cells in the two systems were the same. The apparent half-maximum uptake rate-associated concentrations (Kt') of attached cells, however, were considerably greater than those of suspended cells and depended on cell density and on percolation velocity. A mathematical model was developed to explain the observed differences in terms of substrate transport to the cells. This model was based on the assumptions that the intrinsic half-maximum uptake rate-associated concentration (Kt) was unchanged and that deviations of Kt' from Kt resulted from the stereometry and the hydrodynamics around the cells. Our calculations showed that (i) diffusion to suspended cells and to single attached cells is efficient and therefore only slightly affects Kt'; (ii) diffusion to cells located on crowded surfaces is considerably lower than that to single attached cells and greatly increases Kt', which depends on the cell density; (iii) the convective-diffusive transport to attached cells that occurs in a percolated column is influenced by the liquid flow and results in dependency of Kt' on the flow rate; and (iv) higher specific affinity of cells correlates with higher susceptibility to diffusion limitation. Properties of the experimental system which limited quantitative proof of exclusively transport-controlled variations of Kt' are discussed. PMID:8085817

The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior.

PubMed

Yang, Seungwon; Kim, Hyun-Man

2012-04-01

The proliferation of anchorage-dependent cells of mesenchymal origin requires the attachment of the cells to substrates. Thus, cells that are poorly attached to substrates exhibit retarded cell cycle progression or apoptotic death. A major disadvantage of most polymers used in tissue engineering is their hydrophobicity; hydrophobic surfaces do not allow cells to attach firmly and, therefore, do not allow normal proliferation rates. In this study, we investigated the molecular mechanism underlying the reduced proliferation rate of cells that are poorly attached to substrates. There was an inverse relationship between the activity of the small GTPase RhoA (RhoA) and the cell proliferation rate. RhoA activity correlated inversely with the strength of cell adhesion to the substrates. The high RhoA activity in the cells poorly attached to substrates caused an increase in the activity of Rho-associated kinase (ROCK), a well-known effector of RhoA that upregulated the activity of phosphatase and tensin homolog (PTEN). The resulting activated PTEN downregulated Akt activity, which is essential for cell proliferation. Thus, the cells that were poorly attached to substrates showed low levels of cell proliferation because the RhoA-ROCK-PTEN pathway was hyperactive. In addition, RhoA activity seemed to be related to focal adhesion kinase (FAK) activity. Weak FAK activity in these poorly attached cells failed to downregulate the high RhoA activity that restrained cell proliferation. Interestingly, reducing the expression of any component of the RhoA-ROCK-PTEN pathway rescued the proliferation rate without physico-chemical surface modifications. Based on these results, we suggest that the RhoA-ROCK-PTEN pathway acts as a molecular switch to control cell proliferation and determine anchorage dependence. In cells that are poorly attached to substrates, its inhibition is sufficient to restore cell proliferation without the need for physico-chemical modification of the material surface. Copyright © 2012 Elsevier Ltd. All rights reserved.

Study of the Ability of Bifidobacteria of Human Origin to Prevent and Treat Rotavirus Infection Using Colonic Cell and Mouse Models

PubMed Central

Darveau, André; Fliss, Ismaïl

2016-01-01

Rotavirus is the leading cause of severe acute gastroenteritis among children worldwide. Despite effective vaccines, inexpensive alternatives such as probiotics are needed. The aim of this study was to assess the ability of probiotic candidate Bifidobacterium thermophilum RBL67 to inhibit rotavirus infection. Bacterial adhesion to intestinal cells and interference with viral attachment were evaluated in vitro. B. thermophilum RBL67 displayed adhesion indexes of 625 ± 84 and 1958 ± 318 on Caco-2 and HT-29 cells respectively and was comparable or superior to four other bifidobacteria, including B. longum ATCC 15707 and B. pseudolongum ATCC 25526 strains. Incubation of B. thermophilum RBL67 for 30 min before (exclusion) and simultaneously (competition) with human rotavirus strain Wa decreased virus attachment by 2.0 ± 0.1 and 1.5 ± 0.1 log10 (by 99.0% and 96.8% respectively). Displacement of virus already present was negligible. In CD-1 suckling mice fed B. thermophilum RBL67 challenged with simian rotavirus SA-11, pre-infection feeding with RBL 67 was more effective than post-infection feeding, reducing the duration of diarrhea, limiting epithelial lesions, reducing viral replication in the intestine, accelerating recovery, and stimulating the humoral specific IgG and IgM response, without inducing any adverse effect. B. thermophilum RBL67 had little effect on intestinal IgA titer. These results suggest that humoral immunoglobulin might provide protection against the virus and that B. thermophilum RBL67 has potential as a probiotic able to inhibit rotavirus infection and ultimately reduce its spread. PMID:27727323

Triple Antibiotic Polymer Nanofibers for Intracanal Drug Delivery: Effects on Dual Species Biofilm and Cell Function

PubMed Central

Pankajakshan, Divya; Albuquerque, Maria T.P.; Evans, Joshua D.; Kamocka, Malgorzata M.; Gregory, Richard L.; Bottino, Marco C.

2016-01-01

Introduction Root canal disinfection and the establishment of an intracanal microenvironment conducive to the proliferation/differentiation of stem cells play a significant role in regenerative endodontics. This study was designed to (1) investigate the antimicrobial efficacy of triple antibiotic–containing nanofibers against a dual-species biofilm and (2) evaluate the ability of dental pulp stem cells (DPSCs) to adhere to and proliferate on dentin upon nanofiber exposure. Methods Seven-day-old dual-species biofilm established on dentin specimens was exposed for 3 days to the following: saline (control), antibiotic-free nanofibers (control), and triple antibiotic–containing nanofibers or a saturated triple antibiotic paste (TAP) solution (50 mg/mL in phosphate buffer solution). Bacterial viability was assessed using the LIVE/DEAD assay (Molecular Probes, Inc, Eugene, OR) and confocal laser scanning microscopy. For cyto-compatibility studies, dentin specimens after nanofiber or TAP (1 g/mL in phosphate buffer solution) exposure were evaluated for cell adhesion and spreading by actin-phalloidin staining. DPSC proliferation was assessed on days 1, 3, and 7. Statistics were performed, and significance was set at the 5% level. Results Confocal laser scanning microscopy showed significant bacterial death upon antibiotic-containing nanofiber exposure, differing significantly (P < .05) from antibiotic-free fibers and the control (saline). DPSCs showed enhanced adhesion/spreading on dentin specimens treated with antibiotic-containing nanofibers when compared with its TAP counterparts. The DPSC proliferation rate was similar on days 1 and 3 in antibiotic-free nanofibers, triple antibiotic–containing nanofibers, and TAP-treated dentin. Proliferation was higher (9-fold) on dentin treated with antibiotic-containing nanofibers on day 7 compared with TAP. Conclusions Triple antibiotic–containing polymer nanofibers led to significant bacterial death, whereas they did not affect DPSC attachment and proliferation on dentin. PMID:27663615

Strength of silk attachment to Ilex chinensis leaves in the tea bagworm Eumeta minuscula (Lepidoptera, Psychidae)

PubMed Central

Lovtsova, Julia; Gorb, Elena; Dai, Zhendong; Ji, Aihong; Zhao, Zhihui; Jiang, Nan; Gorb, Stanislav N.

2017-01-01

Silks play an important role in the life of various arthropods. A highly neglected prerequisite to make versatile use of silks is sufficient attachment to substrates. Although there have been some studies on the structure and mechanics of silk anchorages of spiders, for insects only anecdotal reports on attachment-associated spinning behaviour exist. Here, we experimentally studied the silk attachment of the pupae and last instar caterpillars of the tea bagworm Eumeta minuscula (Butler 1881) (Lepidoptera, Psychidae) to the leaves of its host plant Ilex chinensis. We found that the bagworms spin attachment discs, which share some structural features with those of spiders, like a plaque consisting of numerous overlaid, looped glue-coated silk fibres and the medially attaching suspension thread. Although the glue, which coats the fibres, cannot spread and adhere very well to the leaf surface, high pull-off forces were measured, yielding a mean safety factor (force divided by the animal weight) of 385.6. Presumably, the bagworms achieve this by removal of the leaf epidermis prior to silk attachment, which exposes the underlying tissue that represents a much better bonding site. This ensures a reliable attachment during the immobile, vulnerable pupal stage. This is the first study on the biomechanics and structure of silk attachments to substrates in insects. PMID:28250101

Hybrid Spreading Mechanisms and T Cell Activation Shape the Dynamics of HIV-1 Infection

PubMed Central

Zhang, Changwang; Zhou, Shi; Groppelli, Elisabetta; Pellegrino, Pierre; Williams, Ian; Borrow, Persephone; Chain, Benjamin M.; Jolly, Clare

2015-01-01

HIV-1 can disseminate between susceptible cells by two mechanisms: cell-free infection following fluid-phase diffusion of virions and by highly-efficient direct cell-to-cell transmission at immune cell contacts. The contribution of this hybrid spreading mechanism, which is also a characteristic of some important computer worm outbreaks, to HIV-1 progression in vivo remains unknown. Here we present a new mathematical model that explicitly incorporates the ability of HIV-1 to use hybrid spreading mechanisms and evaluate the consequences for HIV-1 pathogenenesis. The model captures the major phases of the HIV-1 infection course of a cohort of treatment naive patients and also accurately predicts the results of the Short Pulse Anti-Retroviral Therapy at Seroconversion (SPARTAC) trial. Using this model we find that hybrid spreading is critical to seed and establish infection, and that cell-to-cell spread and increased CD4+ T cell activation are important for HIV-1 progression. Notably, the model predicts that cell-to-cell spread becomes increasingly effective as infection progresses and thus may present a considerable treatment barrier. Deriving predictions of various treatments’ influence on HIV-1 progression highlights the importance of earlier intervention and suggests that treatments effectively targeting cell-to-cell HIV-1 spread can delay progression to AIDS. This study suggests that hybrid spreading is a fundamental feature of HIV infection, and provides the mathematical framework incorporating this feature with which to evaluate future therapeutic strategies. PMID:25837979

Revealing the dependence of cell spreading kinetics on its spreading morphology using microcontact printed fibronectin patterns

PubMed Central

Huang, Cheng-Kuang; Donald, Athene

2015-01-01

Since the dawn of in vitro cell cultures, how cells interact and proliferate within a given external environment has always been an important issue in the study of cell biology. It is now well known that mammalian cells typically exhibit a three-phase sigmoid spreading on encountering a substrate. To further this understanding, we examined the influence of cell shape towards the second rapid expansion phase of spreading. Specifically, 3T3 fibroblasts were seeded onto silicon elastomer films made from polydimethylsiloxane (PDMS), and micro-contact printed with fibronectin stripes of various dimensions. PDMS is adopted in our study for its biocompatibility, its ease in producing very smooth surfaces, and in the fabrication of micro-contact printing stamps. The substrate patterns are compared with respect to their influence on cell spreading over time. Our studies reveal, during the early rapid expansion phase, 3T3 fibroblasts are found to spread radially following a law; meanwhile, they proliferated in a lengthwise fashion on the striped patterns, following a law. We account for the observed differences in kinetics through a simple geometric analysis which predicted similar trends. In particular, a t2 law for radial spreading cells, and a t1 law for lengthwise spreading cells. PMID:25551146

Effect of Porosity of Alumina and Zirconia Ceramics toward Pre-Osteoblast Response

PubMed Central

Hadjicharalambous, Chrystalleni; Prymak, Oleg; Loza, Kateryna; Buyakov, Ales; Kulkov, Sergei; Chatzinikolaidou, Maria

2015-01-01

It is acknowledged that cellular responses are highly affected by biomaterial porosity. The investigation of this effect is important for the development of implanted biomaterials that integrate with bone tissue. Zirconia and alumina ceramics exhibit outstanding mechanical properties and are among the most popular implant materials used in orthopedics, but few data exist regarding the effect of porosity on cellular responses to these materials. The present study investigates the effect of porosity on the attachment and proliferation of pre-osteoblastic cells on zirconia and alumina. For each composition, ceramics of three different porosities are fabricated by sintering, and characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy and X-ray powder diffraction. Cell proliferation is quantified, and microscopy is employed to qualitatively support the proliferation results and evaluate cell morphology. Cell adhesion and metabolic activity are found comparable among low porosity zirconia and alumina. In contrast, higher porosity favors better cell spreading on zirconia and improves growth, but does not significantly affect cell response on alumina. Between the highest porosity materials, cell response on zirconia is found superior to alumina. Results show that an average pore size of ~150 μm and ~50% porosity can be considered beneficial to cellular growth on zirconia ceramics. PMID:26579516

Agrobacterium tumefaciens mutants affected in attachment to plant cells.

PubMed Central

Douglas, C J; Halperin, W; Nester, E W

1982-01-01

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated. Images PMID:6292165

Free energy analysis of cell spreading.

PubMed

McEvoy, Eóin; Deshpande, Vikram S; McGarry, Patrick

2017-10-01

In this study we present a steady-state adaptation of the thermodynamically motivated stress fiber (SF) model of Vigliotti et al. (2015). We implement this steady-state formulation in a non-local finite element setting where we also consider global conservation of the total number of cytoskeletal proteins within the cell, global conservation of the number of binding integrins on the cell membrane, and adhesion limiting ligand density on the substrate surface. We present a number of simulations of cell spreading in which we consider a limited subset of the possible deformed spread-states assumed by the cell in order to examine the hypothesis that free energy minimization drives the process of cell spreading. Simulations suggest that cell spreading can be viewed as a competition between (i) decreasing cytoskeletal free energy due to strain induced assembly of cytoskeletal proteins into contractile SFs, and (ii) increasing elastic free energy due to stretching of the mechanically passive components of the cell. The computed minimum free energy spread area is shown to be lower for a cell on a compliant substrate than on a rigid substrate. Furthermore, a low substrate ligand density is found to limit cell spreading. The predicted dependence of cell spread area on substrate stiffness and ligand density is in agreement with the experiments of Engler et al. (2003). We also simulate the experiments of Théry et al. (2006), whereby initially circular cells deform and adhere to "V-shaped" and "Y-shaped" ligand patches. Analysis of a number of different spread states reveals that deformed configurations with the lowest free energy exhibit a SF distribution that corresponds to experimental observations, i.e. a high concentration of highly aligned SFs occurs along free edges, with lower SF concentrations in the interior of the cell. In summary, the results of this study suggest that cell spreading is driven by free energy minimization based on a competition between decreasing cytoskeletal free energy and increasing passive elastic free energy. Copyright © 2017 Elsevier Ltd. All rights reserved.

Engineered microtopographies and surface chemistries direct cell attachment and function

NASA Astrophysics Data System (ADS)

Magin, Chelsea Marie

Harrison, in 1914, first recognized that cells respond to physicochemical cues such as substratum topography when he observed that fibroblasts elongated while cultured on spider silk. Recently, techniques developed in the micro-electronics industry have been used to create molds for producing microscaled topographies with various shapes and spatial arrangements. Although these patterning techniques are well-established, very little is known about the mechanisms underlying cell sensing and response to microtopographies. In this work cellular micro-environments with varying surface topographies and chemistries were evaluated with marine organisms and mammalian cells to investigate cellular sensing and response. Biofouling---the accumulation of micro-organisms, plants, and animals on submerged surfaces---is an environmental and economic concern. Engineered topographies, replicated in polydimethylsiloxane elastomer (PDMSe) and functionalized poly(ethylene glycol)-dimethacrylate (PEGDMA) hydrogels, were evaluated for inhibition of marine fouling organism attachment. Microtopographies replicated in PDMSe inhibited attachment of the marine bacterium, Cobetia marina up to 99% versus smooth. The average normalized attachment densities of cells of C. marina and zoospores of the green algae Ulva on PDMSe topographies scaled inversely with the Engineered Roughness Index (ERIII), a representation of surface energy. Attachment densities of Ulva from four assays and C. marina from two growth phases to PDMSe surfaces scaled inversely with one equation: ERI II multiplied by the Reynolds number of the organism (Re) (R 2 = 0.77). The same microtopographies created in PDMSe reduced the initial attachment density and attachment strength of cells of the diatoms Navicula incerta and Seminavis robusta compared to smooth PDMSe. The average normalized attachment density of Navicula after exposure to shear stress (48 Pa) was correlated with the contact area between the diatom and a topographically modified surface (R2=0.82). Functionalized PEGDMA hydrogels significantly reduced attachment and attachment strength of Navicula and C. marina. These hydrogels also reduced attachment of zoospores of Ulva compared to PDMSe. Attachment of Ulva to microtopographies in PDMSe and PEGDMA-co-HEMA negatively correlated with ERIII*Re (R2 = 0.94 and R2 = 0.99, respectively). Incorporating a surface energy term into this equation created a correlation between the attachment densities of cells from two evolutionarily diverse groups on substrates of two surface chemistries with an equation that describes the various microtopographies and surface chemistries in terms of surface energy (R2 = 0.80). The current Attachment Model can now be used to design engineered antifouling surface microtopographies and chemistries that inhibit the attachment of organisms from three evoluntionarily diverse groups. Hydrogels based on PEGDMA were also chosen as a substratum material for mammalian cell culture. Capturing endothelial progenitor cells (EPCs) and inducing differentiation into the endothelial cell (EC) phenotype is the ideal way to re-endothelialize a small-diameter vascular graft. Substratum elasticity has been reported to direct stem cell differentiation into specific lineages. Functionalized PEGDMA hydrogels provided good compliance, high fidelity of topographic features and sites for surface modification with biomolecules. Fibronectin grafting and topography both increased EC attachment. This combination of adjustable elasticity, surface chemistry and topography has the potential to promote the capture and differentiation of EPCs into a confluent EC monolayer. Engineered microtopographies replicated in PDMSe directed elongation and alignment of human coronary artery endothelial cells (HCAECs) and human coronary artery smooth muscle cells (HCASMCs) compared to smooth surfaces. Engineered cellular micro-environments were created with specific surface energies defined by chemistry and topography to successfully direct cell attachment and function.

Ruxolitinib and Polycation Combination Treatment Overcomes Multiple Mechanisms of Resistance of Pancreatic Cancer Cells to Oncolytic Vesicular Stomatitis Virus

PubMed Central

Felt, Sébastien A.; Droby, Gaith N.

2017-01-01

ABSTRACT Vesicular stomatitis virus (VSV) is a promising oncolytic virus (OV). Although VSV is effective against a majority of pancreatic ductal adenocarcinoma cell (PDAC) cell lines, some PDAC cell lines are highly resistant to VSV, and the mechanisms of resistance are still unclear. JAK1/2 inhibitors (such as ruxolitinib and JAK inhibitor I) strongly stimulate VSV replication and oncolysis in all resistant cell lines but only partially improve the susceptibility of resistant PDACs to VSV. VSV tumor tropism is generally dependent on the permissiveness of malignant cells to viral replication rather than on receptor specificity, with several ubiquitously expressed cell surface molecules playing a role in VSV attachment to host cells. However, as VSV attachment to PDAC cells has never been tested before, here we examined if it was possibly inhibited in resistant PDAC cells. Our data show a dramatically weaker attachment of VSV to HPAF-II cells, the most resistant human PDAC cell line. Although sequence analysis of low-density lipoprotein (LDL) receptor (LDLR) mRNA did not reveal any amino acid substitutions in this cell line, HPAF-II cells displayed the lowest level of LDLR expression and dramatically lower LDL uptake. Treatment of cells with various statins strongly increased LDLR expression levels but did not improve VSV attachment or LDL uptake in HPAF-II cells. However, LDLR-independent attachment of VSV to HPAF-II cells was dramatically improved by treating cells with Polybrene or DEAE-dextran. Moreover, combining VSV with ruxolitinib and Polybrene or DEAE-dextran successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. IMPORTANCE Oncolytic virus (OV) therapy is an anticancer approach that uses viruses that selectively infect and kill cancer cells. This study focuses on oncolytic vesicular stomatitis virus (VSV) against pancreatic ductal adenocarcinoma (PDAC) cells. Although VSV is effective against most PDAC cells, some are highly resistant to VSV, and the mechanisms are still unclear. Here we examined if VSV attachment to cells was inhibited in resistant PDAC cells. Our data show very inefficient attachment of VSV to the most resistant human PDAC cell line, HPAF-II. However, VSV attachment to HPAF-II cells was dramatically improved by treating cells with polycations. Moreover, combining VSV with polycations and ruxolitinib (which inhibits antiviral signaling) successfully broke the resistance of HPAF-II cells to VSV by simultaneously improving VSV attachment and replication. We envision that this novel triple-combination approach could be used in the future to treat PDAC tumors that are highly resistant to OV therapy. PMID:28566376

The Role of Depression and Attachment Styles in Predicting Students' Addiction to Cell Phones.

PubMed

Ghasempour, Abdollah; Mahmoodi-Aghdam, Mansour

2015-01-01

The present study aimed at investigating the role of depression and attachment styles in predicting cell phone addiction. In this descriptive correlational study, a sample including 100 students of Payame Noor University (PNU), Reyneh Center, Iran, in the academic year of 2013-2014 was selected using volunteer sampling. Participants were asked to complete the adult attachment inventory (AAI), Beck depression inventory-13 (BDI-13) and the cell phone overuse scale (COS). Results of the stepwise multiple regression analysis showed that depression and avoidant attachment style were the best predictors of students' cell phone addiction (R(2) = 0.23). The results of this study highlighted the predictive value of depression and avoidant attachment style concerning students' cell phone addiction.

Assessing the role of spatial correlations during collective cell spreading

PubMed Central

Treloar, Katrina K.; Simpson, Matthew J.; Binder, Benjamin J.; McElwain, D. L. Sean; Baker, Ruth E.

2014-01-01

Spreading cell fronts are essential features of development, repair and disease processes. Many mathematical models used to describe the motion of cell fronts, such as Fisher's equation, invoke a mean–field assumption which implies that there is no spatial structure, such as cell clustering, present. Here, we examine the presence of spatial structure using a combination of in vitro circular barrier assays, discrete random walk simulations and pair correlation functions. In particular, we analyse discrete simulation data using pair correlation functions to show that spatial structure can form in a spreading population of cells either through sufficiently strong cell–to–cell adhesion or sufficiently rapid cell proliferation. We analyse images from a circular barrier assay describing the spreading of a population of MM127 melanoma cells using the same pair correlation functions. Our results indicate that the spreading melanoma cell populations remain very close to spatially uniform, suggesting that the strength of cell–to–cell adhesion and the rate of cell proliferation are both sufficiently small so as not to induce any spatial patterning in the spreading populations. PMID:25026987

Topical application of polyethylenimine as a candidate for novel prophylactic therapeutics against genital herpes caused by herpes simplex virus.

PubMed

Hayashi, Kyoko; Onoue, Hiroki; Sasaki, Kohei; Lee, Jung-Bum; Kumar, Penmetcha K R; Gopinath, Subash C B; Maitani, Yoshie; Kai, Takashi; Hayashi, Toshimitsu

2014-03-01

Herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) cause genital herpes, which can enhance the acquisition of human immunodeficiency virus. The development of anti-HSV agents with novel mechanisms of action is urgently required in the topical therapy of genital herpes. In this study, the in vitro and in vivo anti-HSV effects of Epomin SP-012(®), a highly cationic polyethylenimine, were evaluated. When the in vitro antiviral effects of SP-012 were assessed, this compound showed potent activity against HSV-1 and HSV-2. It inhibited the attachment of HSV-2 to host cells and cell-to-cell spread of infection in a concentration-dependent manner and exerted a virucidal effect. No SP-012-resistant HSV-2 was found when the virus was successively passaged in the presence of SP-012. In a mouse genital herpes model, topically administered SP-012 inhibited the progression of the disease caused by HSV infection. These data illustrate that SP-012 may be a novel class of HSV inhibitor that would be acceptable for long-term topical application.

Harvesting keratolimbal allografts from corneoscleral buttons: a novel application of cyanoacrylate adhesive.

PubMed

Lim, L T; Bhatt, P R; Ramaesh, K

2008-11-01

To describe an alternative and novel technique using cyanoacrylate glue to achieve successful limbal tissue dissection, from an organ culture media stored corneoscleral button, without an artificial anterior chamber. A donor corneoscleral button (leftover from penetrating keratoplasty) was divided into two equal semicircular halves. A thick layer of tissue adhesive (N-butyl-2-cyanoacrylate) was spread on a sterile rubber block (the under surface of the donor punch). One half of the donor corneoscleral rim was placed epithelial side up on the adhesive and allowed to attach firmly to the block. This composite provided stability to the donor rim allowing lamellar dissection of the limbal tissue to be performed without damaging the limbal epithelium. Regular, partial-thickness limbal tissue was obtained. There was no histological evidence of glue or cellular toxicity of the harvested limbal stem cells. This harvested tissue had been grafted successfully in patients with limbal stem cell deficiency also undergoing keratoplasty. Tissue adhesive can be a simple, effective and useful tool in the dissection and harvesting of corneal limbal stem cell allografts from corneoscleral buttons stored in organ culture media.

Preparation, characterization, and in vitro osteoblast functions of a nano-hydroxyapatite/polyetheretherketone biocomposite as orthopedic implant material

PubMed Central

Ma, Rui; Tang, Songchao; Tan, Honglue; Lin, Wentao; Wang, Yugang; Wei, Jie; Zhao, Liming; Tang, Tingting

2014-01-01

A bioactive composite was prepared by incorporating 40 wt% nano-hydroxyapatite (nHA) into polyetheretherketone (PEEK) through a process of compounding, injection, and molding. The mechanical and surface properties of the nHA/PEEK composite were characterized, and the in vitro osteoblast functions in the composite were investigated. The mechanical properties (elastic modulus and compressive strength) of the nHA/PEEK composite increased significantly, while the tensile strength decreased slightly as compared with PEEK. Further, the addition of nHA into PEEK increased the surface roughness and hydrophilicity of the nHA/PEEK composite. In cell tests, compared with PEEK and ultra-high-molecular-weight polyethylene, it was found that the nHA/PEEK composite could promote the functions of MC3T3-E1 cells, including cell attachment, spreading, proliferation, alkaline phosphatase activity, calcium nodule formation, and expression of osteogenic differentiation-related genes. Incorporation of nHA into PEEK greatly improved the bioperformance of PEEK. The nHA/PEEK composite might be a promising orthopedic implant material. PMID:25170265

Photopolymerizable chitosan-collagen hydrogels for bone tissue engineering.

PubMed

Arakawa, Christopher; Ng, Ronald; Tan, Steven; Kim, Soyon; Wu, Benjamin; Lee, Min

2017-01-01

Photopolymerizable hydrogels derived from naturally occurring polymers have attracted significant interest in tissue-engineering applications due to their excellent biocompatibility, hydrophilic nature favourable for cell ingrowth and ability to be cured in situ through a minimally invasive procedure. In this study, we developed a composite hydrogel consisting of photocrosslinkable methacrylated glycol chitosan (MeGC) and semi-interpenetrating collagen (Col) with a riboflavin photoinitiator under blue light. The incorporation of Col in MeGC hydrogels enhanced the compressive modulus and slowed the degradation rate of the hydrogels. MeGC-Col composite hydrogels significantly enhanced cellular attachment, spreading, proliferation and osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) seeded on the hydrogels compared with pure MeGC hydrogels, as observed by upregulated alkaline phosphatase (ALP) activity as well as increased mineralization. Similarly, when cells were encapsulated within hydrogels, BMSCs exhibited greater proliferation, ALP activity and mineral deposits in the presence of Col. These findings demonstrate that MeGC-Col composite hydrogels may be useful in promoting bone regeneration. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Ca-P spots modified zirconia by liquid precursor infiltration and the effect on osteoblast-like cell responses.

PubMed

Li, Yongmei; Liu, Yan; Zhang, Zutai; Zhuge, Ruishen; Ding, Ning; Tian, Yueming

2018-01-26

Ca-P spots modified zirconia by liquid precursor infiltration and the cell responses were investigated. Pre-sintered zirconia specimens were immersed in Ca-P precursor solution. After dense sintering, scanning electron microscopy showed Ca-P spots were formed on the zirconia and anchored with zirconia substrates. The distribution density was increased with the extension of immersion time. Energy dispersive spectrometer confirmed the stoichiometric Ca/P ratio was about 1.67. After hydrothermal treatment, Ca-P spots turned into rod crystals where diffraction peaks of tricalcium phosphate and hydroxyapatite were detected by X-ray diffraction, and Ca 2+ and PO 4 3- release decreased slightly (p>0.05). There was no significant decrease on three-point bending strength (p>0.05). Osteoblast-like MC3T3-E1 cells attached and spread well and showed higher proliferation on Ca-P spots modified zirconia (p<0.05), though its initial alkaline phosphatase activity was not significant high (p>0.05). In conclusion, Ca-P liquid precursor infiltration is a potential method to modify the zirconia ceramics for improving bioactivity.

Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

PubMed Central

Martin, Danyelle N.; Perelson, Alan S.; Dahari, Harel

2015-01-01

ABSTRACT It has been proposed that viral cell-to-cell transmission plays a role in establishing and maintaining chronic infections. Thus, understanding the mechanisms and kinetics of cell-to-cell spread is fundamental to elucidating the dynamics of infection and may provide insight into factors that determine chronicity. Because hepatitis C virus (HCV) spreads from cell to cell and has a chronicity rate of up to 80% in exposed individuals, we examined the dynamics of HCV cell-to-cell spread in vitro and quantified the effect of inhibiting individual host factors. Using a multidisciplinary approach, we performed HCV spread assays and assessed the appropriateness of different stochastic models for describing HCV focus expansion. To evaluate the effect of blocking specific host cell factors on HCV cell-to-cell transmission, assays were performed in the presence of blocking antibodies and/or small-molecule inhibitors targeting different cellular HCV entry factors. In all experiments, HCV-positive cells were identified by immunohistochemical staining and the number of HCV-positive cells per focus was assessed to determine focus size. We found that HCV focus expansion can best be explained by mathematical models assuming focus size-dependent growth. Consistent with previous reports suggesting that some factors impact HCV cell-to-cell spread to different extents, modeling results estimate a hierarchy of efficacies for blocking HCV cell-to-cell spread when targeting different host factors (e.g., CLDN1 > NPC1L1 > TfR1). This approach can be adapted to describe focus expansion dynamics under a variety of experimental conditions as a means to quantify cell-to-cell transmission and assess the impact of cellular factors, viral factors, and antivirals. IMPORTANCE The ability of viruses to efficiently spread by direct cell-to-cell transmission is thought to play an important role in the establishment and maintenance of viral persistence. As such, elucidating the dynamics of cell-to-cell spread and quantifying the effect of blocking the factors involved has important implications for the design of potent antiviral strategies and controlling viral escape. Mathematical modeling has been widely used to understand HCV infection dynamics and treatment response; however, these models typically assume only cell-free virus infection mechanisms. Here, we used stochastic models describing focus expansion as a means to understand and quantify the dynamics of HCV cell-to-cell spread in vitro and determined the degree to which cell-to-cell spread is reduced when individual HCV entry factors are blocked. The results demonstrate the ability of this approach to recapitulate and quantify cell-to-cell transmission, as well as the impact of specific factors and potential antivirals. PMID:25833046

Effect of hydrocortisone on cell morphology in C6 cells

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

Berliner, J.A.; Bennett, K.; de Vellis, J.

Hydrocortisone has been found to induce cell spreading in rat glial C6 cells by 24 hours after its addition. This spreading phenomenon is correlated with an increase in the fraction of the peripheral cytoplasm occupied by microfilaments. Cytochalasin B causes disorganization of microfilaments in the peripheral cytoplasm of the cells. Additionally, it also prevents cell spreading in response to hormonal stimulation. High levels of calcium prevent recovery of normal microfilament organization and cell spreading following removal of cytochalasin B, but have no effect on normal microfilament organization alone. Additionally both the hydrocortisone induced spreading of C6 cells and increases inmore » peripheral microfilaments are shown to be dependent on RNA and protein synthesis. The levels of protein co-electrophorescing with actin are not affected by hydrocortisone.« less

Endothelialization of Novel Magnesium-Rare Earth Alloys with Fluoride and Collagen Coating

PubMed Central

Zhao, Nan; Workman, Benjamin; Zhu, Donghui

2014-01-01

Magnesium (Mg) alloys are promising scaffolds for the next generation of cardiovascular stents because of their better biocompatibility and biodegradation compared to traditional metals. However, insufficient mechanical strength and high degradation rate are still the two main limitations for Mg materials. Hydrofluoric acid (HF) treatment and collagen coating were used in this research to improve the endothelialization of two rare earth-based Mg alloys. Results demonstrated that a nanoporous film structure of fluoride with thickness of ~20 μm was formed on the Mg material surface, which improved the corrosion resistance. Primary human coronary artery endothelial cells (HCAECs) had much better attachment, spreading, growth and proliferation (the process of endothelialization) on HF-treated Mg materials compared to bare- or collagen-coated ones. PMID:24670478

Characterization of somatic embryo attached structures in Feijoa sellowiana Berg. (Myrtaceae).

PubMed

Correia, Sandra M; Canhoto, Jorge M

2010-06-01

The presence of an attached organ to somatic embryos of angiosperms connecting the embryo to the supporting tissue has been a subject of controversy. This study shows that 67% of the morphologically normal somatic embryos of Feijoa sellowiana possess this type of organ and that its formation was not affected by culture media composition. Histological and ultrastructural analysis indicated that the attached structures of somatic embryos displayed a great morphological diversity ranging from a few cells to massive and columnar structures. This contrast with the simple suspensors observed in zygotic embryos which were only formed by five cells. As well as the suspensor of zygotic embryos, somatic embryo attached structures undergo a process of degeneration in later stages of embryo development. Other characteristic shared by zygotic suspensors and somatic embryo attached structures was the presence of thick cell walls surrounding the cells. Elongated thin filaments were often associated with the structures attached to somatic embryos, whereas in other cases, tubular cells containing starch grains connected the embryo to the supporting tissue. These characteristics associated with the presence of plasmodesmata in the cells of the attached structures seem to indicate a role on embryo nutrition. However, cell proliferation in the attached structures resulting into new somatic embryos may also suggest a more complex relationship between the embryo and the structures connecting it to the supporting tissue.

Positive and negative regulation by SLP-76/ADAP and Pyk2 of chemokine-stimulated T-lymphocyte adhesion mediated by integrin α4β1.

PubMed

Dios-Esponera, Ana; Isern de Val, Soledad; Sevilla-Movilla, Silvia; García-Verdugo, Rosa; García-Bernal, David; Arellano-Sánchez, Nohemí; Cabañas, Carlos; Teixidó, Joaquin

2015-09-15

Stimulation by chemokines of integrin α4β1-dependent T-lymphocyte adhesion is a crucial step for lymphocyte trafficking. The adaptor Vav1 is required for chemokine-activated T-cell adhesion mediated by α4β1. Conceivably, proteins associating with Vav1 could potentially modulate this adhesion. Correlating with activation by the chemokine CXCL12 of T-lymphocyte attachment to α4β1 ligands, a transient stimulation in the association of Vav1 with SLP-76, Pyk2, and ADAP was observed. Using T-cells depleted for SLP-76, ADAP, or Pyk2, or expressing Pyk2 kinase-inactive forms, we show that SLP-76 and ADAP stimulate chemokine-activated, α4β1-mediated adhesion, whereas Pyk2 opposes T-cell attachment. While CXCL12-promoted generation of high-affinity α4β1 is independent of SLP-76, ADAP, and Pyk2, the strength of α4β1-VCAM-1 interaction and cell spreading on VCAM-1 are targets of regulation by these three proteins. GTPase assays, expression of activated or dominant-negative Rac1, or combined ADAP and Pyk2 silencing indicated that Rac1 activation by CXCL12 is a common mediator response in SLP-76-, ADAP-, and Pyk2-regulated cell adhesion involving α4β1. Our data strongly suggest that chemokine-stimulated associations between Vav1, SLP-76, and ADAP facilitate Rac1 activation and α4β1-mediated adhesion, whereas Pyk2 opposes this adhesion by limiting Rac1 activation. © 2015 Dios-Esponera, Isern de Val, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Silencing by nuclear matrix attachment distinguishes cell-type specificity: association with increased proliferation capacity

PubMed Central

Linnemann, Amelia K.; Krawetz, Stephen A.

2009-01-01

DNA loop organization by nuclear scaffold/matrix attachment is a key regulator of gene expression that may provide a means to modulate phenotype. We have previously shown that attachment of genes to the NaCl-isolated nuclear matrix correlates with their silencing in HeLa cells. In contrast, expressed genes were associated with the lithium 3,5-diiodosalicylate (LIS)-isolated nuclear scaffold. To define their role in determining phenotype matrix attached regions (MARs) on human chromosomes 14–18 were identified as a function of expression in a primary cell line. The locations of MARs in aortic adventitial fibroblast (AoAF) cells were very stable (r = 0.909) and 96% of genes attached at MARs are silent (P < 0.001). Approximately one-third of the genes uniquely expressed in AoAF cells were associated with the HeLa cell nuclear matrix and silenced. Comparatively, 81% were associated with the AoAF cell nuclear scaffold (P < 0.001) and expressed. This suggests that nuclear scaffold/matrix association mediates a portion of cell type-specific gene expression thereby modulating phenotype. Interestingly, nuclear matrix attachment and thus silencing of specific genes that regulate proliferation and maintain the integrity of the HeLa cell genome suggests that transformation may at least in part be achieved through aberrant nuclear matrix attachment. PMID:19276204

Seasonal and pandemic human influenza viruses attach better to human upper respiratory tract epithelium than avian influenza viruses.

PubMed

van Riel, Debby; den Bakker, Michael A; Leijten, Lonneke M E; Chutinimitkul, Salin; Munster, Vincent J; de Wit, Emmie; Rimmelzwaan, Guus F; Fouchier, Ron A M; Osterhaus, Albert D M E; Kuiken, Thijs

2010-04-01

Influenza viruses vary markedly in their efficiency of human-to-human transmission. This variation has been speculated to be determined in part by the tropism of influenza virus for the human upper respiratory tract. To study this tropism, we determined the pattern of virus attachment by virus histochemistry of three human and three avian influenza viruses in human nasal septum, conchae, nasopharynx, paranasal sinuses, and larynx. We found that the human influenza viruses-two seasonal influenza viruses and pandemic H1N1 virus-attached abundantly to ciliated epithelial cells and goblet cells throughout the upper respiratory tract. In contrast, the avian influenza viruses, including the highly pathogenic H5N1 virus, attached only rarely to epithelial cells or goblet cells. Both human and avian viruses attached occasionally to cells of the submucosal glands. The pattern of virus attachment was similar among the different sites of the human upper respiratory tract for each virus tested. We conclude that influenza viruses that are transmitted efficiently among humans attach abundantly to human upper respiratory tract, whereas inefficiently transmitted influenza viruses attach rarely. These results suggest that the ability of an influenza virus to attach to human upper respiratory tract is a critical factor for efficient transmission in the human population.

Nanocarbon-Coated Porous Anodic Alumina for Bionic Devices

PubMed Central

Aramesh, Morteza; Tong, Wei; Fox, Kate; Turnley, Ann; Seo, Dong Han; Prawer, Steven; Ostrikov, Kostya (Ken)

2015-01-01

A highly-stable and biocompatible nanoporous electrode is demonstrated herein. The electrode is based on a porous anodic alumina which is conformally coated with an ultra-thin layer of diamond-like carbon. The nanocarbon coating plays an essential role for the chemical stability and biocompatibility of the electrodes; thus, the coated electrodes are ideally suited for biomedical applications. The corrosion resistance of the proposed electrodes was tested under extreme chemical conditions, such as in boiling acidic/alkali environments. The nanostructured morphology and the surface chemistry of the electrodes were maintained after wet/dry chemical corrosion tests. The non-cytotoxicity of the electrodes was tested by standard toxicity tests using mouse fibroblasts and cortical neurons. Furthermore, the cell–electrode interaction of cortical neurons with nanocarbon coated nanoporous anodic alumina was studied in vitro. Cortical neurons were found to attach and spread to the nanocarbon coated electrodes without using additional biomolecules, whilst no cell attachment was observed on the surface of the bare anodic alumina. Neurite growth appeared to be sensitive to nanotopographical features of the electrodes. The proposed electrodes show a great promise for practical applications such as retinal prostheses and bionic implants in general. PMID:28793486

Downregulation of TFPI in breast cancer cells induces tyrosine phosphorylation signaling and increases metastatic growth by stimulating cell motility

PubMed Central

2011-01-01

Background Increased hemostatic activity is common in many cancer types and often causes additional complications and even death. Circumstantial evidence suggests that tissue factor pathway inhibitor-1 (TFPI) plays a role in cancer development. We recently reported that downregulation of TFPI inhibited apoptosis in a breast cancer cell line. In this study, we investigated the effects of TFPI on self-sustained growth and motility of these cells, and of another invasive breast cancer cell type (MDA-MB-231). Methods Stable cell lines with TFPI (both α and β) and only TFPIβ downregulated were created using RNA interference technology. We investigated the ability of the transduced cells to grow, when seeded at low densities, and to form colonies, along with metastatic characteristics such as adhesion, migration and invasion. Results Downregulation of TFPI was associated with increased self-sustained cell growth. An increase in cell attachment and spreading was observed to collagen type I, together with elevated levels of integrin α2. Downregulation of TFPI also stimulated migration and invasion of cells, and elevated MMP activity was involved in the increased invasion observed. Surprisingly, equivalent results were observed when TFPIβ was downregulated, revealing a novel function of this isoform in cancer metastasis. Conclusions Our results suggest an anti-metastatic effect of TFPI and may provide a novel therapeutic approach in cancer. PMID:21849050

Structural basis of efficient contagion: measles variations on a theme by parainfluenza viruses.

PubMed

Mateo, Mathieu; Navaratnarajah, Chanakha K; Cattaneo, Roberto

2014-04-01

A quartet of attachment proteins and a trio of fusion protein subunits play the cell entry concert of parainfluenza viruses. While many of these viruses bind sialic acid to enter cells, wild type measles binds exclusively two tissue-specific proteins, the lymphatic receptor signaling lymphocytic activation molecule (SLAM), and the epithelial receptor nectin-4. SLAM binds near the stalk-head junction of the hemagglutinin. Nectin-4 binds a hydrophobic groove located between blades 4 and 5 of the hemagglutinin β-propeller head. The mutated vaccine strain hemagglutinin binds in addition the ubiquitous protein CD46, which explains attenuation. The measles virus entry concert has four movements. Andante misterioso: the virus takes over the immune system. Allegro con brio: it rapidly spreads in the upper airway's epithelia. 'Targeting' fugue: the versatile orchestra takes off. Presto furioso: the virus exits the host with thunder. Be careful: music is contagious. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparation and Reinforcement of Dual-Porous Biocompatible Cellulose Scaffolds for Tissue Engineering.

PubMed

Pircher, Nicole; Fischhuber, David; Carbajal, Leticia; Strauß, Christine; Nedelec, Jean-Marie; Kasper, Cornelia; Rosenau, Thomas; Liebner, Falk

2015-09-01

1Biocompatible cellulose-based aerogels composed of nanoporous struts, which embed interconnected voids of controlled micron-size, have been prepared employing temporary templates of fused porogens, reinforcement by interpenetrating PMMA networks and supercritical carbon dioxide drying. Different combinations of cellulose solvent (Ca(SCN) 2 /H 2 O/LiCl or [EMIm][OAc]/DMSO) and anti-solvent (EtOH), porogen type (paraffin wax or PMMA spheres) and porogen size (various fractions in the range of 100-500 μm) as well as intensity of PMMA reinforcement have been investigated to tailor the materials for cell scaffolding applications. All aerogels exhibited an open and dual porosity (micronporosity >100 μm and nanoporosity extending to the low micrometer range). Mechanical properties of the dual-porous aerogels under compressive stress were considerably improved by introduction of interpenetrating PMMA networks. The effect of the reinforcing polymer on attachment, spreading, and proliferation of NIH 3T3 fibroblast cells, cultivated on selected dual-porous aerogels to pre-evaluate their biocompatibility was similarly positive.

Dual-functioning peptides discovered by phage display increase the magnitude and specificity of BMSC attachment to mineralized biomaterials.

PubMed

Ramaraju, Harsha; Miller, Sharon J; Kohn, David H

2017-07-01

Design of biomaterials for cell-based therapies requires presentation of specific physical and chemical cues to cells, analogous to cues provided by native extracellular matrices (ECM). We previously identified a peptide sequence with high affinity towards apatite (VTKHLNQISQSY, VTK) using phage display. The aims of this study were to identify a human MSC-specific peptide sequence through phage display, combine it with the apatite-specific sequence, and verify the specificity of the combined dual-functioning peptide to both apatite and human bone marrow stromal cells. In this study, a combinatorial phage display identified the cell binding sequence (DPIYALSWSGMA, DPI) which was combined with the mineral binding sequence to generate the dual peptide DPI-VTK. DPI-VTK demonstrated significantly greater binding affinity (1/K D ) to apatite surfaces compared to VTK, phosphorylated VTK (VTK phos ), DPI-VTK phos , RGD-VTK, and peptide-free apatite surfaces (p < 0.01), while significantly increasing hBMSC adhesion strength (τ 50 , p < 0.01). MSCs demonstrated significantly greater adhesion strength to DPI-VTK compared to other cell types, while attachment of MC3T3 pre-osteoblasts and murine fibroblasts was limited (p < 0.01). MSCs on DPI-VTK coated surfaces also demonstrated increased spreading compared to pre-osteoblasts and fibroblasts. MSCs cultured on DPI-VTK coated apatite films exhibited significantly greater proliferation compared to controls (p < 0.001). Moreover, early and late stage osteogenic differentiation markers were elevated on DPI-VTK coated apatite films compared to controls. Taken together, phage display can identify non-obvious cell and material specific peptides to increase human MSC adhesion strength to specific biomaterial surfaces and subsequently increase cell proliferation and differentiation. These new peptides expand biomaterial design methodology for cell-based regeneration of bone defects. This strategy of combining cell and material binding phage display derived peptides is broadly applicable to a variety of systems requiring targeted adhesion of specific cell populations, and may be generalized to the engineering of any adhesion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Modulation of osteoblast attachment and growth in vitro by inertial forces

NASA Astrophysics Data System (ADS)

Kacena, Melissa Ann

1999-11-01

Spaceflight exploration and associated experiments show that human bones lose in density during inertial unloading, due principally to their demineralization. This research project examines the effect of gravity on osteoblast attachment and function in various inertial environments. Chicken calvarial osteoblasts were cultured under the following inertial conditions: spaceflight, simulated shuttle launch accelerations and vibrations, centrifugation, clino-rotation, and inversion. Cultures exposed to these conditions were compared with cultures grown in the laboratory as static 1G controls. Electron and light microscopy revealed the number of total osteoblasts attached to their substrate. Biochemical assays discerned changes in viable cell number, alkaline phosphatase levels, and mineralization. Immunohistochemical assays were used to investigate differences in cytoskeletal and extracellular matrix protein concentrations in the cultures, the percentage of proliferative cells, and cell viability. Compared to controls, spaceflight results indicated that the number of attached osteoblast cells was reduced. Launch simulation data indicated that the associated accelerations and vibrations may contribute to the reduction of attached osteoblasts in spaceflight cultures. Following centrifugation, the number of attached cells was unaltered; however, immunostaining of actin, fibronectin, and vinculin did show alterations in cultures exposed to hypergravity. Confluent cultures that were right side up, inverted, and clino-rotated contained a comparable number of attached cells and functioned similarly on the basis of measured alkaline phosphatase and bound calcium content. Sparse clino-rotated or inverted cultures showed an immediate response of diminished viable osteoblast numbers, but this effect disappeared with time and all remaining attached cells functioned similarly (APase and bound calcium). On the basis of these data osteoblast attachment and function in confluent cultures is minimally, if at all, affected by alterations in inertial environments. However, in sparse cultures about half as many cells are found attached initially. The remaining attached cells appear to multiply and function normally. These results suggest that the effects of spaceflight on bone are thus not likely to be caused by direct intrinsic effects of gravity on single osteoblasts that can be simulated in laboratory experiments in vitro experiments.

Cell behavior on gallium nitride surfaces: peptide affinity attachment versus covalent functionalization.

PubMed

Foster, Corey M; Collazo, Ramon; Sitar, Zlatko; Ivanisevic, Albena

2013-07-02

Gallium nitride is a wide band gap semiconductor that demonstrates a unique set of optical and electrical properties as well as aqueous stability and biocompatibility. This combination of properties makes gallium nitride a strong candidate for use in chemical and biological applications such as sensors and neural interfaces. Molecular modification can be used to enhance the functionality and properties of the gallium nitride surface. Here, gallium nitride surfaces were functionalized with a PC12 cell adhesion promoting peptide using covalent and affinity driven attachment methods. The covalent scheme proceeded by Grignard reaction and olefin metathesis while the affinity driven scheme utilized the recognition peptide isolated through phage display. This study shows that the method of attaching the adhesion peptide influences PC12 cell adhesion and differentiation as measured by cell density and morphological analysis. Covalent attachment promoted monolayer and dispersed cell adhesion while affinity driven attachment promoted multilayer cell agglomeration. Higher cell density was observed on surfaces modified using the recognition peptide. The results suggest that the covalent and affinity driven attachment methods are both suitable for promoting PC12 cell adhesion to the gallium nitride surface, though each method may be preferentially suited for distinct applications.

Escherichia coli attachment to model particulates: The effects of bacterial cell characteristics and particulate properties.

PubMed

Liang, Xiao; Liao, Chunyu; Soupir, Michelle L; Jarboe, Laura R; Thompson, Michael L; Dixon, Philip M

2017-01-01

E. coli bacteria move in streams freely in a planktonic state or attached to suspended particulates. Attachment is a dynamic process, and the fraction of attached microorganisms is thought to be affected by both bacterial characteristics and particulate properties. In this study, we investigated how the properties of cell surfaces and stream particulates influence attachment. Attachment assays were conducted for 77 E. coli strains and three model particulates (ferrihydrite, Ca-montmorillonite, or corn stover) under environmentally relevant conditions. Surface area, particle size distribution, and total carbon content were determined for each type of particulate. Among the three particulates, attachment fractions to corn stover were significantly larger than the attachments to 2-line ferrihydrite (p-value = 0.0036) and Ca-montmorillonite (p-value = 0.022). Furthermore, attachment to Ca-montmorillonite and corn stover was successfully modeled by a Generalized Additive Model (GAM) using cell characteristics as predictor variables. The natural logarithm of the net charge on the bacterial surface had a significant, positive, and linear impact on the attachment of E. coli bacteria to Ca-montmorillonite (p-value = 0.013), but it did not significantly impact the attachment to corn stover (p-value = 0.36). The large diversities in cell characteristics among 77 E. coli strains, particulate properties, and attachment fractions clearly demonstrated the inadequacy of using a static parameter or linear coefficient to predict the attachment behavior of E. coli in stream water quality models.

Effect of Pore Size and Porosity on the Biomechanical Properties and Cytocompatibility of Porous NiTi Alloys

PubMed Central

Jian, Yu-Tao; Yang, Yue; Tian, Tian; Stanford, Clark; Zhang, Xin-Ping; Zhao, Ke

2015-01-01

Five types of porous Nickel-Titanium (NiTi) alloy samples of different porosities and pore sizes were fabricated. According to compressive and fracture strengths, three groups of porous NiTi alloy samples underwent further cytocompatibility experiments. Porous NiTi alloys exhibited a lower Young’s modulus (2.0 GPa ~ 0.8 GPa). Both compressive strength (108.8 MPa ~ 56.2 MPa) and fracture strength (64.6 MPa ~ 41.6 MPa) decreased gradually with increasing mean pore size (MPS). Cells grew and spread well on all porous NiTi alloy samples. Cells attached more strongly on control group and blank group than on all porous NiTi alloy samples (p < 0.05). Cell adhesion on porous NiTi alloys was correlated negatively to MPS (277.2 μm ~ 566.5 μm; p < 0.05). More cells proliferated on control group and blank group than on all porous NiTi alloy samples (p < 0.05). Cellular ALP activity on all porous NiTi alloy samples was higher than on control group and blank group (p < 0.05). The porous NiTi alloys with optimized pore size could be a potential orthopedic material. PMID:26047515

Novel bilayer wound dressing based on electrospun gelatin/keratin nanofibrous mats for skin wound repair.

PubMed

Yao, Chun-Hsu; Lee, Chia-Yu; Huang, Chiung-Hua; Chen, Yueh-Sheng; Chen, Kuo-Yu

2017-10-01

A bilayer membrane (GKU) with a commercial polyurethane wound dressing as an outer layer and electrospun gelatin/keratin nanofibrous mat as an inner layer was fabricated as a novel wound dressing. Scanning electron micrographs showed that gelatin/keratin nanofibers had a uniform morphology and bead-free structure with average fiber diameter of 160.4nm. 3-(4,5-Dimethylthiazolyl)-2,5-diphenyltetrazolium bromide assay using L929 fibroblast cells indicated that the residues released from the gelatin/keratin composite nanofibrous mat accelerated cell proliferation. Cell attachment experiments revealed that adhered cells spread better and migrated deeper into the gelatin/keratin nanofibrous mat than that into the gelatin nanofibrous mat. In animal studies, compared with the bilayer membrane without keratin, gauze and commercial wound dressing, Comfeel®, GKU membrane gave much more number of blood vessels and a greater reduction in wound area at 4days, and better wound repair at 14days with a thicker epidermis and larger number of newly formed hair follicles. GKU membrane, thus, could be a good candidate for wound dressing applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Modeling universal dynamics of cell spreading on elastic substrates.

PubMed

Fan, Houfu; Li, Shaofan

2015-11-01

A three-dimensional (3D) multiscale moving contact line model is combined with a soft matter cell model to study the universal dynamics of cell spreading over elastic substrates. We have studied both the early stage and the late stage cell spreading by taking into account the actin tension effect. In this work, the cell is modeled as an active nematic droplet, and the substrate is modeled as a St. Venant Kirchhoff elastic medium. A complete 3D simulation of cell spreading has been carried out. The simulation results show that the spreading area versus spreading time at different stages obeys specific power laws, which is in good agreement with experimental data and theoretical prediction reported in the literature. Moreover, the simulation results show that the substrate elasticity may affect force dipole distribution inside the cell. The advantage of this approach is that it combines the hydrodynamics of actin retrograde flow with moving contact line model so that it can naturally include actin tension effect resulting from actin polymerization and actomyosin contraction, and thus it might be capable of simulating complex cellular scale phenomenon, such as cell spreading or even crawling.

Effect of C-implantation on Nerve-Cell Attachment to Polystyrene Films

NASA Astrophysics Data System (ADS)

Sommani, Piyanuch; Tsuji, Hiroshi; Kitamura, Tsuyoshi; Hattori, Mitsutaka; Yamada, Tetsuya; Sato, Hiroko; Gotoh, Yasuhito; Ishikawa, Junzo

The surfaces of the polystyrene films spin-coated on glass were modified by carbon negative-ion implantation with various ion doses from 1×1014 to 3×1016 ions/cm2 at 5 and 10 keV. The implantation conditions with and without a pattering mask were for investigation of the cell-attachment properties and for evaluation of surface physical properties of contact angle, respectively. The contact angles of modified surface were investigated by pure water drop and air bubble method. The lowest angle value of the implanted films at 5 and 10 keV were approximately 72° at 3×1015 ions/cm2 after dipping in the de-ionized water for 2 hours. The lowering of contact angles on C-implanted surfaces when increase the ion dose is due to formation of the OH and C-O bonds. Nerve-cell-attachment properties of modified surface were investigated by the nerve-like cell of rat adrenal pheochromocytoma (PC12h) in vitro. After 2 days culture of the PC12h cells, no cells attached on the polystyrene films implanted with low ion dose from 1×1014 to 3×1014 ions/cm2. On the polystyrene films implanted with the dose order of 1015 ions/cm2, the cells selectively attached only on the implanted region. Whereas on the surfaces implanted with high dose such as 1×1016 and 3×1016 ions/cm2 mostly cells attached on the implanted region, and some attached on the unimplanted region, as well as cells were abnormal in shape and large size. Therefore, the suitable dose implantation for the selective-attachment of nerve-cells on the polystyrene films implanted at 5 and 10 keV were obtained around the dose order of 1015 ions/cm2, and the best condition for the selective attachment properties was at 3×1015 ions/cm2 corresponding to the lowest contact angle.

Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae.

PubMed

Hover, Tal; Maya, Tal; Ron, Sapir; Sandovsky, Hani; Shadkchan, Yana; Kijner, Nitzan; Mitiagin, Yulia; Fichtman, Boris; Harel, Amnon; Shanks, Robert M Q; Bruna, Roberto E; García-Véscovi, Eleonora; Osherov, Nir

2016-05-01

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well.S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Mechanisms of Bacterial (Serratia marcescens) Attachment to, Migration along, and Killing of Fungal Hyphae

PubMed Central

Hover, Tal; Maya, Tal; Ron, Sapir; Sandovsky, Hani; Shadkchan, Yana; Kijner, Nitzan; Mitiagin, Yulia; Fichtman, Boris; Harel, Amnon; Shanks, Robert M. Q.; Bruna, Roberto E.; García-Véscovi, Eleonora

2016-01-01

We have found a remarkable capacity for the ubiquitous Gram-negative rod bacterium Serratia marcescens to migrate along and kill the mycelia of zygomycete molds. This migration was restricted to zygomycete molds and several basidiomycete species. No migration was seen on any molds of the phylum Ascomycota. S. marcescens migration did not require fungal viability or surrounding growth medium, as bacteria migrated along aerial hyphae as well. S. marcescens did not exhibit growth tropism toward zygomycete mycelium. Bacterial migration along hyphae proceeded only when the hyphae grew into the bacterial colony. S. marcescens cells initially migrated along the hyphae, forming attached microcolonies that grew and coalesced to generate a biofilm that covered and killed the mycelium. Flagellum-defective strains of S. marcescens were able to migrate along zygomycete hyphae, although they were significantly slower than the wild-type strain and were delayed in fungal killing. Bacterial attachment to the mycelium does not necessitate type 1 fimbrial adhesion, since mutants defective in this adhesin migrated equally well as or faster than the wild-type strain. Killing does not depend on the secretion of S. marcescens chitinases, as mutants in which all three chitinase genes were deleted retained wild-type killing abilities. A better understanding of the mechanisms by which S. marcescens binds to, spreads on, and kills fungal hyphae might serve as an excellent model system for such interactions in general; fungal killing could be employed in agricultural fungal biocontrol. PMID:26896140

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions

PubMed Central

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja

2018-01-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/106 cells after cold storage, 5 ± 3 nmol/106 cells after rewarming vs. control 29 ± 6 nmol/106 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec−1 per 106 cells after rewarming vs. control 232 ± 83 pmol sec−1 per 106 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/106 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production—as elicited by an inhibitor of the respiratory chain, antimycin A—can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions. PMID:29390882

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions.

PubMed

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja; Rauen, Ursula

2017-12-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/10 6 cells after cold storage, 5 ± 3 nmol/10 6 cells after rewarming vs. control 29 ± 6 nmol/10 6 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec -1 per 10 6 cells after rewarming vs. control 232 ± 83 pmol sec -1 per 10 6 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/10 6 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production-as elicited by an inhibitor of the respiratory chain, antimycin A-can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions.

Embroidered polymer-collagen hybrid scaffold variants for ligament tissue engineering.

PubMed

Hoyer, M; Drechsel, N; Meyer, M; Meier, C; Hinüber, C; Breier, A; Hahner, J; Heinrich, G; Rentsch, C; Garbe, L-A; Ertel, W; Schulze-Tanzil, G; Lohan, A

2014-10-01

Embroidery techniques and patterns used for scaffold production allow the adaption of biomechanical scaffold properties. The integration of collagen into embroidered polylactide-co-caprolactone [P(LA-CL)] and polydioxanone (PDS) scaffolds could stimulate neo-tissue formation by anterior cruciate ligament (ACL) cells. Therefore, the aim of this study was to test embroidered P(LA-CL) and PDS scaffolds as hybrid scaffolds in combination with collagen hydrogel, sponge or foam for ligament tissue engineering. ACL cells were cultured on embroidered P(LA-CL) and PDS scaffolds without or with collagen supplementation. Cell adherence, vitality, morphology and ECM synthesis were analyzed. Irrespective of thread size, ACL cells seeded on P(LA-CL) scaffolds without collagen adhered and spread over the threads, whereas the cells formed clusters on PDS and larger areas remained cell-free. Using the collagen hydrogel, the scaffold colonization was limited by the gel instability. The collagen sponge layers integrated into the scaffolds were hardly penetrated by the cells. Collagen foams increased scaffold colonization in P(LA-CL) but did not facilitate direct cell-thread contacts in the PDS scaffolds. The results suggest embroidered P(LA-CL) scaffolds as a more promising basis for tissue engineering an ACL substitute than PDS due to superior cell attachment. Supplementation with a collagen foam presents a promising functionalization strategy. Copyright © 2014 Elsevier B.V. All rights reserved.

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

PubMed Central

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. PMID:22710062

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

PubMed

Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

Enhancing cell migration in shape-memory alginate-collagen composite scaffolds: In vitro and ex vivo assessment for intervertebral disc repair.

PubMed

Guillaume, Olivier; Naqvi, Syeda Masooma; Lennon, Kerri; Buckley, Conor Timothy

2015-04-01

Lower lumbar disc disorders pose a significant problem in an aging society with substantial socioeconomic consequences. Both inner tissue (nucleus pulposus) and outer tissue (annulus fibrosus) of the intervertebral disc are affected by such debilitating disorders and can lead to disc herniation and lower back pain. In this study, we developed an alginate-collagen composite porous scaffold with shape-memory properties to fill defects occurring in annulus fibrosus tissue of degenerated intervertebral discs, which has the potential to be administered using minimal invasive surgery. In the first part of this work, we assessed how collagen incorporation on preformed alginate scaffolds influences the physical properties of the final composite scaffold. We also evaluated the ability of annulus fibrosus cells to attach, migrate, and proliferate on the composite alginate-collagen scaffolds compared to control scaffolds (alginate only). In vitro experiments, performed in intervertebral disc-like microenvironmental conditions (low glucose and low oxygen concentrations), revealed that for alginate only scaffolds, annulus fibrosus cells agglomerated in clusters with limited infiltration and migration capacity. In comparison, for alginate-collagen scaffolds, annulus fibrosus cells readily attached and colonized constructs, while preserving their typical fibroblastic-like cell morphology with spreading behavior and intense cytoskeleton expression. In a second part of this study, we investigated the effects of alginate-collagen scaffold when seeded with bone marrow derived mesenchymal stem cells. In vitro, we observed that alginate-collagen porous scaffolds supported cell proliferation and extracellular matrix deposition (collagen type I), with secretion amplified by the local release of transforming growth factor-β3. In addition, when cultured in ex vivo organ defect model, alginate-collagen scaffolds maintained viability of transplanted mesenchymal stem cells for up to 5 weeks. Taken together, these findings illustrate the advantages of incorporating collagen as a means to enhance cell migration and proliferation in porous scaffolds which could be used to augment tissue repair strategies. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Cell-micropatterning by micromolding in capillary technique based on UV polymerization

NASA Astrophysics Data System (ADS)

Park, Min J.; Choi, Won M.; Park, O. O.

2006-01-01

Although optical lithography or photolithography is one of the most well-established techniques for micro, nano-fabrication, its usage with proteins and cells is restricted by steps that must be carried out in harsh organic solvents. Here, we present simple methods for cell-micropatterning using poly(dimethylsiloxane) (PDMS) as a mold. Cell non-adhesive surface or nonfouling surface providing a physico-chemical barrier to cell attachment was introduced for biomaterial pattering, where cells fail to interact with the surface over desired periods of time determined by each application. Poly(ethylene glycol) (PEG) was selected as nonfouling material to inhibit protein adsorption from biological media. The fouling resistance of PEG polymer is often explained by a steric repulsion interaction, resulting from the compression of PEG chains as proteins approach the surface. We also chose fibronectin to direct cell attachment because it is an extracellular matrix protein that is involved in the adhesion and spreading of anchorage-dependent cells. In our experiment, we propose two methods by application of micromolding in capillary (MIMIC) method based on UV polymerization to obtain a surface of alternating PEG and fibronectin. First to fabricate PEG microstructure via MIMIC method, a pre-patterned PDMS mold is placed on a desired substrate, and then the relief structure in the mold forms a network of empty channels. A drop of ethylene glycol monomer solution containing initiator for UV polymerization is placed at the open ends of the network of channels, which is then polymerized by exposure to UV light at room temperature. Once PEG microstructure is fabricated, incubation of the patterned surface in a fibronectin-containing solution allows back-filling of only the bare regions with fibronectin via adsorption. In the alternative method, a substrate is first incubated in a fibronectin-containing solution, leading to the adsorption of fibronectin over the entire surface, and the fibronectin-adsorbed substrate is then micropatterned with the PEG by MIMIC based on UV polymerization. Both methods create reproducible alternating PEG and fibronectin patterns applicable to cell-surface interactions on the microscale.

Attachment Anxiety is Linked to Alterations in Cortisol Production and Cellular Immunity

PubMed Central

Jaremka, Lisa M.; Glaser, Ronald; Loving, Timothy J.; Malarkey, William B.; Stowell, Jeffrey R.; Kiecolt-Glaser, Janice K.

2013-01-01

Although evidence suggests that attachment anxiety may increase risk for health problems, the mechanisms are not well understood. Married couples (N = 85, Mage = 38.67) provided saliva samples over three days and blood samples on two occasions. Participants with higher attachment anxiety produced more cortisol and had fewer numbers of CD3+ T-cells, CD45+ T-cells, CD3+CD4+ helper T-cells, and CD3+CD8+ cytotoxic T-cells than those with lower attachment anxiety. Higher cortisol was also related to fewer numbers of CD3+, CD45+, CD3+CD4+, and CD3+CD8+, which is mechanistically consistent with research showing that cortisol alters the cellular immune response. These data suggest that attachment anxiety may have physiological costs and provide a glimpse into the pathways through which social relationships impact health. The current study also extends attachment theory in an important new direction by utilizing a psychoneuroimmunological approach to the study of attachment anxiety, stress, and health. PMID:23307944

Sonication reduces the attachment of Salmonella Typhimurium ATCC 14028 cells to bacterial cellulose-based plant cell wall models and cut plant material.

PubMed

Tan, Michelle S F; Rahman, Sadequr; Dykes, Gary A

2017-04-01

This study investigated the removal of bacterial surface structures, particularly flagella, using sonication, and examined its effect on the attachment of Salmonella Typhimurium ATCC 14028 cells to plant cell walls. S. Typhimurium ATCC 14028 cells were subjected to sonication at 20 kHz to remove surface structures without affecting cell viability. Effective removal of flagella was determined by staining flagella of sonicated cells with Ryu's stain and enumerating the flagella remaining by direct microscopic counting. The attachment of sonicated S. Typhimurium cells to bacterial cellulose-based plant cell wall models and cut plant material (potato, apple, lettuce) was then evaluated. Varying concentrations of pectin and/or xyloglucan were used to produce a range of bacterial cellulose-based plant cell wall models. As compared to the non-sonicated controls, sonicated S. Typhimurium cells attached in significantly lower numbers (between 0.5 and 1.0 log CFU/cm 2 ) to all surfaces except to the bacterial cellulose-only composite without pectin and xyloglucan. Since attachment of S. Typhimurium to the bacterial cellulose-only composite was not affected by sonication, this suggests that bacterial surface structures, particularly flagella, could have specific interactions with pectin and xyloglucan. This study indicates that sonication may have potential applications for reducing Salmonella attachment during the processing of fresh produce. Copyright © 2016 Elsevier Ltd. All rights reserved.

Strength of silk attachment to Ilex chinensis leaves in the tea bagworm Eumeta minuscula (Lepidoptera, Psychidae).

PubMed

Wolff, Jonas O; Lovtsova, Julia; Gorb, Elena; Dai, Zhendong; Ji, Aihong; Zhao, Zhihui; Jiang, Nan; Gorb, Stanislav N

2017-03-01

Silks play an important role in the life of various arthropods. A highly neglected prerequisite to make versatile use of silks is sufficient attachment to substrates. Although there have been some studies on the structure and mechanics of silk anchorages of spiders, for insects only anecdotal reports on attachment-associated spinning behaviour exist. Here, we experimentally studied the silk attachment of the pupae and last instar caterpillars of the tea bagworm Eumeta minuscula (Butler 1881) (Lepidoptera, Psychidae) to the leaves of its host plant Ilex chinensis We found that the bagworms spin attachment discs, which share some structural features with those of spiders, like a plaque consisting of numerous overlaid, looped glue-coated silk fibres and the medially attaching suspension thread. Although the glue, which coats the fibres, cannot spread and adhere very well to the leaf surface, high pull-off forces were measured, yielding a mean safety factor (force divided by the animal weight) of 385.6. Presumably, the bagworms achieve this by removal of the leaf epidermis prior to silk attachment, which exposes the underlying tissue that represents a much better bonding site. This ensures a reliable attachment during the immobile, vulnerable pupal stage. This is the first study on the biomechanics and structure of silk attachments to substrates in insects. © 2017 The Author(s).

Direct Simulation of Evolution and Control of Three-Dimensional Instabilities in Attachment-Line Boundary Layers

NASA Technical Reports Server (NTRS)

Joslin, Ronald D.

1995-01-01

The spatial evolution of three-dimensional disturbances in an attachment-line boundary layer is computed by direct numerical simulation of the unsteady, incompressible Navier-Stokes equations. Disturbances are introduced into the boundary layer by harmonic sources that involve unsteady suction and blowing through the wall. Various harmonic- source generators are implemented on or near the attachment line, and the disturbance evolutions are compared. Previous two-dimensional simulation results and nonparallel theory are compared with the present results. The three-dimensional simulation results for disturbances with quasi-two-dimensional features indicate growth rates of only a few percent larger than pure two-dimensional results; however, the results are close enough to enable the use of the more computationally efficient, two-dimensional approach. However, true three-dimensional disturbances are more likely in practice and are more stable than two-dimensional disturbances. Disturbances generated off (but near) the attachment line spread both away from and toward the attachment line as they evolve. The evolution pattern is comparable to wave packets in at-plate boundary-layer flows. Suction stabilizes the quasi-two-dimensional attachment-line instabilities, and blowing destabilizes these instabilities; these results qualitatively agree with the theory. Furthermore, suction stabilizes the disturbances that develop off the attachment line. Clearly, disturbances that are generated near the attachment line can supply energy to attachment-line instabilities, but suction can be used to stabilize these instabilities.

Cell contact as an independent factor modulating cardiac myocyte hypertrophy and survival in long-term primary culture

NASA Technical Reports Server (NTRS)

Clark, W. A.; Decker, M. L.; Behnke-Barclay, M.; Janes, D. M.; Decker, R. S.

1998-01-01

Cardiac myocytes maintained in cell culture develop hypertrophy both in response to mechanical loading as well as to receptor-mediated signaling mechanisms. However, it has been shown that the hypertrophic response to these stimuli may be modulated through effects of intercellular contact achieved by maintaining cells at different plating densities. In this study, we show that the myocyte plating density affects not only the hypertrophic response and features of the differentiated phenotype of isolated adult myocytes, but also plays a significant role influencing myocyte survival in vitro. The native rod-shaped phenotype of freshly isolated adult myocytes persists in an environment which minimizes myocyte attachment and spreading on the substratum. However, these conditions are not optimal for long-term maintenance of cultured adult cardiac myocytes. Conditions which promote myocyte attachment and spreading on the substratum, on the other hand, also promote the re-establishment of new intercellular contacts between myocytes. These contacts appear to play a significant role in the development of spontaneous activity, which enhances the redevelopment of highly differentiated contractile, junctional, and sarcoplasmic reticulum structures in the cultured adult cardiomyocyte. Although it has previously been shown that adult cardiac myocytes are typically quiescent in culture, the addition of beta-adrenergic agonists stimulates beating and myocyte hypertrophy, and thereby serves to increase the level of intercellular contact as well. However, in densely-plated cultures with intrinsically high levels of intercellular contact, spontaneous contractile activity develops without the addition of beta-adrenergic agonists. In this study, we compare the function, morphology, and natural history of adult feline cardiomyocytes which have been maintained in cultures with different levels of intercellular contact, with and without the addition of beta-adrenergic agonists. Intercellular contact, communication, and transmission of contractile forces between myocytes appears to play a primary role in remodeling the 2-dimensional cell layer into a parallel alignment of elongated myocytes with highly developed intercalated disk-like junctions. This highly differentiated state is very stable, and cultures which achieve this state exhibit significantly greater longevity than more sparsely plated myocytes. These myocytes typically continue beating, and survive from 6 to more than 12 weeks in culture. When this level of contact and differentiation are not achieved, even among beta-adrenergic stimulated myocytes, contractile activity is not sustained, myofibrils atrophy, there is little or no development of junctional complexes, and the period of myocyte viability is typically no more than 5 weeks in vitro.

Bioreactor and methods for producing synchronous cells

NASA Technical Reports Server (NTRS)

Helmstetter, Charles E. (Inventor); Thornton, Maureen (Inventor); Gonda, Steve (Inventor)

2005-01-01

Apparatus and methods are directed to a perfusion culture system in which a rotating bioreactor is used to grow cells in a liquid culture medium, while these cells are attached to an adhesive-treated porous surface. As a result of this arrangement and its rotation, the attached cells divide, with one cell remaining attached to the substrate, while the other cell, a newborn cell is released. These newborn cells are of approximately the same age, that are collected upon leaving the bioreactor. The populations of newborn cells collected are of synchronous and are minimally, if at all, disturbed metabolically.

A numerical analysis of forces exerted by laminar flow on spreading cells in a parallel plate flow chamber assay.

PubMed

Olivier, L A; Truskey, G A

1993-10-01

Exposure of spreading anchorage-dependent cells to laminar flow is a common technique to measure the strength of cell adhesion. Since cells protrude into the flow stream, the force exerted by the fluid on the cells is a function of cell shape. To assess the relationship between cell shape and the hydrodynamic force on adherent cells, we obtained numerical solutions of the velocity and stress fields around bovine aortic endothelial cells during various stages of spreading and calculated the force required to detach the cells. Morphometric parameters were obtained from light and scanning electron microscopy measurements. Cells were assumed to have a constant volume, but the surface area increased during spreading until the membrane was stretched taut. Two-dimensional models of steady flow were generated using the software packages ANSYS (mesh generation) and FIDAP (problem solution). The validity of the numerical results was tested by comparison with published results for a semicircle in contact with the surface. The drag force and torque were greatest for round cells making initial contact with the surface. During spreading, the drag force and torque declined by factors of 2 and 20, respectively. The calculated forces and moments were used in adhesion models to predict the wall shear stress at which the cells detached. Based upon published values for the bond force and receptor number, round cells should detach at shear stresses between 2.5 and 6 dyn/cm(2), whereas substantially higher stresses are needed to detach spreading and fully spread cells. Results from the simulations indicate that (1) the drag force varies little with cell shape whereas the torque is very sensitive to cell shape, and (2) the increase in the strength of adhesion during spreading is due to increased contact area and receptor densities within the contact area. (c) 1993 John Wiley & Sons, Inc.

Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins.

PubMed

Stone, Jacquelyn A; Vemulapati, Bhadra M; Bradel-Tretheway, Birgit; Aguilar, Hector C

2016-12-01

The paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions. Nipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Establishment and characterization of a new cell line (SSP-9) derived from Atlantic salmon Salmo salar that expresses type I ifn.

PubMed

Rodriguez Saint-Jean, S; González, C; Monrás, M; Romero, A; Ballesteros, N; Enríquez, R; Perez-Prieto, S

2014-11-01

In the present work, the establishment and biological characterization of a new cell line, SSP-9, derived from the pronephros of the Atlantic salmon Salmo salar, are reported. These cells grew well in Leibovitz's (L15) medium supplemented with 10% foetal calf serum at temperatures from 15 to 25° C, and they have been sub-cultured over 100 passages to produce a continuous cell line with an epithelial-like morphology. The SSP-9 cells attached and spread efficiently at different plating densities, retaining 80% of cell viability after storage in liquid nitrogen. When karyotyped, the cells had 40-52 chromosomes, with a modal number of 48. Viral susceptibility tests showed that SSP-9 cells were susceptible to infectious pancreatic necrosis virus and infectious haematopoietic necrosis virus, producing infectious virus and regular cytopathic effects. Moreover, these cells could be stimulated by poly I:C, showing significant up-regulation in the expression of the genes that regulate immune responses, such as ifn and mx-1. SSP-9 cells constitutively express genes characteristic of macrophages, such as major histocompatibility complex (mhc-II) and interleukin 12b (il-12b), and flow cytometry assays confirmed that SSP-9 cells can be permanently transfected with plasmids expressing a reporter gene. Accordingly, this new cell line is apparently suitable for transgenic manipulation, and to study host cell-virus interactions and immune processes. © 2014 The Fisheries Society of the British Isles.

Ultrafast Spreading Effect Induced Rapid Cell Trapping into Porous Scaffold with Superhydrophilic Surface.

PubMed

Wang, Chenmiao; Qiao, Chunyan; Song, Wenlong; Sun, Hongchen

2015-08-19

In this contribution, superhydrophilic chitosan-based scaffolds with ultrafast spreading property were fabricated and used to improve the trapped efficiency of cells. The ultrafast spreading property allowed cells to be trapped into the internal 3D porous structures of the prepared scaffolds more quickly and effectively. Cell adhesion, growth, and proliferation were also improved, which could be attributed to the combination of UV irradiation and ultrafast spreading property. The construction of ultrafast spreading property on the scaffold surface will offer a novel way to design more effective scaffold in tissue engineering that could largely shorten the therapeutic time for patients.

Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

PubMed

Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

1997-03-01

The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

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

NASA Astrophysics Data System (ADS)

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

2011-02-01

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

Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

PubMed

Ho, Hoang-Yen; Moffat, Ryan C; Patel, Rupal V; Awah, Franklin N; Baloue, Kaitrin; Crowe, David L

2010-09-01

Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Laminin and Fibronectin in Cell Adhesion: Enhanced Adhesion of Cells from Regenerating Liver to Laminin

NASA Astrophysics Data System (ADS)

Carlsson, Roland; Engvall, Eva; Freeman, Aaron; Ruoslahti, Erkki

1981-04-01

Laminin, a basement membrane glycoprotein isolated from cultures of mouse endodermal cells and rat yolk sac carcinoma cells, promoted the attachment of liver cells obtained from regenerating mouse liver. Cells from normal mouse liver attached readily to dishes coated with fibronectin but attached poorly to surfaces coated with laminin. Both proteins efficiently promoted the attachment of cells from livers undergoing regeneration. After regeneration, the attachment to laminin returned to the low levels found in animals not subjected to partial hepatectomy but attachment to fibronectin remained high. Immunofluorescent staining of sections of normal liver with antilaminin revealed the presence of laminin in or adjacent to the walls of the bile ducts and blood vessels. After induction of regeneration by partial hepatectomy, increased amounts of laminin appeared in the sinusoidal areas. After carbon tetrachloride poisoning, staining for laminin was especially pronounced in the necrotic and postnecrotic areas around the central veins. This additional expression of laminin was transient. It reached a maximum around 5-6 days after the injury and then gradually disappeared. These findings show that laminin is an adhesive protein. The increase of laminin in regenerating liver and the adhesiveness of cells from such livers to laminin suggest a role for laminin in the maintenance of a proper tissue organization during liver regeneration.

Modeling the mechanics of cells in the cell-spreading process driven by traction forces

NASA Astrophysics Data System (ADS)

Fang, Yuqiang; Lai, King W. C.

2016-04-01

Mechanical properties of cells and their mechanical interaction with the extracellular environments are main factors influencing cellular function, thus indicating the progression of cells in different disease states. By considering the mechanical interactions between cell adhesion molecules and the extracellular environment, we developed a cell mechanical model that can characterize the mechanical changes in cells during cell spreading. A cell model was established that consisted of various main subcellular components, including cortical cytoskeleton, nuclear envelope, actin filaments, intermediate filaments, and microtubules. We demonstrated the structural changes in subcellular components and the changes in spreading areas during cell spreading driven by traction forces. The simulation of nanoindentation tests was conducted by integrating the indenting force to the cell model. The force-indentation curve of the cells at different spreading states was simulated, and the results showed that cell stiffness increased with increasing traction forces, which were consistent with the experimental results. The proposed cell mechanical model provides a strategy to investigate the mechanical interactions of cells with the extracellular environments through the adhesion molecules and to reveal the cell mechanical properties at the subcellular level as cells shift from the suspended state to the adherent state.

Modeling the mechanics of cells in the cell-spreading process driven by traction forces.

PubMed

Fang, Yuqiang; Lai, King W C

2016-04-01

Mechanical properties of cells and their mechanical interaction with the extracellular environments are main factors influencing cellular function, thus indicating the progression of cells in different disease states. By considering the mechanical interactions between cell adhesion molecules and the extracellular environment, we developed a cell mechanical model that can characterize the mechanical changes in cells during cell spreading. A cell model was established that consisted of various main subcellular components, including cortical cytoskeleton, nuclear envelope, actin filaments, intermediate filaments, and microtubules. We demonstrated the structural changes in subcellular components and the changes in spreading areas during cell spreading driven by traction forces. The simulation of nanoindentation tests was conducted by integrating the indenting force to the cell model. The force-indentation curve of the cells at different spreading states was simulated, and the results showed that cell stiffness increased with increasing traction forces, which were consistent with the experimental results. The proposed cell mechanical model provides a strategy to investigate the mechanical interactions of cells with the extracellular environments through the adhesion molecules and to reveal the cell mechanical properties at the subcellular level as cells shift from the suspended state to the adherent state.

The development of peptide-based interfacial biomaterials for generating biological functionality on the surface of bioinert materials.

PubMed

Meyers, Steven R; Khoo, Xiaojuan; Huang, Xin; Walsh, Elisabeth B; Grinstaff, Mark W; Kenan, Daniel J

2009-01-01

Biomaterials used in implants have traditionally been selected based on their mechanical properties, chemical stability, and biocompatibility. However, the durability and clinical efficacy of implantable biomedical devices remain limited in part due to the absence of appropriate biological interactions at the implant interface and the lack of integration into adjacent tissues. Herein, we describe a robust peptide-based coating technology capable of modifying the surface of existing biomaterials and medical devices through the non-covalent binding of modular biofunctional peptides. These peptides contain at least one material binding sequence and at least one biologically active sequence and thus are termed, "Interfacial Biomaterials" (IFBMs). IFBMs can simultaneously bind the biomaterial surface while endowing it with desired biological functionalities at the interface between the material and biological realms. We demonstrate the capabilities of model IFBMs to convert native polystyrene, a bioinert surface, into a bioactive surface that can support a range of cell activities. We further distinguish between simple cell attachment with insufficient integrin interactions, which in some cases can adversely impact downstream biology, versus biologically appropriate adhesion, cell spreading, and cell survival mediated by IFBMs. Moreover, we show that we can use the coating technology to create spatially resolved patterns of fluorophores and cells on substrates and that these patterns retain their borders in culture.

Biomimetic L-aspartic acid-derived functional poly(ester amide)s for vascular tissue engineering.

PubMed

Knight, Darryl K; Gillies, Elizabeth R; Mequanint, Kibret

2014-08-01

Functionalization of polymeric biomaterials permits the conjugation of cell signaling molecules capable of directing cell function. In this study, l-phenylalanine and l-aspartic acid were used to synthesize poly(ester amide)s (PEAs) with pendant carboxylic acid groups through an interfacial polycondensation approach. Human coronary artery smooth muscle cell (HCASMC) attachment, spreading and proliferation was observed on all PEA films. Vinculin expression at the cell periphery suggested that HCASMCs formed focal adhesions on the functional PEAs, while the absence of smooth muscle α-actin (SMαA) expression implied the cells adopted a proliferative phenotype. The PEAs were also electrospun to yield nanoscale three-dimensional (3-D) scaffolds with average fiber diameters ranging from 130 to 294nm. Immunoblotting studies suggested a potential increase in SMαA and calponin expression from HCASMCs cultured on 3-D fibrous scaffolds when compared to 2-D films. X-ray photoelectron spectroscopy and immunofluorescence demonstrated the conjugation of transforming growth factor-β1 to the surface of the functional PEA through the pendant carboxylic acid groups. Taken together, this study demonstrates that PEAs containing aspartic acid are viable biomaterials for further investigation in vascular tissue engineering. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Self healing hydrogels composed of amyloid nano fibrils for cell culture and stem cell differentiation.

PubMed

Jacob, Reeba S; Ghosh, Dhiman; Singh, Pradeep K; Basu, Santanu K; Jha, Narendra Nath; Das, Subhadeep; Sukul, Pradip K; Patil, Sachin; Sathaye, Sadhana; Kumar, Ashutosh; Chowdhury, Arindam; Malik, Sudip; Sen, Shamik; Maji, Samir K

2015-06-01

Amyloids are highly ordered protein/peptide aggregates associated with human diseases as well as various native biological functions. Given the diverse range of physiochemical properties of amyloids, we hypothesized that higher order amyloid self-assembly could be used for fabricating novel hydrogels for biomaterial applications. For proof of concept, we designed a series of peptides based on the high aggregation prone C-terminus of Aβ42, which is associated with Alzheimer's disease. These Fmoc protected peptides self assemble to β sheet rich nanofibrils, forming hydrogels that are thermoreversible, non-toxic and thixotropic. Mechanistic studies indicate that while hydrophobic, π-π interactions and hydrogen bonding drive amyloid network formation to form supramolecular gel structure, the exposed hydrophobic surface of amyloid fibrils may render thixotropicity to these gels. We have demonstrated the utility of these hydrogels in supporting cell attachment and spreading across a diverse range of cell types. Finally, by tuning the stiffness of these gels through modulation of peptide concentration and salt concentration these hydrogels could be used as scaffolds that can drive differentiation of mesenchymal stem cells. Taken together, our results indicate that small size, ease of custom synthesis, thixotropic nature makes these amyloid-based hydrogels ideally suited for biomaterial/nanotechnology applications. Copyright © 2015 Elsevier Ltd. All rights reserved.

Porous titania surfaces on titanium with hierarchical macro- and mesoporosities for enhancing cell adhesion, proliferation and mineralization.

PubMed

Han, Guang; Müller, Werner E G; Wang, Xiaohong; Lilja, Louise; Shen, Zhijian

2015-02-01

Titanium received a macroporous titania surface layer by anodization, which contains open pores with average pore diameter around 5 μm. An additional mesoporous titania top layer following the contour of the macropores, of 100-200 nm thickness and with a pore diameter of 10nm, was formed by using the evaporation-induced self-assembly (EISA) method with titanium (IV) tetraethoxide as the precursor. A coherent laminar titania surface layer was thus obtained, creating a hierarchical macro- and mesoporous surface that was characterized by high-resolution electron microscopy. The interfacial bonding between the surface layers and the titanium matrix was characterized by the scratch test that confirmed a stable and strong bonding of titania surface layers on titanium. The wettability to water and the effects on the osteosarcoma cell line (SaOS-2) proliferation and mineralization of the formed titania surface layers were studied systematically by cell culture and scanning electron microscopy. The results proved that the porous titania surface with hierarchical macro- and mesoporosities was hydrophilic that significantly promoted cell attachment and spreading. A synergistic role of the hierarchical macro- and mesoporosities was revealed in terms of enhancing cell adhesion, proliferation and mineralization, compared with the titania surface with solo scale topography. Copyright © 2014 Elsevier B.V. All rights reserved.

3D cell entrapment in crosslinked thiolated gelatin-poly(ethylene glycol) diacrylate hydrogels

PubMed Central

Fu, Yao; Xu, Kedi; Zheng, Xiaoxiang; Giacomin, A. Jeffrey; Mix, Adam W.; Kao, Weiyuan John

2012-01-01

The combined use of natural ECM components and synthetic materials offers an attractive alternative to fabricate hydrogel-based tissue engineering scaffolds to study cell-matrix interactions in three-dimensions (3D). A facile method was developed to modify gelatin with cysteine via a bifunctional PEG linker, thus introducing free thiol groups to gelatin chains. A covalently crosslinked gelatin hydrogel was fabricated using thiolated gelatin and poly(ethylene glycol) diacrylate (PEGdA) via thiol-ene reaction. Unmodified gelatin was physically incorporated in a PEGdA-only matrix for comparison. We sought to understand the effect of crosslinking modality on hydrogel physicochemical properties and the impact on 3D cell entrapment. Compared to physically incorporated gelatin hydrogels, covalently crosslinked gelatin hydrogels displayed higher maximum weight swelling ratio (Qmax), higher water content, significantly lower cumulative gelatin dissolution up to 7 days, and lower gel stiffness. Furthermore, fibroblasts encapsulated within covalently crosslinked gelatin hydrogels showed extensive cytoplasmic spreading and the formation of cellular networks over 28 days. In contrast, fibroblasts encapsulated in the physically incorporated gelatin hydrogels remained spheroidal. Hence, crosslinking ECM protein with synthetic matrix creates a stable scaffold with tunable mechanical properties and with long-term cell anchorage points, thus supporting cell attachment and growth in the 3D environment. PMID:21955690

Beam-energy-spread minimization using cell-timing optimization

NASA Astrophysics Data System (ADS)

Rose, C. R.; Ekdahl, C.; Schulze, M.

2012-04-01

Beam energy spread, and related beam motion, increase the difficulty in tuning for multipulse radiographic experiments at the dual-axis radiographic hydrodynamic test facility’s axis-II linear induction accelerator (LIA). In this article, we describe an optimization method to reduce the energy spread by adjusting the timing of the cell voltages (both unloaded and loaded), either advancing or retarding, such that the injector voltage and summed cell voltages in the LIA result in a flatter energy profile. We developed a nonlinear optimization routine which accepts as inputs the 74 cell-voltage, injector voltage, and beam current waveforms. It optimizes cell timing per user-selected groups of cells and outputs timing adjustments, one for each of the selected groups. To verify the theory, we acquired and present data for both unloaded and loaded cell-timing optimizations. For the unloaded cells, the preoptimization baseline energy spread was reduced by 34% and 31% for two shots as compared to baseline. For the loaded-cell case, the measured energy spread was reduced by 49% compared to baseline.

Rac1 mediates cytokine-stimulated hemocyte spreading via prostaglandin biosynthesis in the beet armyworm, Spodoptera exigua

USDA-ARS?s Scientific Manuscript database

Cell spreading is an integral component of insect hemocytic immune reactions to infections and invasions. Cell spreading is accomplished by cytoskeleton rearrangement, which is activated by three major immune mediators, biogenic monoamines, plasmatocyte-spreading peptide (PSP), and eicosanoids, part...

Impact of cell shape in hierarchically structured plant surfaces on the attachment of male Colorado potato beetles (Leptinotarsa decemlineata)

PubMed Central

Seidel, Robin; Bohn, Holger Florian; Speck, Thomas

2012-01-01

Summary Plant surfaces showing hierarchical structuring are frequently found in plant organs such as leaves, petals, fruits and stems. In our study we focus on the level of cell shape and on the level of superimposed microstructuring, leading to hierarchical surfaces if both levels are present. While it has been shown that epicuticular wax crystals and cuticular folds strongly reduce insect attachment, and that smooth papillate epidermal cells in petals improve the grip of pollinators, the impact of hierarchical surface structuring of plant surfaces possessing convex or papillate cells on insect attachment remains unclear. We performed traction experiments with male Colorado potato beetles on nine different plant surfaces with different structures. The selected plant surfaces showed epidermal cells with either tabular, convex or papillate cell shape, covered either with flat films of wax, epicuticular wax crystals or with cuticular folds. On surfaces possessing either superimposed wax crystals or cuticular folds we found traction forces to be almost one order of magnitude lower than on surfaces covered only with flat films of wax. Independent of superimposed microstructures we found that convex and papillate epidermal cell shapes slightly enhance the attachment ability of the beetles. Thus, in plant surfaces, cell shape and superimposed microstructuring yield contrary effects on the attachment of the Colorado potato beetle, with convex or papillate cells enhancing attachment and both wax crystals or cuticular folds reducing attachment. However, the overall magnitude of traction force mainly depends on the presence or absence of superimposed microstructuring. PMID:22428097

From micro to nano contacts in biological attachment devices

PubMed Central

Arzt, Eduard; Gorb, Stanislav; Spolenak, Ralph

2003-01-01

Animals with widely varying body weight, such as flies, spiders, and geckos, can adhere to and move along vertical walls and even ceilings. This ability is caused by very efficient attachment mechanisms in which patterned surface structures interact with the profile of the substrate. An extensive microscopic study has shown a strong inverse scaling effect in these attachment devices. Whereas μm dimensions of the terminal elements of the setae are sufficient for flies and beetles, geckos must resort to sub-μm devices to ensure adhesion. This general trend is quantitatively explained by applying the principles of contact mechanics, according to which splitting up the contact into finer subcontacts increases adhesion. This principle is widely spread in design of natural adhesive systems and may also be transferred into practical applications. PMID:12960386

Dual Function of the pUL7-pUL51 Tegument Protein Complex in Herpes Simplex Virus 1 Infection.

PubMed

Albecka, Anna; Owen, Danielle J; Ivanova, Lyudmila; Brun, Juliane; Liman, Rukayya; Davies, Laura; Ahmed, M Firoz; Colaco, Susanna; Hollinshead, Michael; Graham, Stephen C; Crump, Colin M

2017-01-15

The tegument of herpesviruses is a highly complex structural layer between the nucleocapsid and the envelope of virions. Tegument proteins play both structural and regulatory functions during replication and spread, but the interactions and functions of many of these proteins are poorly understood. Here we focus on two tegument proteins from herpes simplex virus 1 (HSV-1), pUL7 and pUL51, which have homologues in all other herpesviruses. We have now identified that HSV-1 pUL7 and pUL51 form a stable and direct protein-protein interaction, their expression levels rely on the presence of each other, and they function as a complex in infected cells. We demonstrate that expression of the pUL7-pUL51 complex is important for efficient HSV-1 assembly and plaque formation. Furthermore, we also discovered that the pUL7-pUL51 complex localizes to focal adhesions at the plasma membrane in both infected cells and in the absence of other viral proteins. The expression of pUL7-pUL51 is important to stabilize focal adhesions and maintain cell morphology in infected cells and cells infected with viruses lacking pUL7 and/or pUL51 round up more rapidly than cells infected with wild-type HSV-1. Our data suggest that, in addition to the previously reported functions in virus assembly and spread for pUL51, the pUL7-pUL51 complex is important for maintaining the attachment of infected cells to their surroundings through modulating the activity of focal adhesion complexes. Herpesviridae is a large family of highly successful human and animal pathogens. Virions of these viruses are composed of many different proteins, most of which are contained within the tegument, a complex structural layer between the nucleocapsid and the envelope within virus particles. Tegument proteins have important roles in assembling virus particles as well as modifying host cells to promote virus replication and spread. However, little is known about the function of many tegument proteins during virus replication. Our study focuses on two tegument proteins from herpes simplex virus 1 that are conserved in all herpesviruses: pUL7 and pUL51. We demonstrate that these proteins directly interact and form a functional complex that is important for both virus assembly and modulation of host cell morphology. Further, we identify for the first time that these conserved herpesvirus tegument proteins localize to focal adhesions in addition to cytoplasmic juxtanuclear membranes within infected cells. Copyright © 2017 Albecka et al.

Attachment of Escherichia coli O157:H7 grown in tryptic soy broth and nutrient broth to apple and lettuce surfaces as related to cell hydrophobicity, surface charge, and capsule production.

PubMed

Hassan, A N; Frank, J F

2004-10-01

This study investigated the effect of growth in tryptic soy broth (TSB) and nutrient broth (NB) on the ability Escherichia coli O157:H7 to attach to lettuce and apple surfaces. In addition, cell surface hydrophobicity, charge and capsule production were determined on cells grown in these media. Cells grown in NB attached less to lettuce and apple surfaces than did those grown in TSB. TSB, but not NB, supported capsule production by E. coli O157:H7. Cells grown in TSB were more hydrophilic than those grown in NB. No difference was found in the electrokinetic properties of cells grown in these media. Electrostatic and hydrophobic interactions and surface proteins did not appear to play an important role in the attachment of E. coli O157:H7 to these surfaces. Of the factors studied, only capsule production was associated with attachment ability. Copyright 2003 Elsevier B.V.

Identification of Cell Adhesive Sequences in the N-terminal Region of the Laminin α2 Chain*

PubMed Central

Hozumi, Kentaro; Ishikawa, Masaya; Hayashi, Takemitsu; Yamada, Yuji; Katagiri, Fumihiko; Kikkawa, Yamato; Nomizu, Motoyoshi

2012-01-01

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117–128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin β1 and anti-integrin α2β1 antibodies. These results suggest that A2-8 promotes an integrin α2β1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2β1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2β1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2β1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions. PMID:22654118

Development of functional biointerfaces by surface modification of polydimethylsiloxane with bioactive chlorogenic acid.

PubMed

Wu, Ming; He, Jia; Ren, Xiao; Cai, Wen-Sheng; Fang, Yong-Chun; Feng, Xi-Zeng

2014-04-01

The effect of physicochemical surface properties and chemical structure on the attachment and viability of bacteria and mammalian cells has been extensively studied for the development of biologically relevant applications. In this study, we report a new approach that uses chlorogenic acid (CA) to modify the surface wettability, anti-bacterial activity and cell adhesion properties of polydimethylsiloxane (PDMS). The chemical structure of the surface was obtained by X-ray photoelectron spectroscopy (XPS), the roughness was measured by atomic force microscopy (AFM), and the water contact angle was evaluated for PDMS substrates both before and after CA modification. Molecular modelling showed that the modification was predominately driven by van der Waals and electrostatic interactions. The exposed quinic-acid moiety improved the hydrophilicity of CA-modified PDMS substrates. The adhesion and viability of E. coli and HeLa cells were investigated using fluorescence and phase contrast microscopy. Few viable bacterial cells were found on CA-coated PDMS surfaces compared with unmodified PDMS surfaces. Moreover, HeLa cells exhibited enhanced adhesion and increased spreading on the modified PDMS surface. Thus, CA-coated PDMS surfaces reduced the ratio of viable bacterial cells and increased the adhesion of HeLa cells. These results contribute to the purposeful design of anti-bacterial surfaces for medical device use. Copyright © 2013 Elsevier B.V. All rights reserved.

Post-mitotic human dermal fibroblasts efficiently support the growth of human follicular keratinocytes.

PubMed

Limat, A; Hunziker, T; Boillat, C; Bayreuther, K; Noser, F

1989-05-01

For growth at low seeding densities, keratinocytes isolated from human tissues like epidermis or hair follicles are dependent on mesenchyme-derived feeder cells such as the 3T3-cell employed so far. As an alternative method, the present study describes the use of post-mitotic human dermal fibroblasts sublethally irradiated or mitomycin C-treated. Special emphasis was put on efficient growth of primary keratinocyte cultures plated at very low seeding densities. Thus, outer root sheath cells isolated from two anagen human hair follicles and plated in a 35-mm culture dish (3 - 6 X 10(2) attached cells) grew to confluence within 3 weeks (6 - 8 X 10(5) cells). Similar results were obtained for interfollicular keratinocytes. A crucial point for the function of these fibroblast feeder cells is plating at appropriate densities, considering their tremendous increase in cell size at the post-mitotic state. Plating densities of 4 - 5 X 10(3/cm2 allow full spreading of the feeder cells and do not impede the settling and expansion of the keratinocytes. Major advantages of this system include easier handling and better reproducibility than using 3T3-cells. Moreover, homologous fibroblast feeders mimic more closely the physiologic situation and therefore might provide a valuable tool for studying interactions between human mesenchymal and epithelial cells. Finally, potential hazards of using transformed feeder cells from a different species in keratinocyte cultures raised for wound covering in humans could be thus avoided.

ROCK Inhibition Promotes Attachment, Proliferation, and Wound Closure in Human Embryonic Stem Cell–Derived Retinal Pigmented Epithelium

PubMed Central

Croze, Roxanne H.; Thi, William J.; Clegg, Dennis O.

2016-01-01

Purpose Nonexudative (dry) age-related macular degeneration (AMD), a leading cause of blindness in the elderly, is associated with the loss of retinal pigmented epithelium (RPE) cells and the development of geographic atrophy, which are areas devoid of RPE cells and photoreceptors. One possible treatment option would be to stimulate RPE attachment and proliferation to replace dying/dysfunctional RPE and bring about wound repair. Clinical trials are underway testing injections of RPE cells derived from pluripotent stem cells to determine their safety and efficacy in treating AMD. However, the factors regulating RPE responses to AMD-associated lesions are not well understood. Here, we use cell culture to investigate the role of RhoA coiled coil kinases (ROCKs) in human embryonic stem cell–derived RPE (hESC-RPE) attachment, proliferation, and wound closure. Methods H9 hESC were spontaneously differentiated into RPE cells. hESC-RPE cells were treated with a pan ROCK1/2 or a ROCK2 only inhibitor; attachment, and proliferation and cell size within an in vitro scratch assay were examined. Results Pharmacological inhibition of ROCKs promoted hESC-RPE attachment and proliferation, and increased the rate of closure of in vitro wounds. ROCK inhibition decreased phosphorylation of cofilin and myosin light chain, suggesting that regulation of the cytoskeleton underlies the mechanism of action of ROCK inhibition. Conclusions ROCK inhibition promotes attachment, proliferation, and wound closure in H9 hESC-RPE cells. ROCK isoforms may have different roles in wound healing. Translational Relevance Modulation of the ROCK-cytoskeletal axis has potential in stimulating wound repair in transplanted RPE cells and attachment in cellular therapies. PMID:27917311

Direct Simulation of Evolution and Control of Nonlinear Instabilities in Attachment-Line Boundary Layers

NASA Technical Reports Server (NTRS)

Joslin, Ronald D.

2004-01-01

The unsteady, incompressible Navier-Stokes equations are used for the direct numerical simulation (DNS) of spatially evolving disturbances in a three-dimensional (3-D) attachment-line boundary layer. Two-dimensional (2-D) disturbances are introduced either by forcing at the in ow or by harmonic-source generators at the wall; 3-D disturbances are introduced by harmonic-source generators at the wall. The DNS results are in good agreement with both 2-D non-parallel theory (for small-amplitude disturbances) and weakly nonlinear theory (for finite-amplitude disturbances), which validates the two theories. The 2-D DNS results indicate that nonlinear disturbance growth occurs near branch II of the neutral stability curve; however, steady suction can be used to stabilize this disturbance growth. For 3-D instabilities that are generated o the attachment line, spreading both toward and away from the attachment line causes energy transfer to the attachment-line and downstream instabilities; suction stabilizes these instabilities. Furthermore, 3-D instabilities are more stable than 2-D or quasi-2-D instabilities.

The chemotaxis-like Che1 pathway has an indirect role in adhesive cell properties of Azospirillum brasilense.

PubMed

Siuti, Piro; Green, Calvin; Edwards, Amanda Nicole; Doktycz, Mitchel J; Alexandre, Gladys

2011-10-01

The Azospirillum brasilense chemotaxis-like Che1 signal transduction pathway was recently shown to modulate changes in adhesive cell surface properties that, in turn, affect cell-to-cell aggregation and flocculation behaviors rather than flagellar-mediated chemotaxis. Attachment to surfaces and root colonization may be functions related to flocculation. Here, the conditions under which A. brasilense wild-type Sp7 and che1 mutant strains attach to abiotic and biotic surfaces were examined using in vitro attachment and biofilm assays combined with atomic force microscopy and confocal microscopy. The nitrogen source available for growth is found to be a major modulator of surface attachment by A. brasilense and could be promoted in vitro by lectins, suggesting that it depends on interaction with surface-exposed residues within the extracellular matrix of cells. However, Che1-dependent signaling is shown to contribute indirectly to surface attachment, indicating that distinct mechanisms are likely underlying flocculation and attachment to surfaces in A. brasilense. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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

PubMed

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

2006-06-01

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

Human metapneumovirus Induces Reorganization of the Actin Cytoskeleton for Direct Cell-to-Cell Spread

PubMed Central

El Najjar, Farah; Cifuentes-Muñoz, Nicolás; Zhu, Haining; Buchholz, Ursula J.; Moncman, Carole L.; Dutch, Rebecca Ellis

2016-01-01

Paramyxovirus spread generally involves assembly of individual viral particles which then infect target cells. We show that infection of human bronchial airway cells with human metapneumovirus (HMPV), a recently identified paramyxovirus which causes significant respiratory disease, results in formation of intercellular extensions and extensive networks of branched cell-associated filaments. Formation of these structures is dependent on actin, but not microtubule, polymerization. Interestingly, using a co-culture assay we show that conditions which block regular infection by HMPV particles, including addition of neutralizing antibodies or removal of cell surface heparan sulfate, did not prevent viral spread from infected to new target cells. In contrast, inhibition of actin polymerization or alterations to Rho GTPase signaling pathways significantly decreased cell-to-cell spread. Furthermore, viral proteins and viral RNA were detected in intercellular extensions, suggesting direct transfer of viral genetic material to new target cells. While roles for paramyxovirus matrix and fusion proteins in membrane deformation have been previously demonstrated, we show that the HMPV phosphoprotein extensively co-localized with actin and induced formation of cellular extensions when transiently expressed, supporting a new model in which a paramyxovirus phosphoprotein is a key player in assembly and spread. Our results reveal a novel mechanism for HMPV direct cell-to-cell spread and provide insights into dissemination of respiratory viruses. PMID:27683250

Selective fibronectin adsorption against albumin and enhanced stem cell attachment on helium atmospheric pressure glow discharge treated titanium

NASA Astrophysics Data System (ADS)

Han, Inho; Vagaska, Barbora; Joo Park, Bong; Lee, Mi Hee; Jin Lee, Seung; Park, Jong-Chul

2011-06-01

Successful tissue integration of implanted medical devices depends on appropriate initial cellular response. In this study, the effect of helium atmospheric pressure glow discharge (He-APGD) treatment of titanium on selective protein adsorption and the initial attachment processes and focal adhesion formation of osteoprogenitor cells and stem cells were examined. Titanium disks were treated in a self-designed He-APGD system. Initial attachment of MC3T3-E1 mouse pre-osteoblasts and human mesenchymal stem cells (MSCs) was evaluated by MTT assay and plasma membrane staining followed by morphometric analysis. Fibronectin adsorption was investigated by Enzyme-Linked ImmunoSorbant Assay. MSCs cell attachment to treated and non-treated titanium disks coated with different proteins was verified also in serum-free culture. Organization of actin cytoskeleton and focal adhesions was evaluated microscopically. He-APGD treatment effectively modified the titanium surfaces by creating a super-hydrophilic surface, which promoted selectively higher adsorption of fibronectin, a protein of critical importance for cell/biomaterial interaction. In two different types of cells, the He-APGD treatment enhanced the number of attaching cells as well as their attachment area. Moreover, cells had higher organization of actin cytoskeleton and focal adhesions. Faster acceptance of the material by the progenitor cells in the early phases of tissue integration after the implantation may significantly reduce the overall healing time; therefore, titanium treatment with He-APGD seems to be an effective method of surface modification of titanium for improving its tissue inductive properties.

[Metapneumovirus expands the understanding of Paramyxovirus cell fusion--a review].

PubMed

Liu, Xiaoyu; Zhang, Xiaodong; Wei, Yongwei

2014-04-04

For most viruses in Paramyxoviridae, cell fusion requires both attachment protein and fusion protein. The attachment protein is responsible for the binding to its cognate receptors, while the interaction between fusion protein and attachment protein triggers the fusion protein which is responsible for the fusion. However, the Metapneumovirus fusion in Pneumovirinae subfamily displayed different mechanism where the attachment protein is not required. The cell fusion is accomplished by fusion protein alone without the help of the attachment protein. Recent studies indicate that low pH is required for cell fusion promoted by some hMPV strains. The fusion protein of aMPV type A is highly fusogenic, whereas that of type B is low. The original fusion models for Paramyxovirus cannot explain the phenomenon above. The mechanism to regulate the cell fusion of Metapneumovirus is poorly understood. It is becoming a hot spot for the study of cell fusion triggered by Paramyxovirus where it enlarged the traditional scope of Paramyxovirus fusion. In this review, we discuss the new achievements and advances in the understanding of cell fusion triggered by Metapneumovirus.

Adhesion inhibition of Mycoplasma iowae to chicken lymphoma DT40 cells by monoclonal antibodies reacting with a 65-kD polypeptide.

PubMed

Fiorentin, L; Panangala, V S; Zhang, Y; Toivio-Kinnucan, M

1998-01-01

Tissue- and cell-specific attachment of mycoplasmas is a key aspect of the host-parasite relationship. In this study, monoclonal antibodies (MAbs) recognizing surface membrane polypeptides with molecular masses of 46 kD (p46) and 65 kD (p65), respectively, were examined in a microtiter cell attachment (agglutination) inhibition assay. MAbs MI3, MI6, and MI12 reacting with p65 polypeptide of Mycoplasma iowae inhibited attachment of the organisms to chicken lymphoma (DT 40) cells. One MAb (MI2) that reacted with p65 in immunoblots did not inhibit cell attachment, possibly because of the intrinsic native conformation of the epitope(s) in intact mycoplasmas as opposed to the linear state (sodium dodecyl sulfate denatured) in immunoblots. More pronounced M. iowae adherence inhibition was demonstrated by polyclonal turkey and mouse anti-M. iowae antisera compared with MAbs. Immunogold labelling followed by electron microscopy allowed us to localize the MAb-recognized epitopes on the membrane surface of M. iowae. On the basis of the cell attachment inhibition of M. iowae by specific MAbs (MI3, MI6, and MI12), we propose that the p65 polypeptide plays a role in cytadherence. The ability of polyclonal antisera to inhibit attachment of M. iowae more efficiently than the MAbs suggests that additional epitopes within p65 and/or other proteins are involved in cell attachment.

Swimming and twitching motility are essential for attachment and virulence of Pantoea ananatis in onion seedlings.

PubMed

Weller-Stuart, Tania; Toth, Ian; De Maayer, Pieter; Coutinho, Teresa

2017-06-01

Pantoea ananatis is a widespread phytopathogen with a broad host range. Despite its ability to infect economically important crops, such as maize, rice and onion, relatively little is known about how this bacterium infects and colonizes host tissue or spreads within and between hosts. To study the role of motility in pathogenicity, we analysed both swimming and twitching motility in P. ananatis LMG 20103. Genetic recombineering was used to construct four mutants affected in motility. Two flagellar mutants were disrupted in the flgK and motA genes, required for flagellar assembly and flagellar rotation, respectively. Similarly, two twitching motility mutants were generated, impaired in the structure (pilA) and functioning (pilT) of the type IV pili. The role of swimming and twitching motility during the infection cycle of P. ananatis in onion seedlings was determined by comparing the mutant- and wild-type strains using several in vitro and in planta assays. From the results obtained, it was evident that flagella aid P. ananatis in locating and attaching to onion leaf surfaces, as well as in pathogenicity, whereas twitching motility is instrumental in the spread of the bacteria on the surface once attachment has occurred. Both swimming and twitching motility contribute towards the ability of P. ananatis to cause disease in onions. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin.

PubMed

Jenssen, Håvard; Sandvik, Kjersti; Andersen, Jeanette H; Hancock, Robert E W; Gutteberg, Tore J

2008-09-01

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.

Substrate stress relaxation regulates cell spreading

NASA Astrophysics Data System (ADS)

Chaudhuri, Ovijit; Gu, Luo; Darnell, Max; Klumpers, Darinka; Bencherif, Sidi A.; Weaver, James C.; Huebsch, Nathaniel; Mooney, David J.

2015-02-01

Studies of cellular mechanotransduction have converged upon the idea that cells sense extracellular matrix (ECM) elasticity by gauging resistance to the traction forces they exert on the ECM. However, these studies typically utilize purely elastic materials as substrates, whereas physiological ECMs are viscoelastic, and exhibit stress relaxation, so that cellular traction forces exerted by cells remodel the ECM. Here we investigate the influence of ECM stress relaxation on cell behaviour through computational modelling and cellular experiments. Surprisingly, both our computational model and experiments find that spreading for cells cultured on soft substrates that exhibit stress relaxation is greater than cells spreading on elastic substrates of the same modulus, but similar to that of cells spreading on stiffer elastic substrates. These findings challenge the current view of how cells sense and respond to the ECM.

Intercellular spreading of Porphyromonas gingivalis infection in primary gingival epithelial cells.

PubMed

Yilmaz, Ozlem; Verbeke, Philippe; Lamont, Richard J; Ojcius, David M

2006-01-01

Porphyromonas gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues. The organism successfully invades, multiplies in, and survives for extended periods in primary gingival epithelial cells (GECs). It is unknown whether P. gingivalis resides in the cytoplasm of infected cells throughout the infection or can spread to adjacent cells over time. We developed a technique based on flow cytofluorometry and fluorescence microscopy to study propagation of the organism at different stages of infection of GECs. Results showed that P. gingivalis spreads cell to cell and that the amount of spreading increases gradually over time. There was a very low level of propagation of bacteria to uninfected cells early in the infection (3 h postinfection), but there were 20-fold and 45-fold increases in the propagation rate after 24 h and 48 h, respectively, of infection. Immunofluorescence microscopy of infected cells suggested that intercellular translocation of P. gingivalis may be mediated through actin-based membrane protrusions, bypassing the need for release of bacteria into extracellular medium. Consistent with these observations, cytochalasin D treatment of infected cells resulted in significant inhibition of bacterial spreading. This study shows for the first time that P. gingivalis disseminates from cell to cell without passing through the extracellular space. This mechanism of spreading may allow P. gingivalis to colonize oral tissues without exposure to the humoral immune response.

Double-chimera proteins to enhance recruitment of endothelial cells and their progenitor cells.

PubMed

Behjati, M; Kazemi, M; Hashemi, M; Zarkesh-Esfahanai, S H; Bahrami, E; Hashemi-Beni, B; Ahmadi, R

2013-08-20

Enhanced attraction of selective vascular reparative cells is of great importance in order to increase vascular patency after endovascular treatments. We aimed to evaluate efficient attachment of endothelial cells and their progenitors on surfaces coated with mixture of specific antibodies, L-selectin and VE-cadherin, with prohibited platelet attachment. The most efficient conditions for coating of L-selectin-Fc chimera and VE-cadherin-Fc chimera proteins were first determined by protein coating on ELISA plates. The whole processes were repeated on titanium substrates, which are commonly used to coat stents. Endothelial progenitor cells (EPCs) and human umbilical vein endothelial cells (HUVECs) were isolated and characterized by flow cytometry. Cell attachment, growth, proliferation, viability and surface cytotoxicity were evaluated using nuclear staining and MTT assay. Platelet and cell attachment were evaluated using scanning electron microscopy. Optimal concentration of each protein for surface coating was 50 ng/ml. The efficacy of protein coating was both heat and pH independent. Calcium ions had significant impact on simultaneous dual-protein coating (P<0.05). Coating stability data revealed more than one year stability for these coated proteins at 4°C. L-selectin and VE-cadherin (ratio of 50:50) coated surface showed highest EPC and HUVEC attachment, viability and proliferation compared to single protein coated and non-coated titanium surfaces (P<0.05). This double coated surface did not show any cytotoxic effect. Surfaces coated with L-selectin and VE-cadherin are friendly surface for EPC and endothelial cell attachment with less platelet attachment. These desirable factors make the L-selectin and VE-cadherin coated surfaces perfect candidate endovascular device. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Intracellular signaling required for CCL25-stimulated T cell adhesion mediated by the integrin alpha4beta1.

PubMed

Parmo-Cabañas, Marisa; García-Bernal, David; García-Verdugo, Rosa; Kremer, Leonor; Márquez, Gabriel; Teixidó, Joaquin

2007-08-01

The alpha4beta1 integrin is expressed on thymocytes and mediates cell attachment to its ligands CS-1/fibronectin (CS-1/FN) and VCAM-1 in the thymus. The chemokine CCL25 is highly expressed in the thymus, where it binds to its receptor CCR9 on thymocytes promoting migration and activation. We show here that alpha4beta1 and CCR9 are coexpressed mainly on double- and single-positive thymocytes and that CCL25 strongly stimulates CD4(+)CD8(+) and CD4(+)CD8(-) adhesion to CS-1/FN and VCAM-1. CCL25 rapidly activated the GTPases Rac and Rap1 on thymocytes, and this activation was required for stimulation of adhesion, as detected using the CCR9(+)/alpha4beta1(+) human T cell line Molt-4. To study the role on CCL25-stimulated adhesion of the Rac downstream effector Wiskott-Aldrich syndrome protein family verproline-homologous protein 2 (WAVE2) as well as of Rap1-GTP-interacting proteins, regulator of adhesion and cell polarization enriched in lymphoid tissues (RAPL) and Rap1-GTP-interacting adapter molecule (RIAM), we knocked down their expression and tested transfectant attachment to alpha4beta1 ligands. We found that WAVE2 and RAPL but not RIAM were required for efficient triggering by CCL25 of T cell adhesion to CS-1/FN and VCAM-1. Although Rac and Rap1 activation was required during early steps of T cell adhesion stimulated by CCL25, WAVE2 was needed for the development of actin-dependent T cell spreading subsequent to adhesion strengthening but not during initial alpha4beta1-ligand interactions. These results suggest that regulation by CCL25 of adhesion of thymocyte subpopulations mediated by alpha4beta1 could contribute to control their trafficking in the thymus during maturation, and identify Rac-WAVE2 and Rap1-RAPL as pathways whose activation is required in inside-out signaling, leading to stimulated adhesion.

Quantifying rates of cell migration and cell proliferation in co-culture barrier assays reveals how skin and melanoma cells interact during melanoma spreading and invasion.

PubMed

Haridas, Parvathi; Penington, Catherine J; McGovern, Jacqui A; McElwain, D L Sean; Simpson, Matthew J

2017-06-21

Malignant spreading involves the migration of cancer cells amongst other native cell types. For example, in vivo melanoma invasion involves individual melanoma cells migrating through native skin, which is composed of several distinct subpopulations of cells. Here, we aim to quantify how interactions between melanoma and fibroblast cells affect the collective spreading of a heterogeneous population of these cells in vitro. We perform a suite of circular barrier assays that includes: (i) monoculture assays with fibroblast cells; (ii) monoculture assays with SK-MEL-28 melanoma cells; and (iii) a series of co-culture assays initiated with three different ratios of SK-MEL-28 melanoma cells and fibroblast cells. Using immunostaining, detailed cell density histograms are constructed to illustrate how the two subpopulations of cells are spatially arranged within the spreading heterogeneous population. Calibrating the solution of a continuum partial differential equation to the experimental results from the monoculture assays allows us to estimate the cell diffusivity and the cell proliferation rate for the melanoma and the fibroblast cells, separately. Using the parameter estimates from the monoculture assays, we then make a prediction of the spatial spreading in the co-culture assays. Results show that the parameter estimates obtained from the monoculture assays lead to a reasonably accurate prediction of the spatial arrangement of the two subpopulations in the co-culture assays. Overall, the spatial pattern of spreading of the melanoma cells and the fibroblast cells is very similar in monoculture and co-culture conditions. Therefore, we find no clear evidence of any interactions other than cell-to-cell contact and crowding effects. Copyright © 2017 Elsevier Ltd. All rights reserved.

Effect of acid tolerance response (ATR) on attachment of Listeria monocytogenes Scott A to stainless steel under extended exposure to acid or/and salt stress and resistance of sessile cells to subsequent strong acid challenge.

PubMed

Chorianopoulos, Nikos; Giaouris, Efstathios; Grigoraki, Ioanna; Skandamis, Panagiotis; Nychas, George-John

2011-02-28

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate (ca. 10⁸ CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly (p<0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment. Copyright © 2011 Elsevier B.V. All rights reserved.

Controlling attachment and growth of Listeria monocytogenes in polyvinyl chloride model floor drains using a peroxide chemical, chitosan-arginine, or heat.

PubMed

Berrang, Mark E; Hofacre, Charles L; Frank, Joseph F

2014-12-01

Listeria monocytogenes can colonize a poultry processing plant as a resident in floor drains. Limiting growth and attachment to drain surfaces may help lessen the potential for cross-contamination of product. The objective of this study was to compare a hydrogen peroxide-peroxyacetic acid-based chemical to chitosan-arginine or heat to prevent attachment of or destroy existing L. monocytogenes on the inner surface of model floor drains. L. monocytogenes was introduced to result in about 10(9) planktonic and attached cells within untreated polyvinyl chloride model drain pipes. Treatments (0.13 % peroxide-based sanitizer, 0.1 % chitosan-arginine, or 15 s of hot water at 95 to 100°C) were applied immediately after inoculation or after 24 h of incubation. Following treatment, all pipes were incubated for an additional 24 h; planktonic and attached cells were enumerated by plate count. All treatments significantly (P < 0.05) lowered numbers of planktonic and attached cells recovered. Chitosan-arginine resulted in approximately a 6-log reduction in planktonic cells when applied prior to incubation and a 3-log reduction after the inoculum had a chance to grow. Both heat and peroxide significantly outperformed chitosan-arginine (8- to 9-log reduction) and were equally effective before and after incubation. Heat was the only treatment that eliminated planktonic L. monocytogenes. All treatments were less effective against attached cells. Chitosan-arginine provided about a 4.5-log decrease in attached cells when applied before incubation and no significant decrease when applied after growth. Like with planktonic cells, peroxide-peroxyacetic acid and heat were equally effective before or after incubation, causing decreases ranging from 7 to 8.5 log for attached L. monocytogenes. Applied at the most efficacious time, any of these techniques may lessen the potential for L. monocytogenes to remain as a long-term resident in processing plant floor drains.

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

PubMed Central

Taylor, Matthew P.; Kratchmarov, Radomir; Enquist, Lynn W.

2013-01-01

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread. PMID:23978901

Limnoithona sinensis as refuge for bacteria: protection from UV radiation and chlorine disinfection in drinking water treatment.

PubMed

Lin, Tao; Cai, Bo; Chen, Wei

2014-11-01

In this study, we tested the potential of Limnoithona sinensis to provide its attached bacteria refuge against disinfection. The experimental results indicated that in water devoid of zooplankton, both UV radiation and chlorine disinfection significantly decreased the viability of free-living bacteria. In the presence of L. sinensis, however, the attached bacteria could survive and rapidly recover from disinfection. This demonstrated that L. sinensis provided protection from external damage to various aquatic bacteria that were attached to its body. The surviving bacteria remained on L. sinensis after disinfection exposure, which enabled a rapid increase in the bacterial population followed by their subsequent release into the surrounding water. Compared with UV radiation, chlorine disinfection was more effective in terms of inactivating attached bacteria. Both UV radiation and chlorine disinfection had little effect in terms of preventing the spread of undesirable bacteria, due to the incomplete inactivation of the bacteria associated with L. sinensis.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

PubMed

Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

2014-03-01

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

Morphological Survey of Microbial Mats Near Deep-Sea Thermal Vents †

PubMed Central

Jannasch, Holger W.; Wirsen, Carl O.

1981-01-01

A microscopic survey is presented of the most commonly observed and morphologically conspicuous microorganisms found attached to natural surfaces or to artificial materials deposited in the immediate vicinity of thermal submarine vents at the Galapagos Rift ocean spreading zone at a depth of 2,550 meters. Of special interest were the following findings: (i) all surfaces intermittently exposed to H2S-containing hydrothermal fluid were covered by layers, ca. 5 to 10 μm thick, of procaryotic, gram-negative cells interspaced with amorphous metal (Mn-Fe) deposits; (ii) although some of the cells were encased by dense metal deposits, there was little apparent correlation between metal deposition and the occurrence of microbial mats, (iii) highly differentiated forms appeared to be analogues of certain cyanobacteria, (iv) isolates from massive mats of a prosthecate bacterium could be identified as Hyphomicrobium spp., (v) intracellular membrane systems similar to those found in methylotrophic and nitrifying bacteria were observed in approximately 20% of the cells composing the mats, (vi) thiosulfate enrichments made from mat material resulted in isolations of different types of sulfur-oxidizing bacteria including the obligately chemolithotrophic genus Thiomicrospira. Images PMID:16345722

Dynamics of Cell Area and Force during Spreading

PubMed Central

Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-01-01

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. PMID:25517168

Spreading of Neutrophils: From Activation to Migration

PubMed Central

Sengupta, Kheya; Aranda-Espinoza, Helim; Smith, Lee; Janmey, Paul; Hammer, Daniel

2006-01-01

Neutrophils rely on rapid changes in morphology to ward off invaders. Time-resolved dynamics of spreading human neutrophils after activation by the chemoattractant fMLF (formyl methionyl leucyl phenylalanine) was observed by RICM (reflection interference contrast microscopy). An image-processing algorithm was developed to identify the changes in the overall cell shape and the zones of close contact with the substrate. We show that in the case of neutrophils, cell spreading immediately after exposure of fMLF is anisotropic and directional. The dependence of spreading area, A, of the cell as a function of time, t, shows several distinct regimes, each of which can be fitted as power laws (A ∼ tb). The different spreading regimes correspond to distinct values of the exponent b and are related to the adhesion state of the cell. Treatment with cytochalasin-B eliminated the anisotropy in the spreading. PMID:17012330

Novel peptide-based platform for the dual presentation of biologically active peptide motifs on biomaterials.

PubMed

Mas-Moruno, Carlos; Fraioli, Roberta; Albericio, Fernando; Manero, José María; Gil, F Javier

2014-05-14

Biofunctionalization of metallic materials with cell adhesive molecules derived from the extracellular matrix is a feasible approach to improve cell-material interactions and enhance the biointegration of implant materials (e.g., osseointegration of bone implants). However, classical biomimetic strategies may prove insufficient to elicit complex and multiple biological signals required in the processes of tissue regeneration. Thus, newer strategies are focusing on installing multifunctionality on biomaterials. In this work, we introduce a novel peptide-based divalent platform with the capacity to simultaneously present distinct bioactive peptide motifs in a chemically controlled fashion. As a proof of concept, the integrin-binding sequences RGD and PHSRN were selected and introduced in the platform. The biofunctionalization of titanium with this platform showed a positive trend towards increased numbers of cell attachment, and statistically higher values of spreading and proliferation of osteoblast-like cells compared to control noncoated samples. Moreover, it displayed statistically comparable or improved cell responses compared to samples coated with the single peptides or with an equimolar mixture of the two motifs. Osteoblast-like cells produced higher levels of alkaline phosphatase on surfaces functionalized with the platform than on control titanium; however, these values were not statistically significant. This study demonstrates that these peptidic structures are versatile tools to convey multiple biofunctionality to biomaterials in a chemically defined manner.

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture

PubMed Central

Schmal, Olga; Seifert, Jan; Schäffer, Tilman E.; Walter, Christina B.; Aicher, Wilhelm K.; Klein, Gerd

2016-01-01

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard. PMID:26839560

Hematopoietic Stem and Progenitor Cell Expansion in Contact with Mesenchymal Stromal Cells in a Hanging Drop Model Uncovers Disadvantages of 3D Culture.

PubMed

Schmal, Olga; Seifert, Jan; Schäffer, Tilman E; Walter, Christina B; Aicher, Wilhelm K; Klein, Gerd

2016-01-01

Efficient ex vivo expansion of hematopoietic stem cells with a concomitant preservation of stemness and self-renewal potential is still an unresolved ambition. Increased numbers of methods approaching this issue using three-dimensional (3D) cultures were reported. Here, we describe a simplified 3D hanging drop model for the coculture of cord blood-derived CD34(+) hematopoietic stem and progenitor cells (HSPCs) with bone marrow-derived mesenchymal stromal cells (MSCs). When seeded as a mixed cell suspension, MSCs segregated into tight spheroids. Despite the high expression of niche-specific extracellular matrix components by spheroid-forming MSCs, HSPCs did not migrate into the spheroids in the initial phase of coculture, indicating strong homotypic interactions of MSCs. After one week, however, HSPC attachment increased considerably, leading to spheroid collapse as demonstrated by electron microscopy and immunofluorescence staining. In terms of HSPC proliferation, the conventional 2D coculture system was superior to the hanging drop model. Furthermore, expansion of primitive hematopoietic progenitors was more favored in 2D than in 3D, as analyzed in colony-forming assays. Conclusively, our data demonstrate that MSCs, when arranged with a spread (monolayer) shape, exhibit better HSPC supportive qualities than spheroid-forming MSCs. Therefore, 3D systems are not necessarily superior to traditional 2D culture in this regard.

Glyoxal Crosslinking of Cell-Seeded Chitosan/Collagen Hydrogels for Bone Regeneration

PubMed Central

Wang, Limin; Stegemann, Jan P.

2011-01-01

Chitosan and collagen are natural biomaterials that have been used extensively in tissue engineering, both separately and as composite materials. Most methods to fabricate chitosan/collagen composites use freeze drying and chemical crosslinking to create stable porous scaffolds, which subsequently can be seeded with cells. In this study, we directly embedded human bone marrow stem cells (hBMSC) in chitosan/collagen materials by initiating gelation using β-glycerophosphate at physiological temperature and pH. We further examined the use of glyoxal, a dialdehyde with relatively low toxicity, to crosslink these materials and characterized the resulting changes in matrix and cell properties. The cytocompatibility of glyoxal and the crosslinked gels were investigated in terms of hBMSC metabolic activity, viability, proliferation, and osteogenic differentiation. These studies revealed that glyoxal was cytocompatible at concentrations below about 1 mM for periods of exposure up to 15 h, though the degree of cell spreading and proliferation were dependent on matrix composition. Glyoxal-crosslinked matrices were stiffer and compacted less than uncrosslinked controls. It was further demonstrated that hBMSC can attach and proliferate in 3D matrices composed of 50/50 chitosan/collagen, and that these materials supported osteogenic differentiation in response to stimulation. Such glyoxal-crosslinked chitosan/collagen composite materials may find utility as cell delivery vehicles for enhancing the repair of bone defects. PMID:21345389

Protection and attachment of Vibrio cholerae mediated by the toxin-coregulated pilus in the infant mouse model.

PubMed

Krebs, Shelly J; Taylor, Ronald K

2011-10-01

Colonization of the human small intestine by Vibrio cholerae is an essential step in pathogenesis that requires the type IV toxin-coregulated pilus (TCP). To date, three functions of TCP have been characterized: it serves as the CTXΦ receptor, secretes the colonization factor TcpF, and functions in microcolony formation by mediating bacterium-bacterium interactions. Although type IV pili in other pathogenic bacteria have been characterized as playing a major role in attachment to epithelial cells, there are very few studies to suggest that TCP acts as an attachment factor. Taking this into consideration, we investigated the function of TCP in attachment to Caco-2 cells and found that mutants lacking TCP were defective in attachment compared to the wild type. Overexpression of ToxT, the activator of TCP, significantly increased attachment of wild-type V. cholerae to Caco-2 cells. Using field-emission scanning electron microscopy (FESEM), we also observed TCP-mediated attachment to the small intestines of infected infant mice by using antibodies specific to TCP and V. cholerae. Remarkably, we also visualized matrices comprised of TCP appearing to engulf V. cholerae during infection, and we demonstrated that these matrices protected the bacteria from a component of bile, disclosing a possible new role of this pilus in protection of the bacterial cells from antimicrobial agents. This study provides new insights into TCP's function in V. cholerae colonization of the small intestine, describing additional roles in mediating attachment and protection of V. cholerae bacterial cells.

Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein.

PubMed

Sato, Yuma; Watanabe, Shumpei; Fukuda, Yoshinari; Hashiguchi, Takao; Yanagi, Yusuke; Ohno, Shinji

2018-03-15

Measles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV. IMPORTANCE Measles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE. Copyright © 2018 American Society for Microbiology.

77 FR 30000 - Combined Notice of Filings #1

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-21

.... Applicants: Golden Spread Electric Cooperative, Inc., Denver City Energy Associates, L.P., Great Point Power Denver City LP, LLC, LSP- Denver City, LLC, GPP Investors I, LLC, QUIXX Mustang Station, LLC Description... ER08- 1419-004--Attachment O to be effective 7/26/2010. Filed Date: 5/11/12 Accession Number: 20120511...

The effect of repeated sodium hypochlorite exposure on chlorine resistance development in Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

The emergence and spread of microorganisms with reduced susceptibility to antimicrobial agents is a major public health problem. Chlorine water has been widely used to reduce attached bacteria on the surface of food or for sanitizing the processing facilities. However, there are limited reports conc...

Morphological Heterogeneity and Attachment of Phaeobacter inhibens.

PubMed

Segev, Einat; Tellez, Adèle; Vlamakis, Hera; Kolter, Roberto

2015-01-01

The Roseobacter clade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters.

Morphological Heterogeneity and Attachment of Phaeobacter inhibens

PubMed Central

Segev, Einat; Tellez, Adèle; Vlamakis, Hera; Kolter, Roberto

2015-01-01

The Roseobacter clade is a key group of bacteria in the ocean exhibiting diverse metabolic repertoires and a wide range of symbiotic life-styles. Many Roseobacters possess remarkable capabilities of attachment to both biotic and abiotic surfaces. When attached to each other, these bacteria form multi-cellular structures called rosettes. Phaeobacter inhibens, a well-studied Roseobacter, exhibits various cell sizes and morphologies that are either associated with rosettes or occur as single cells. Here we describe the distribution of P. inhibens morphologies and rosettes within a population. We detect an N-acetylglucosamine-containing polysaccharide on the poles of some cells and at the center of all rosettes. We demonstrate that rosettes are formed by the attachment of individual cells at the polysaccharide-containing pole rather than by cell division. Finally, we show that P. inhibens attachment to abiotic surfaces is hindered by the presence of DNA from itself, but not from other bacteria. Taken together, our findings demonstrate that cell adhesiveness is likely to play a significant role in the life cycle of P. inhibens as well as other Roseobacters. PMID:26560130

Stable kinetochore-microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells.

PubMed

Tauchman, Eric C; Boehm, Frederick J; DeLuca, Jennifer G

2015-12-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore-microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore-microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal.

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

PubMed

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.

Stable kinetochore–microtubule attachment is sufficient to silence the spindle assembly checkpoint in human cells

PubMed Central

Tauchman, Eric C.; Boehm, Frederick J.; DeLuca, Jennifer G.

2015-01-01

During mitosis, duplicated sister chromatids attach to microtubules emanating from opposing sides of the bipolar spindle through large protein complexes called kinetochores. In the absence of stable kinetochore–microtubule attachments, a cell surveillance mechanism known as the spindle assembly checkpoint (SAC) produces an inhibitory signal that prevents anaphase onset. Precisely how the inhibitory SAC signal is extinguished in response to microtubule attachment remains unresolved. To address this, we induced formation of hyper-stable kinetochore–microtubule attachments in human cells using a non-phosphorylatable version of the protein Hec1, a core component of the attachment machinery. We find that stable attachments are sufficient to silence the SAC in the absence of sister kinetochore bi-orientation and strikingly in the absence of detectable microtubule pulling forces or tension. Furthermore, we find that SAC satisfaction occurs despite the absence of large changes in intra-kinetochore distance, suggesting that substantial kinetochore stretching is not required for quenching the SAC signal. PMID:26620470

Adherence of carp leucocytes to adults and cercariae of the blood fluke Sanguinicola inermis.

PubMed

Richards, D T; Hoole, D; Lewis, J W; Ewens, E; Arme, C

1996-03-01

Live adult and cercarial stages of Sanguinicola inermis Plehn, 1905 (Trematoda:Sanguinicolidae) were maintained in vitro in the presence of carp (Cyprinus carpio L.) leucocytes. Cells and parasites were fixed at intervals from 0.25 to 48 h and examined using light microscopy, SEM and TEM. Within 12 h of exposure, leucocytes were found attached to cercariae although, by 24 h, fewer cells were found attached to postcercarial, juvenile adult stages that had shed their tails. Neutrophils and macrophages were found attached to the damaged tegument of cercarie that had not transformed by 48 h. Few cells were attached to the tegument of adult flukes that were alive when fixed. However, there was extensive tegumental damage and numerous cells were attached to adult flukes that had died before fixation. The results are discussed with reference to parasite survival within the vascular system of the host.

Simultaneous investigation of intracellular Ca2+ increase and morphological events upon fertilization in the sand dollar egg.

PubMed

Hamaguchi, Y; Hamaguchi, M S

1990-06-01

An increase in intracellular Ca2+ concentration ([Ca2+]) and morphological were simultaneously observed by epifluorescence and differential interference contrast (DIC) microscopy during fertilization of the sand dollar, Clypeaster japonicus. [Ca2+], which was detected by a Ca2+ indicator, Fluo-3, initially increased just beneath the sperm-attached site on the egg surface 8.6 sec after attachment. The increase spread into the egg as a concentric sphere to the egg center and, thereafter, propagated in the egg cytoplasm as a planar wave rather than a spherical wave. It reached the site opposite the initiation site across the egg 24.2 sec after initiation. The fertilization envelope (FE) began to elevate 10.3 sec after the initiation of the increase in [Ca2+] and 21.2 sec after sperm attachment.

The effect of nicotine on reproduction and attachment of human gingival fibroblasts in vitro.

PubMed

Peacock, M E; Sutherland, D E; Schuster, G S; Brennan, W A; O'Neal, R B; Strong, S L; Van Dyke, T E

1993-07-01

The ability of fibroblasts to reproduce and attach to teeth is of paramount importance in re-establishing the lost connective tissue attachment after periodontal therapy. This study examined the effect of nicotine, a major component of the particulate phase of tobacco smoke, on human gingival fibroblast (HGF) reproduction and attachment to tissue culture surfaces. Pooled HGF cultures made from explants of gingival biopsies were utilized between passages 5 and 10 and plated in 96-well plates at 1.0 x 10(4) cells per well. Cell numbers were determined using 3-(4,5-dimethylthiazol-2-y)-2,5-diphenyl tetrazolium bromide (MTT), which is a reflection of mitochondrial dehydrogenase activity. The concentrations of nicotine used were 0.025, 0.05, 0.1, 0.2, and 0.4 microM, the average serum concentration for a smoker being approximately 0.1 microM. The effect of continuous nicotine exposure on HGF reproduction was determined by incubating cell cultures and media containing nicotine for up to 48 hours. Residual toxicity was determined by preincubating cells with nicotine for 1 or 6 hours. HGF suspensions and increasing concentrations of nicotine were added together to determine the effect on attachment. Results showed an enhanced effect of nicotine on HGF attachment, with increasing numbers of cells attaching with increasing nicotine concentrations, compared to the control. Low concentrations of nicotine had a stimulatory effect on cell replication, while higher concentrations of nicotine appear to have no significant effect on HGF reproduction. The responses of cells to some concentrations of nicotine may persist after its removal.

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

PubMed

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

2013-03-01

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

Priming the Secure Attachment Schema Affects the Emotional Face Processing Bias in Attachment Anxiety: An fMRI Research

PubMed Central

Tang, Qingting; Chen, Xu; Hu, Jia; Liu, Ying

2017-01-01

Our study explored how priming with a secure base schema affects the processing of emotional facial stimuli in individuals with attachment anxiety. We enrolled 42 undergraduate students between 18 and 27 years of age, and divided them into two groups: attachment anxiety and attachment secure. All participants were primed under two conditions, the secure priming using references to the partner, and neutral priming using neutral references. We performed repeated attachment security priming combined with a dual-task paradigm and functional magnetic resonance imaging. Participants’ reaction times in terms of responding to the facial stimuli were also measured. Attachment security priming can facilitate an individual’s processing of positive emotional faces; for instance, the presentation of the partner’s name was associated with stronger activities in a wide range of brain regions and faster reaction times for positive facial expressions in the subjects. The current finding of higher activity in the left-hemisphere regions for secure priming rather than neutral priming is consistent with the prediction that attachment security priming triggers the spread of the activation of a positive emotional state. However, the difference in brain activity during processing of both, positive and negative emotional facial stimuli between the two priming conditions appeared in the attachment anxiety group alone. This study indicates that the effect of attachment secure priming on the processing of emotional facial stimuli could be mediated by chronic attachment anxiety. In addition, it highlights the association between higher-order processes of the attachment system (secure attachment schema priming) and early-stage information processing system (attention), given the increased attention toward the effects of secure base schema on the processing of emotion- and attachment-related information among the insecure population. Thus, the following study has applications in providing directions for clinical treatment of mood disorders in attachment anxiety. PMID:28473796

Priming the Secure Attachment Schema Affects the Emotional Face Processing Bias in Attachment Anxiety: An fMRI Research.

PubMed

Tang, Qingting; Chen, Xu; Hu, Jia; Liu, Ying

2017-01-01

Our study explored how priming with a secure base schema affects the processing of emotional facial stimuli in individuals with attachment anxiety. We enrolled 42 undergraduate students between 18 and 27 years of age, and divided them into two groups: attachment anxiety and attachment secure. All participants were primed under two conditions, the secure priming using references to the partner, and neutral priming using neutral references. We performed repeated attachment security priming combined with a dual-task paradigm and functional magnetic resonance imaging. Participants' reaction times in terms of responding to the facial stimuli were also measured. Attachment security priming can facilitate an individual's processing of positive emotional faces; for instance, the presentation of the partner's name was associated with stronger activities in a wide range of brain regions and faster reaction times for positive facial expressions in the subjects. The current finding of higher activity in the left-hemisphere regions for secure priming rather than neutral priming is consistent with the prediction that attachment security priming triggers the spread of the activation of a positive emotional state. However, the difference in brain activity during processing of both, positive and negative emotional facial stimuli between the two priming conditions appeared in the attachment anxiety group alone. This study indicates that the effect of attachment secure priming on the processing of emotional facial stimuli could be mediated by chronic attachment anxiety. In addition, it highlights the association between higher-order processes of the attachment system (secure attachment schema priming) and early-stage information processing system (attention), given the increased attention toward the effects of secure base schema on the processing of emotion- and attachment-related information among the insecure population. Thus, the following study has applications in providing directions for clinical treatment of mood disorders in attachment anxiety.

Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels

PubMed Central

Yue, Kan; Santiago, Grissel Trujillo-de; Alvarez, Mario Moisés; Tamayol, Ali; Annabi, Nasim; Khademhosseini, Ali

2015-01-01

Gelatin methacryloyl (GelMA) hydrogels have been widely used for various biomedical applications due to their suitable biological properties and tunable physical characteristics. Three dimensional (3D) GelMA hydrogels closely resemble some essential properties of native extracellular matrix (ECM) due to the presence of cell-attaching and matrix metalloproteinase responsive peptide motifs, which allow cells to proliferate and spread in GelMA-based scaffolds. GelMA is also versatile from a processing perspective. It crosslinks when exposed to light irradiation to form hydrogels with tunable mechanical properties which mimic the native ECM. It can also be microfabricated using different methodologies including micromolding, photomasking, bioprinting, self-assembly, and microfluidic techniques to generate constructs with controlled architectures. Hybrid hydrogel systems can also be formed by mixing GelMA with nanoparticles such as carbon nanotubes and graphene oxide, and other polymers to form networks with desired combined properties and characteristics for specific biological applications. Recent research has demonstrated the proficiency of GelMA-based hydrogels in a wide range of applications including engineering of bone, cartilage, cardiac, and vascular tissues, among others. Other applications of GelMA hydrogels, besides tissue engineering, include fundamental single-single cell research, cell signaling, drug and gene delivery, and bio-sensing. PMID:26414409

Development of a novel biomaterial with an important osteoinductive capacity for hard tissue engineering.

PubMed

Simu, Meda-Romana; Pall, Emoke; Radu, Teodora; Miclaus, Maria; Culic, Bogdan; Mesaros, Anca-Stefania; Muntean, Alexandrina; Filip, Gabriela Adriana

2018-06-01

In this study we designed a composite biomaterial based on a high viscosity soft propolis extract (70% propolis) and shell clam, with antiseptic and osteoinductive qualities, that can be used in dentistry, orthopedics and other areas where hard tissue regeneration is needed. We assessed it in interaction with stabilized human cells isolated from dental papilla of wisdom teeth (D1MSCs). We performed detailed characterization of the obtained material by Scanning Electronic Microscopy (SEM), X-Ray Diffraction (XRD), Energy Dispersive X-Ray Spectroscopy (EDX), Fourier Transform Infrared Spectroscopy (FTIR) techniques. SEM investigation revealed the roughness and porosity of the shell, which acted like a scaffold, as it allowed cells to penetrate the pores, proliferate on the surface, spread and grow in the depressions provided by the substrate. in vitro cell viability, proliferation and differentiation assays showed that the newly obtain biomaterial presented low toxicity on D1MSCs and determined the development of numerous osteogenic nodules that were in a higher number even than in the specific induction medium. Our results demonstrated that the shell-propolis based biomaterial promoted and sustained human stem cells attachment, proliferation and differentiation, presenting an important osteoinductive effect essential for mineralized tissue reparation process. Copyright © 2018 Elsevier Ltd. All rights reserved.

Tunneling Nanotubes as a Novel Route of Cell-to-Cell Spread of Herpesviruses.

PubMed

Panasiuk, Mirosława; Rychłowski, Michał; Derewońko, Natalia; Bieńkowska-Szewczyk, Krystyna

2018-05-15

Various types of intercellular connections that are essential for communication between cells are often utilized by pathogens. Recently, a new type of cellular connection, consisting of long, thin, actin-rich membrane extensions named tunneling nanotubes (TNTs), has been shown to play an important role in cell-to-cell spread of HIV and influenza virus. In the present report, we show that TNTs are frequently formed by cells infected by an alphaherpesvirus, bovine herpesvirus 1 (BoHV-1). Viral proteins, such as envelope glycoprotein E (gE), capsid protein VP26, and tegument protein Us3, as well as cellular organelles (mitochondria) were detected by immunofluorescence and live-cell imaging of nanotubes formed by bovine primary fibroblasts and oropharynx cells (KOP cells). Time-lapse confocal studies of live cells infected with fluorescently labeled viruses showed that viral particles were transmitted via TNTs. This transfer also occurred in the presence of neutralizing antibodies, which prevented free entry of BoHV-1. We conclude that TNT formation contributes to successful cell-to-cell spread of BoHV-1 and demonstrate for the first time the participation of membrane nanotubes in intercellular transfer of a herpesvirus in live cells. IMPORTANCE Efficient transmission of viral particles between cells is an important factor in successful infection by herpesviruses. Herpesviruses can spread by the free-entry mode or direct cell-to-cell transfer via cell junctions and long extensions of neuronal cells. In this report, we show for the first time that an alphaherpesvirus can also spread between various types of cells using tunneling nanotubes, intercellular connections that are utilized by HIV and other viruses. Live-cell monitoring revealed that viral transmission occurs between the cells of the same type as well as between epithelial cells and fibroblasts. This newly discovered route of herpesviruses spread may contribute to efficient transmission despite the presence of host immune responses, especially after reactivation from latency that developed after primary infection. Long-range communication provided by TNTs may facilitate the spread of herpesviruses between many tissues and organs of an infected organism. Copyright © 2018 American Society for Microbiology.

Impact of pre-incubation time of silk fibroin scaffolds in culture medium on cell proliferation and attachment.

PubMed

Amirikia, Mehdi; Shariatzadeh, Seyed Mohammad Ali; Jorsaraei, Seyed Gholam Ali; Soleimani Mehranjani, Malek

2017-12-01

Cell behaviours such as proliferation and attachment can be affected by the length of pre-incubation period of the scaffolds in the culture medium for long term. The aim of this study was to investigate the long term pre-incubation of 3D silk fibroin scaffolds in complete culture medium on cell attachment and proliferation. After the preparation of silk fibroin scaffolds by the technique of freeze drying, the scaffolds were pre-incubated in complete culture medium for 2 d, 6 d or 10 d before apical papilla stem cells (SCAP) seeding. Modifications of the scaffold surface and wettability were examined by FE-SEM and water contact angle, respectively. Results showed a decrease both in roughness and water contact angle as pre-incubation time increases. DNA measurement after 18h and 10 d cell seeding showed a significant increase of DNA concentration which represents better attachment and proliferation with pre-incubation time increase. Qualitative examination, live&dead assay or H&E staining method after 30h and 10 d cell seeding respectively, indicated that pre-incubation of scaffolds has time dependent effect on cell proliferation and attachment. This suggests that improvement of cell attachment and proliferation may be mediated by differences in the amount of wettability (decreased water contact angle) after exposure of scaffold to culture medium for long term which, in turn, causes more protein adsorption in the surface of silk fibroin scaffold (decreased roughness). Copyright © 2017. Published by Elsevier Ltd.

Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

PubMed Central

Bailey, Jake V; Salman, Verena; Rouse, Gregory W; Schulz-Vogt, Heide N; Levin, Lisa A; Orphan, Victoria J

2011-01-01

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization. PMID:21697959

Contact-induced mitochondrial polarization supports HIV-1 virological synapse formation.

PubMed

Groppelli, Elisabetta; Starling, Shimona; Jolly, Clare

2015-01-01

Rapid HIV-1 spread between CD4 T lymphocytes occurs at retrovirus-induced immune cell contacts called virological synapses (VS). VS are associated with striking T cell polarization and localized virus budding at the site of contact that facilitates cell-cell spread. In addition to this, spatial clustering of organelles, including mitochondria, to the contact zone has been previously shown. However, whether cell-cell contact specifically induces dynamic T cell remodeling during VS formation and what regulates this process remain unclear. Here, we report that contact between an HIV-1-infected T cell and an uninfected target T cell specifically triggers polarization of mitochondria concomitant with recruitment of the major HIV-1 structural protein Gag to the site of cell-cell contact. Using fixed and live-cell imaging, we show that mitochondrial and Gag polarization in HIV-1-infected T cells occurs within minutes of contact with target T cells, requires the formation of stable cell-cell contacts, and is an active, calcium-dependent process. We also find that perturbation of mitochondrial polarization impairs cell-cell spread of HIV-1 at the VS. Taken together, these data suggest that HIV-1-infected T cells are able to sense and respond to contact with susceptible target cells and undergo dynamic cytoplasmic remodeling to create a synaptic environment that supports efficient HIV-1 VS formation between CD4 T lymphocytes. HIV-1 remains one of the major global health challenges of modern times. The capacity of HIV-1 to cause disease depends on the virus's ability to spread between immune cells, most notably CD4 T lymphocytes. Cell-cell transmission is the most efficient way of HIV-1 spread and occurs at the virological synapse (VS). The VS forms at the site of contact between an infected cell and an uninfected cell and is characterized by polarized assembly and budding of virions and clustering of cellular organelles, including mitochondria. Here, we show that cell-cell contact induces rapid recruitment of mitochondria to the contact site and that this supports efficient VS formation and consequently cell-cell spread. Additionally, we observed that cell-cell contact induces a mitochondrion-dependent increase in intracellular calcium, indicative of cellular signaling. Taken together, our data suggest that VS formation is a regulated process and thus a potential target to block HIV-1 cell-cell spread. Copyright © 2015, Groppelli et al.

Expression of adhesion and extracellular matrix genes in human blastocysts upon attachment in a 2D co-culture system.

PubMed

Aberkane, A; Essahib, W; Spits, C; De Paepe, C; Sermon, K; Adriaenssens, T; Mackens, S; Tournaye, H; Brosens, J J; Van de, Velde H

2018-05-26

What are the changes in human embryos, in terms of morphology and gene expression, upon attachment to endometrial epithelial cells? Apposition and adhesion of human blastocysts to endometrial epithelial cells are predominantly initiated at the embryonic pole and these steps are associated with changes in expression of adhesion and extracellular matrix (ECM) genes in the embryo. Both human and murine embryos have been co-cultured with Ishikawa cells, although embryonic gene expression associated with attachment has not yet been investigated in an in-vitro implantation model. Vitrified human blastocysts were warmed and co-cultured for up to 48 h with Ishikawa cells, a model cell line for receptive endometrial epithelium. Six-days post fertilisation (6dpf) human embryos were co-cultured with Ishikawa cells for 12 h, 24 h (7dpf) or 48 h (8dpf) and attachment rate and morphological development investigated. Expression of 84 adhesion and ECM genes was analysed by quantitative PCR. Immunofluorescence microscopy was used to assess the expression of three informative genes at the protein level. Data are reported on 115 human embryos. Mann-Whitney U was used for statistical analysis between two groups, with P < 0.05 considered significant. The majority of embryos attached to Ishikawa cells at the level of the polar trophectoderm; 41% of co-cultured embryos were loosely attached after 12 h and 86% firmly attached after 24 h. Outgrowth of hCG-positive embryonic cells at 8dpf indicated differentiation of trophectoderm into invasive syncytiotrophoblast. Gene expression analysis was performed on loosely attached and unattached embryos co-cultured with Ishikawa cells for 12 h. In contrast to unattached embryos, loosely attached embryos expressed THBS1, TNC, COL12A1, CTNND2, ITGA3, ITGAV, and LAMA3 and had significantly higher CD44 and TIMP1 transcript levels (P = 0.014 and P = 0.029, respectively). LAMA3, THBS1 and TNC expression was validated at the protein level in firmly attached 7dpf embryos. Thrombospondin 1 (THBS1) resided in the cytoplasm of embryonic cells whereas laminin subunit alpha 3 (LAMA3) and tenascin C (TNC) were expressed on the cell surface of trophectoderm cells. Incubation with a neutralizing TNC antibody did not affect the rate of embryo attachment or hCG secretion. None. This in-vitro study made use of an endometrial adenocarcinoma cell line to mimic receptive luminal epithelium. Also, the number of embryos was limited. Contamination of recovered embryos with Ishikawa cells was unlikely based on their differential gene expression profiles. Taken together, we provide a 'proof of concept' that initiation of the implantation process coincides with the induction of specific embryonic genes. Genome-wide expression profiling of a larger sample set may provide insights into the molecular embryonic pathways underlying successful or failed implantation. A.A. was supported by a grant from the "Instituut voor Innovatie door Wetenschap en Technologie" (IWT, 121716, Flanders, Belgium). This work was supported by the "Wetenschappelijk Fonds Willy Gepts" (WFWG G142 and G170, Universitair Ziekenhuis Brussel). The authors declare no conflict of interest.

The effect of nicotine and cotinine on human gingival fibroblasts attachment to root surfaces.

PubMed

Esfahrood, Zeinab Rezaei; Zamanian, Amirhosein; Torshabi, Maryam; Abrishami, Maryam

2015-09-01

Different compounds of smoking (e.g., nicotine and cotinine) are risk factors for various diseases such as oral cancer and periodontal diseases. Some studies reported the negative effects of nicotine on cell proliferation and differentiation. The present in vitro study assessed the effects of nicotine and cotinine (long-acting metabolite of nicotine) on the attachment and viability of human gingival fibroblast (HGF) cells to tooth root surfaces. A total of 70 teeth specimens were placed into 48-well culture plates and covered with HGF cell suspension, in complete Dulbecco's modified Eagle's medium culture medium containing 1 nM, 1 μm, 1 mM, and 5 mM of nicotine and cotinine concentrations. Cellular attachment and viability measured using an MTT assay and a scanning electron microscope were used for cell morphological evaluation. After 24 h, low (nanomolar and micromolar) and high concentrations (millimolar) of nicotine and cotinine caused a significant reduction in the initial cell adhesion in comparison with the control group, but no significant difference was observed between the nicotine and the cotinine groups (p<0.05). Dentally attached cells with low concentrations of nicotine and cotinine proliferated 48 h after exposure, the same as the control group. However, dentally attached cells with high concentrations of nicotine and cotinine (especially 5 mM) did not proliferate 24 h after exposure (p<0.05). Low concentrations of nicotine and cotinine caused a reduction in the initial cell adhesion. However, no significant adverse effects on the proliferation of attached cells were seen in the longer period. High concentrations of nicotine and cotinine have adverse effects on the cell adhesion and proliferation of HGF cells.

Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER).

PubMed

Feldman, Ross D; Ding, Qingming; Hussain, Yasin; Limbird, Lee E; Pickering, J Geoffrey; Gros, Robert

2016-06-01

Although aldosterone is a known regulator of renal and cardiovascular function, its role as a regulator of cancer growth and spread has not been widely considered. This study tested the hypothesis that aldosterone regulates cancer cell growth/spread via G protein-coupled estrogen receptor (GPER) activation. In vitro in murine renal cortical adenocarcinoma (RENCA) cells, a widely used murine in vitro model for the study of renal cell adenocarcinoma, aldosterone increased RENCA cell proliferation to a maximum of 125 ± 3% of control at a concentration of 10 nM, an effect blocked by the GPER antagonist G15 or by GPER knockdown using short interfering (sh) RNA techniques. Further, aldosterone increased RENCA cell migration to a maximum of 170 ± 20% of control at a concentration of 100 nM, an effect also blocked by G15 or by GPER down-regulation. In vivo, after orthotopic RENCA cell renal transplantation, pulmonary tumor spread was inhibited by pharmacologic blockade of aldosterone effects with spironolactone (percentage of lung occupied by metastasis: control = 68 ± 13, spironolactone = 26 ± 8, P < 0.05) or inhibition of aldosterone synthesis with a high dietary salt diet (percentage of lung: control = 44 ± 6, high salt = 12 ± 3, P < 0.05), without reducing primary tumor size. Additionally, adrenalectomy significantly reduced the extent of pulmonary tumor spread, whereas aldosterone infusion recovered pulmonary metastatic spread toward baseline levels. Finally, inhibition of GPER either with the GPER antagonist G15 or by GPER knockdown comparably inhibited RENCA cell pulmonary metastatic cancer spread. Taken together, these findings provide strong evidence for aldosterone serving a causal role in renal cell cancer regulation via its GPER receptor; thus, antagonism of GPER represents a potential new target for treatment to reduce metastatic spread.-Feldman, R. D., Ding, Q., Hussain, Y., Limbird, L. E., Pickering, J. G., Gros, R. Aldosterone mediates metastatic spread of renal cancer via the G protein-coupled estrogen receptor (GPER). © FASEB.

Serum-free media formulations are cell line-specific and require optimization for microcarrier culture.

PubMed

Tan, Kah Yong; Teo, Kim Leng; Lim, Jessica F Y; Chen, Allen K L; Choolani, Mahesh; Reuveny, Shaul; Chan, Jerry; Oh, Steve Kw

2015-08-01

Mesenchymal stromal cells (MSCs) are being investigated as potential cell therapies for many different indications. Current methods of production rely on traditional monolayer culture on tissue-culture plastic, usually with the use of serum-supplemented growth media. However, the monolayer culturing system has scale-up limitations and may not meet the projected hundreds of billions to trillions batches of cells needed for therapy. Furthermore, serum-free medium offers several advantages over serum-supplemented medium, which may have supply and contaminant issues, leading to many serum-free medium formulations being developed. We cultured seven MSC lines in six different serum-free media and compared their growth between monolayer and microcarrier culture. We show that (i) expansion levels of MSCs in serum-free monolayer cultures may not correlate with expansion in serum-containing media; (ii) optimal culture conditions (serum-free media for monolayer or microcarrier culture) differ for each cell line; (iii) growth in static microcarrier culture does not correlate with growth in stirred spinner culture; (iv) and that early cell attachment and spreading onto microcarriers does not necessarily predict efficiency of cell expansion in agitated microcarrier culture. Current serum-free media developed for monolayer cultures of MSCs may not support MSC proliferation in microcarrier cultures. Further optimization in medium composition will be required for microcarrier suspension culture for each cell line. Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Epitope topography controls bioactivity in supramolecular nanofibers

PubMed Central

Sur, Shantanu; Tantakitti, Faifan; Matson, John B.; Stupp, Samuel I.

2015-01-01

Incorporating bioactivity into artificial scaffolds using peptide epitopes present in the extracellular matrix (ECM) is a well-known approach. A common strategy has involved epitopes that provide cells with attachment points and external cues through interaction with integrin receptors. Although a variety of bioactive sequences have been identified so far, less is known about their optimal display in a scaffold. We report here on the use of self-assembled peptide amphiphile (PA) nanofiber matrices to investigate the impact of spatial presentation of the fibronectin derived epitope RGDS on cell response. Using one, three, or five glycine residues, RGDS epitopes were systematically spaced out from the surface of the rigid nanofibers. We found that cell morphology was strongly affected by the separation of the epitope from the nanofiber surface, with the longest distance yielding the most cell-spreading, bundling of actin filaments, and a round-to-polygonal transformation of cell shape. Cell response to this type of epitope display was also accompanied with activated integrin-mediated signaling and formation of stronger adhesions between cells and substrate. Interestingly, unlike length, changing the molecular flexibility of the linker had minimal influence on cell behavior on the substrate for reasons that remain poorly understood. The use in this study of high persistence length nanofibers rather than common flexible polymers allows us to conclude that epitope topography at the nanoscale structure of a scaffold influences its bioactive properties independent of epitope density and mechanical properties. PMID:25745558

A 2,5-Dihydroxybenzoic Acid–Gelatin Conjugate: The Synthesis, Antiviral Activity and Mechanism of Antiviral Action Against Two Alphaherpesviruses

PubMed Central

Lisov, Alexander; Vrublevskaya, Veronika; Lisova, Zoy; Leontievsky, Alexey; Morenkov, Oleg

2015-01-01

Various natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections. PMID:26501311

Photocrosslinkable biodegradable elastomers based on cinnamate-functionalized polyesters.

PubMed

Zhu, Congcong; Kustra, Stephen R; Bettinger, Christopher J

2013-07-01

Synthetic biodegradable elastomers are an emerging class of materials that play a critical role in supporting innovations in bioabsorbable medical implants. This paper describes the synthesis and characterization of poly(glycerol-co-sebacate)-cinnamate (PGS-CinA), a biodegradable elastomer based on hyperbranched polyesters derivatized with pendant cinnamate groups. PGS-CinA can be prepared via photodimerization in the absence of photoinitiators using monomers that are found in common foods. The resulting network exhibits a Young's modulus of 50.5-152.1kPa and a projected in vitro degradation half-life time between 90 and 140days. PGS-CinA elastomers are intrinsically cell-adherent and support rapid proliferation of fibroblasts. Spreading and proliferation of fibroblasts are loosely governed by the substrate stiffness within the range of Young's moduli in PGS-CinA networks that were prepared. The thermo-mechanical properties, biodegradability and intrinsic support of cell attachment and proliferation suggest that PGS-CinA networks are broadly applicable for use in next generation bioabsorable materials including temporary medical devices and scaffolds for soft tissue engineering. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Role of Nanodiamonds in Drug Delivery and Stem Cell Therapy.

PubMed

Ansari, Shakeel Ahmed; Satar, Rukhsana; Jafri, Mohammad Alam; Rasool, Mahmood; Ahmad, Waseem; Kashif Zaidi, Syed

2016-09-01

The use of nanotechnology in medicine and more specifically drug delivery is set to spread rapidly. Currently many substances are under investigation for drug delivery and more specifically for cancer therapy. Nanodiamonds (NDs) have contributed significantly in the development of highly efficient and successful drug delivery systems, and in stem cell therapy. Drug delivery through NDs is an intricate and complex process that deserves special attention to unravel underlying molecular mechanisms in order to overcome certain bottlenecks associated with it. It has already been established that NDs based drug delivery systems have excellent biocompatibility, nontoxicity, photostability and facile surface functionalization properties. There is mounting evidence that suggests that such conjugated delivery systems well retain the properties of nanoparticles like small size, large surface area to volume ratio that provide greater biocatalytic activity to the attached drug in terms of selectivity, loading and stability. NDs based drug delivery systems may form the basis for the development of effective novel drug delivery vehicles with salient features that may facilitate their utility in fluorescence imaging, target specificity and sustainedrelease.

Preparation and Reinforcement of Dual‐Porous Biocompatible Cellulose Scaffolds for Tissue Engineering

PubMed Central

Pircher, Nicole; Fischhuber, David; Carbajal, Leticia; Strauß, Christine; Nedelec, Jean‐Marie; Kasper, Cornelia; Rosenau, Thomas

2015-01-01

1 Biocompatible cellulose‐based aerogels composed of nanoporous struts, which embed interconnected voids of controlled micron‐size, have been prepared employing temporary templates of fused porogens, reinforcement by interpenetrating PMMA networks and supercritical carbon dioxide drying. Different combinations of cellulose solvent (Ca(SCN)2/H2O/LiCl or [EMIm][OAc]/DMSO) and anti‐solvent (EtOH), porogen type (paraffin wax or PMMA spheres) and porogen size (various fractions in the range of 100–500 μm) as well as intensity of PMMA reinforcement have been investigated to tailor the materials for cell scaffolding applications. All aerogels exhibited an open and dual porosity (micronporosity >100 μm and nanoporosity extending to the low micrometer range). Mechanical properties of the dual‐porous aerogels under compressive stress were considerably improved by introduction of interpenetrating PMMA networks. The effect of the reinforcing polymer on attachment, spreading, and proliferation of NIH 3T3 fibroblast cells, cultivated on selected dual‐porous aerogels to pre‐evaluate their biocompatibility was similarly positive. PMID:26941565

Triple Antibiotic Polymer Nanofibers for Intracanal Drug Delivery: Effects on Dual Species Biofilm and Cell Function.

PubMed

Pankajakshan, Divya; Albuquerque, Maria T P; Evans, Joshua D; Kamocka, Malgorzata M; Gregory, Richard L; Bottino, Marco C

2016-10-01

Root canal disinfection and the establishment of an intracanal microenvironment conducive to the proliferation/differentiation of stem cells play a significant role in regenerative endodontics. This study was designed to (1) investigate the antimicrobial efficacy of triple antibiotic-containing nanofibers against a dual-species biofilm and (2) evaluate the ability of dental pulp stem cells (DPSCs) to adhere to and proliferate on dentin upon nanofiber exposure. Seven-day-old dual-species biofilm established on dentin specimens was exposed for 3 days to the following: saline (control), antibiotic-free nanofibers (control), and triple antibiotic-containing nanofibers or a saturated triple antibiotic paste (TAP) solution (50 mg/mL in phosphate buffer solution). Bacterial viability was assessed using the LIVE/DEAD assay (Molecular Probes, Inc, Eugene, OR) and confocal laser scanning microscopy. For cytocompatibility studies, dentin specimens after nanofiber or TAP (1 g/mL in phosphate buffer solution) exposure were evaluated for cell adhesion and spreading by actin-phalloidin staining. DPSC proliferation was assessed on days 1, 3, and 7. Statistics were performed, and significance was set at the 5% level. Confocal laser scanning microscopy showed significant bacterial death upon antibiotic-containing nanofiber exposure, differing significantly (P < .05) from antibiotic-free fibers and the control (saline). DPSCs showed enhanced adhesion/spreading on dentin specimens treated with antibiotic-containing nanofibers when compared with its TAP counterparts. The DPSC proliferation rate was similar on days 1 and 3 in antibiotic-free nanofibers, triple antibiotic-containing nanofibers, and TAP-treated dentin. Proliferation was higher (9-fold) on dentin treated with antibiotic-containing nanofibers on day 7 compared with TAP. Triple antibiotic-containing polymer nanofibers led to significant bacterial death, whereas they did not affect DPSC attachment and proliferation on dentin. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Generation of airborne Listeria innocua from model floor drains.

PubMed

Berrang, Mark E; Frank, Joseph F

2012-07-01

Listeria monocytogenes can colonize floor drains in poultry processing and further processing facilities, remaining present even after cleaning and disinfection. Therefore, during wash down, workers exercise caution to avoid spraying hoses directly into drains in an effort to prevent the escape and transfer of drain microflora to food contact surfaces. The objective of this study was to examine the extent to which an inadvertent water spray into a colonized floor drain can cause the spread of airborne Listeria. Listeria innocua was used to inoculate a polyvinyl chloride model floor drain, resulting in approximately 10(8) cells per ml of phosphate-buffered saline and 10(4) attached cells per square centimeter of inner surface. Each model drain was subjected to a 2-s spray of tap water at 68.9 kPa from a distance of 1 m. Drains were sprayed while filled and again after emptying. Airborne cells were collected by using sedimentation plates containing Listeria selective agar which were placed on the floor and walls of a contained room at incremental horizontal and vertical distances of 0.6, 1.2, 2.4, or 4.0 m from the drain. Sedimentation plates were exposed for 10 min. A mechanical sampler was used to also collect air by impaction on the surface of Listeria selective agar to determine the number of cells per liter of air. The experiment was conducted in triplicate rooms for each of four replications. L. innocua was detected on sedimentation plates on the floor as far as 4.0 m from the drain and on walls as high as 2.4 m above the floor and 4 m from the drain. A 2-s spray with a water hose into a contaminated drain can cause airborne spread of Listeria, resulting in the potential for cross-contamination of food contact surfaces, equipment, and exposed product.

Heat Transfer to a Thin Solid Combustible in Flame Spreading at Microgravity

NASA Technical Reports Server (NTRS)

Bhattacharjee, S.; Altenkirch, R. A.; Olson, S. L.; Sotos, R. G.

1991-01-01

The heat transfer rate to a thin solid combustible from an attached diffusion flame, spreading across the surface of the combustible in a quiescent, microgravity environment, was determined from measurements made in the drop tower facility at NASA-Lewis Research Center. With first-order Arrhenius pyrolysis kinetics, the solid-phase mass and energy equations along with the measured spread rate and surface temperature profiles were used to calculate the net heat flux to the surface. Results of the measurements are compared to the numerical solution of the complete set of coupled differential equations that describes the temperature, species, and velocity fields in the gas and solid phases. The theory and experiment agree on the major qualitative features of the heat transfer. Some fundamental differences are attributed to the neglect of radiation in the theoretical model.

Dynamics of cell area and force during spreading.

PubMed

Brill-Karniely, Yifat; Nisenholz, Noam; Rajendran, Kavitha; Dang, Quynh; Krishnan, Ramaswamy; Zemel, Assaf

2014-12-16

Experiments on human pulmonary artery endothelial cells are presented to show that cell area and the force exerted on a substrate increase simultaneously, but with different rates during spreading; rapid-force increase systematically occurred several minutes past initial spreading. We examine this theoretically and present three complementary mechanisms that may accompany the development of lamellar stress during spreading and underlie the observed behavior. These include: 1), the dynamics of cytoskeleton assembly at the cell basis; 2), the strengthening of acto-myosin forces in response to the generated lamellar stresses; and 3), the passive strain-stiffening of the cytoskeleton. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Fc-receptor induced cell spreading during frustrated phagocytosis in J774A.1 macrophages

NASA Astrophysics Data System (ADS)

Kovari, Daniel; Curtis, Jennifer; Wei, Wenbin

2014-03-01

Phagocytosis is the process where by cells engulf foreign particles. It is the primary mechanism through which macrophages and neutrophils (white blood cells) eliminate pathogens and debris from the body. The behavior is the result of a cascade of chemical and mechanical cues, which result in the actin-driven expansion of the cell's membrane around its target. For macrophages undergoing Fc-mediated phagocytosis, we show that above a minimum threshold the spreading rate and maximum cell-target contact area are independent of the target's opsonin density. Qualitatively, macrophage phagocytic spreading is similar to the spreading of other cell types (e.g. fibroblasts, lymphocytes, and Dict.d.). Early spreading is most likely the result of ``passive'' alignment of the cell to the target surface. This is followed by an active expansion period driven by actin. Finally upon reaching a maximum contact area, typically 2-3 times the size of ``non-activated'' cells, macrophages often undergo a period of rapid contraction not reported in other cell types. We hypothesize that this, as yet unexplained, transition may be specific to the chemical and mechanical machinery associated with phagocytosis. This work was funded by NSF grant PHYS 0848797 and NSF grant DMR 0820382.

Actin-Related Protein 2 (ARP2) and Virus-Induced Filopodia Facilitate Human Respiratory Syncytial Virus Spread

PubMed Central

McCarty, Thomas; Martin, Scott E.; Le Nouën, Cyril; Buehler, Eugen; Chen, Yu-Chi; Smelkinson, Margery; Ganesan, Sundar; Fischer, Elizabeth R.; Brock, Linda G.; Liang, Bo; Munir, Shirin; Collins, Peter L.; Buchholz, Ursula J.

2016-01-01

Human respiratory syncytial virus (RSV) is an enveloped RNA virus that is the most important viral cause of acute pediatric lower respiratory tract illness worldwide, and lacks a vaccine or effective antiviral drug. The involvement of host factors in the RSV replicative cycle remains poorly characterized. A genome-wide siRNA screen in human lung epithelial A549 cells identified actin-related protein 2 (ARP2) as a host factor involved in RSV infection. ARP2 knockdown did not reduce RSV entry, and did not markedly reduce gene expression during the first 24 hr of infection, but decreased viral gene expression thereafter, an effect that appeared to be due to inhibition of viral spread to neighboring cells. Consistent with reduced spread, there was a 10-fold reduction in the release of infectious progeny virions in ARP2-depleted cells at 72 hr post-infection. In addition, we found that RSV infection induced filopodia formation and increased cell motility in A549 cells and that this phenotype was ARP2 dependent. Filopodia appeared to shuttle RSV to nearby uninfected cells, facilitating virus spread. Expression of the RSV F protein alone from a plasmid or heterologous viral vector in A549 cells induced filopodia, indicating a new role for the RSV F protein, driving filopodia induction and virus spread. Thus, this study identified roles for ARP2 and filopodia in RSV-induced cell motility, RSV production, and RSV cell-to-cell spread. PMID:27926942

RECON-Dependent Inflammation in Hepatocytes Enhances Listeria monocytogenes Cell-to-Cell Spread.

PubMed

McFarland, Adelle P; Burke, Thomas P; Carletti, Alexie A; Glover, Rochelle C; Tabakh, Hannah; Welch, Matthew D; Woodward, Joshua J

2018-05-15

The oxidoreductase RECON is a high-affinity cytosolic sensor of bacterium-derived cyclic dinucleotides (CDNs). CDN binding inhibits RECON's enzymatic activity and subsequently promotes inflammation. In this study, we sought to characterize the effects of RECON on the infection cycle of the intracellular bacterium Listeria monocytogenes , which secretes cyclic di-AMP (c-di-AMP) into the cytosol of infected host cells. Here, we report that during infection of RECON-deficient hepatocytes, which exhibit hyperinflammatory responses, L. monocytogenes exhibits significantly enhanced cell-to-cell spread. Enhanced bacterial spread could not be attributed to alterations in PrfA or ActA, two virulence factors critical for intracellular motility and intercellular spread. Detailed microscopic analyses revealed that in the absence of RECON, L. monocytogenes actin tail lengths were significantly longer and there was a larger number of faster-moving bacteria. Complementation experiments demonstrated that the effects of RECON on L. monocytogenes spread and actin tail lengths were linked to its enzymatic activity. RECON enzyme activity suppresses NF-κB activation and is inhibited by c-di-AMP. Consistent with these previous findings, we found that augmented NF-κB activation in the absence of RECON caused enhanced L. monocytogenes cell-to-cell spread and that L. monocytogenes spread correlated with c-di-AMP secretion. Finally, we discovered that, remarkably, increased NF-κB-dependent inducible nitric oxide synthase expression and nitric oxide production were responsible for promoting L. monocytogenes cell-to-cell spread. The work presented here supports a model whereby L. monocytogenes secretion of c-di-AMP inhibits RECON's enzymatic activity, drives augmented NF-κB activation and nitric oxide production, and ultimately enhances intercellular spread. IMPORTANCE To date, bacterial CDNs in eukaryotes are solely appreciated for their capacity to activate cytosolic sensing pathways in innate immunity. However, it remains unclear whether pathogens that actively secrete CDNs benefit from this process. Here, we provide evidence that secretion of CDNs leads to enhancement of L. monocytogenes cell-to-cell spread. This is a heretofore-unknown role of these molecules and suggests L. monocytogenes may benefit from their secretion in certain contexts. Molecular characterization revealed that, surprisingly, nitric oxide was responsible for the enhanced spread. Pathogens act to prevent nitric oxide production or, like L. monocytogenes , they have evolved to resist its direct antimicrobial effects. This study provides evidence that intracellular bacterial pathogens not only tolerate nitric oxide, which is inevitably encountered during infection, but can also capitalize on the changes this pleiotropic molecule enacts on the host cell. Copyright © 2018 McFarland et al.

Physical impaction injury effects on bacterial cells during spread plating influenced by cell characteristics of the organisms.

PubMed

Thomas, P; Mujawar, M M; Sekhar, A C; Upreti, R

2014-04-01

To understand the factors that contribute to the variations in colony-forming units (CFU) in different bacteria during spread plating. Employing a mix culture of vegetative cells of ten organisms varying in cell characteristics (Gram reaction, cell shape and cell size), spread plating to the extent of just drying the agar surface (50-60 s) was tested in comparison with the alternate spotting-and-tilt-spreading (SATS) approach where 100 μl inoculum was distributed by mere tilting of plate after spotting as 20-25 microdrops. The former imparted a significant reduction in CFU by 20% over the spreader-independent SATS approach. Extending the testing to single organisms, Gram-negative proteobacteria with relatively larger cells (Escherichia, Enterobacter, Agrobacterium, Ralstonia, Pantoea, Pseudomonas and Sphingomonas spp.) showed significant CFU reduction with spread plating except for slow-growing Methylobacterium sp., while those with small rods (Xenophilus sp.) and cocci (Acinetobacter sp.) were less affected. Among Gram-positive nonspore formers, Staphylococcus epidermidis showed significant CFU reduction while Staphylococcus haemolyticus and actinobacteria (Microbacterium, Cellulosimicrobium and Brachybacterium spp.) with small rods/cocci were unaffected. Vegetative cells of Bacillus pumilus and B. subtilis were generally unaffected while others with larger rods (B. thuringiensis, Brevibacillus, Lysinibacillus and Paenibacillus spp.) were significantly affected. A simulated plating study coupled with live-dead bacterial staining endorsed the chances of cell disruption with spreader impaction in afflicted organisms. Significant reduction in CFU could occur during spread plating due to physical impaction injury to bacterial cells depending on the spreader usage and the variable effects on different organisms are determined by Gram reaction, cell size and cell shape. The inoculum spreader could impart physical disruption of vegetative cells against a hard surface. Possibility of CFU reduction in sensitive organisms and the skewed selection of hardier organisms during spread plating, and the recommendation of SATS as an easier and safer alternative for CFU enumerations. © 2013 The Society for Applied Microbiology.

Minimum line width of ion beam-modified polystyrene by negative carbon ions for nerve-cell attachment and neurite extension

NASA Astrophysics Data System (ADS)

Sommani, P.; Tsuji, H.; Sato, H.; Kitamura, T.; Hattori, M.; Gotoh, Y.; Ishikawa, J.

2007-04-01

The minimum line width of the negative-ion-modified polystyrene (PS) for guidance and immobilizations of nerve-cell body and neurite extension have been investigated. Carbon negative ions were implanted into PS at fluence of 3 × 1015 ions/cm2 and energy of 5-20 keV through the various triangle apertures of the micro-pattern mask. After in vitro culture of the nerve-like cells of rat adrenal pheochromocytoma (PC12h), results showed that the minimum line widths for a single cell attachment and for neurite extension were 5-7 and 3-5 μm, respectively. While the minimum line width for attachment of cell group with long neurite was about 20 μm. The suitable widths for a large number of cells and for neurite extension were 20 and 5 μm, respectively. Therefore, the guidance for a clear separation of the attachment size of cell body and neurite extension could be achieved by the different modified line widths.

Fluorescence lifetime microscopy for monitoring cell adhesion using metal induced energy transfer

NASA Astrophysics Data System (ADS)

Hwang, Wonsang; Seo, JinWon; Song, Jun ho; Kim, DongEun; Won, YoungJae; Choi, In-Hong; Yoo, Kyung-Hwa; Kim, Dug Young

2018-02-01

A precise control and a reliable monitoring tool for the adhesion properties of a cell are very important in atherosclerosis studies. If endothelial cells in contact with the intracellular membrane are not attached securely, low-density lipoprotein (LDL) particles can enter into the inner membrane. It is therefore necessary to measure conditions under which endothelial cell detachment occurs. When a cell is attached to a metal thin film, the lifetime of a fluorescence probe attached to the membrane of the cell is reduced by the metal-induced energy transfer (MIET). Fluorescence lifetime imaging microscopy (FLIM) is used to monitor the attachment condition of a cell to a metal surface using FRET. However, this requires high numerical aperture (NA) objective lens because axial confocal resolution must be smaller than the cell thickness. This requirement limits the field of view of the measurement specimen. In this study we provides a new method which can measure adhesion properties of endothelial cells even with a low NA objective lens by resolving two lifetime components in FLIM.

Cell-to-Cell Transmission Can Overcome Multiple Donor and Target Cell Barriers Imposed on Cell-Free HIV

PubMed Central

Ilinskaya, Anna; Dorjbal, Batsukh; Truong, Rosaline; Derse, David; Uchil, Pradeep D.; Heidecker, Gisela; Mothes, Walther

2013-01-01

Virus transmission can occur either by a cell-free mode through the extracellular space or by cell-to-cell transmission involving direct cell-to-cell contact. The factors that determine whether a virus spreads by either pathway are poorly understood. Here, we assessed the relative contribution of cell-free and cell-to-cell transmission to the spreading of the human immunodeficiency virus (HIV). We demonstrate that HIV can spread by a cell-free pathway if all the steps of the viral replication cycle are efficiently supported in highly permissive cells. However, when the cell-free path was systematically hindered at various steps, HIV transmission became contact-dependent. Cell-to-cell transmission overcame barriers introduced in the donor cell at the level of gene expression and surface retention by the restriction factor tetherin. Moreover, neutralizing antibodies that efficiently inhibit cell-free HIV were less effective against cell-to-cell transmitted virus. HIV cell-to-cell transmission also efficiently infected target T cells that were relatively poorly susceptible to cell-free HIV. Importantly, we demonstrate that the donor and target cell types influence critically the extent by which cell-to-cell transmission can overcome each barrier. Mechanistically, cell-to-cell transmission promoted HIV spread to more cells and infected target cells with a higher proviral content than observed for cell-free virus. Our data demonstrate that the frequently observed contact-dependent spread of HIV is the result of specific features in donor and target cell types, thus offering an explanation for conflicting reports on the extent of cell-to-cell transmission of HIV. PMID:23308151

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

PubMed Central

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

2013-01-01

Background: Root surface debridement (RSD) is necessary to create an environment suitable for reattachment of the periodontium. Root surface conditioning may aid the formation of a biocompatible surface suitable for cell reattachment. BioPure™ MTAD (mixture of Doxycycline, citric acid and a detergent) is an endodontic irrigant with antibacterial properties and the ability to remove smear layer. It was hypothesized that MTAD may be useful for root surface conditioning. The efficacy of MTAD as a conditioner was measured by examining fibroblast attachment to root surfaces. Materials and Methods: Thirty-two specimens of human teeth with advanced periodontal disease were used. The surfaces were root planed until smooth. Half of the specimens were treated with 0.9% saline and the other samples with Biopure MTAD. As a negative control group, five further samples were left unscaled with surface calculus. Human gingival fibroblast cells HGF1-PI1 were cultured and poured over the tooth specimens and incubated. After fixation, the samples were sputter-coated with gold and examined with a SEM. The morphology and number of attached, fixed viable cells were examined. The data was analysed using the Mann-Whitney-U statistical test. Results: There was no significant difference between the numbers of attached cells in the experimental group treated with MTAD and the control group treated with saline. Little or no attached cells were seen in the negative control group. Conclusion: RSD created an environment suitable for cell growth and attachment in a laboratory setting. The use of MTAD did not promote the attachment and growth of cells on the surface of human roots following RSD. PMID:23869124

Establishment of highly metastatic KRAS mutant lung cancer cell sublines in long-term three-dimensional low attachment cultures

PubMed Central

Nakano, Tomoyuki; Kanai, Yoshihiko; Amano, Yusuke; Yoshimoto, Taichiro; Matsubara, Daisuke; Shibano, Tomoki; Tamura, Tomoko; Oguni, Sachiko; Katashiba, Shizuka; Ito, Takeshi; Murakami, Yoshinori; Fukayama, Masashi; Murakami, Takashi; Endo, Shunsuke; Niki, Toshiro

2017-01-01

Decreased cell-substratum adhesion is crucially involved in metastasis. Previous studies demonstrated that lung cancer with floating cell clusters in histology is more likely to develop metastasis. In the present study, we investigated whether cancer cells in long-term, three-dimensional low attachment cultures acquire high metastatic potential; these cells were then used to examine the mechanisms underlying metastasis. Two KRAS-mutated adenocarcinoma cell lines (A549 and H441) were cultured and selected on ultra-low attachment culture dishes, and the resulting cells were defined as FL (for floating) sublines. Cancer cells were inoculated into NOD/SCID mice via an intracardiac injection, and metastasis was evaluated using luciferase-based imaging and histopathology. In vitro cell growth (in attachment or suspension cultures), migration, and invasion were assayed. A whole genomic analysis was performed to identify key molecular alterations in FL sublines. Upon detachment on low-binding dishes, parental cells initially formed rounded spheroids with limited growth activity. However, over time in cultures, cells gradually formed smaller spheroids that grew slowly, and, after 3–4 months, we obtained FL sublines that regained prominent growth potential in suspension cultures. On ordinary dishes, FL cells reattached and exhibited a more spindle-shaped morphology than parental cells. No marked differences were observed in cell growth with attachment, migration, or invasion between FL sublines and parental cell lines; however, FL cells exhibited markedly increased growth potential under suspended conditions in vitro and stronger metastatic abilities in vivo. A genomic analysis identified epithelial-mesenchymal transition (EMT) and c-Myc amplification in A549-FL and H441-FL cells, respectively, as candidate mechanisms for metastasis. The growth potential of FL cells was markedly inhibited by lentiviral ZEB1 knockdown in A549-FL cells and by the inhibition of c-Myc through lentiviral knockdown or the pharmacological inhibitor JQ1 in H441-FL cells. Long-term three-dimensional low attachment cultures may become a useful method for investigating the mechanisms underlying metastasis mediated by decreased cell-substratum adhesion. PMID:28786996

Reduction of Fe(III) colloids by Shewanella putrefaciens: A kinetic model

NASA Astrophysics Data System (ADS)

Bonneville, Steeve; Behrends, Thilo; van Cappellen, Philippe; Hyacinthe, Christelle; Röling, Wilfred F. M.

2006-12-01

A kinetic model for the microbial reduction of Fe(III) oxyhydroxide colloids in the presence of excess electron donor is presented. The model assumes a two-step mechanism: (1) attachment of Fe(III) colloids to the cell surface and (2) reduction of Fe(III) centers at the surface of attached colloids. The validity of the model is tested using Shewanella putrefaciens and nanohematite as model dissimilatory iron reducing bacteria and Fe(III) colloidal particles, respectively. Attachment of nanohematite to the bacteria is formally described by a Langmuir isotherm. Initial iron reduction rates are shown to correlate linearly with the relative coverage of the cell surface by nanohematite particles, hence supporting a direct electron transfer from membrane-bound reductases to mineral particles attached to the cells. Using internally consistent parameter values for the maximum attachment capacity of Fe(III) colloids to the cells, Mmax, the attachment constant, KP, and the first-order Fe(III) reduction rate constant, k, the model reproduces the initial reduction rates of a variety of fine-grained Fe(III) oxyhydroxides by S. putrefaciens. The model explains the observed dependency of the apparent Fe(III) half-saturation constant, Km∗, on the solid to cell ratio, and it predicts that initial iron reduction rates exhibit saturation with respect to both the cell density and the abundance of the Fe(III) oxyhydroxide substrate.

Tetrahydrocannabinol-induced suppression of macrophage spreading and phagocytic activity in vitro.

PubMed

Lopez-Cepero, M; Friedman, M; Klein, T; Friedman, H

1986-06-01

The effects of tetrahydrocannabinol (THC) on several parameters of macrophage function in vitro were assessed. Delta 9 THC added to cultures of normal mouse peritoneal cells in vitro affected the ability of the cells to spread on glass surfaces and also had some effect on their ability to phagocytize yeast. These effects were dose related. A concentration of 20 micrograms of THC almost completely inhibited macrophage spreading, but it also decreased viability and the total number of these cells. Doses of 10 or 5 micrograms of THC also inhibited spreading but had little effect on cell viability or number. A dose of 1.0 microgram of THC had some inhibitory effect on spreading and the lowest dose affecting spreading appeared to be about 0.05 micrograms per culture. Higher doses of THC were necessary to inhibit phagocytosis of yeast particles as determined by direct microscopic examination or use of radiolabeled yeast as the test particles. These results indicate that several readily measured functions of macrophages may be suppressed by THC.

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings

PubMed Central

Vilardell, A. M.; Cinca, N.; Jokinen, A.; Garcia-Giralt, N.; Dosta, S.; Cano, I. G.; Guilemany, J. M.

2016-01-01

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule–molecule interactions but also molecule–material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 μm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time. PMID:27618911

The development of peptide-based interfacial biomaterials for generating biological functionality on the surface of bioinert materials

PubMed Central

Meyers, Steven R.; Khoo, Xiaojuan; Huang, Xin; Walsh, Elisabeth B.; Grinstaff, Mark W.; Kenan, Daniel J.

2013-01-01

Biomaterials used in implants have traditionally been selected based on their mechanical properties, chemical stability, and biocompatibility. However, the durability and clinical efficacy of implantable biomedical devices remains limited in part due to the absence of appropriate biological interactions at the implant interface and the lack of integration into adjacent tissues. Herein, we describe a robust peptide-based coating technology capable of modifying the surface of existing biomaterials and medical devices through the non-covalent binding of modular biofunctional peptides. These peptides contain at least one material binding sequence and at least one biologically active sequence and thus are termed, “Interfacial Biomaterials” (IFBMs). IFBMs can simultaneously bind the biomaterial surface while endowing it with desired biological functionalities at the interface between the material and biological realms. We demonstrate the capabilities of model IFBMs to convert native polystyrene, a bioinert surface, into a bioactive surface that can support a range of cell activities. We further distinguish between simple cell attachment with insufficient integrin interactions, which in some cases can adversely impact downstream biology, versus biologically appropriate adhesion, cell spreading, and cell survival mediated by IFBMs. Moreover, we show that we can use the coating technology to create spatially resolved patterns of fluorophores and cells on substrates and that these patterns retain their borders in culture. PMID:18929406

The role of particle-to-cell interactions in dictating nanoparticle aided magnetophoretic separation of microalgal cells.

PubMed

Toh, Pey Yi; Ng, Bee Wah; Ahmad, Abdul Latif; Chieh, Derek Chan Juinn; Lim, JitKang

2014-11-07

Successful application of a magnetophoretic separation technique for harvesting biological cells often relies on the need to tag the cells with magnetic nanoparticles. This study investigates the underlying principle behind the attachment of iron oxide nanoparticles (IONPs) onto microalgal cells, Chlorella sp. and Nannochloropsis sp., in both freshwater and seawater, by taking into account the contributions of various colloidal forces involved. The complex interplay between van der Waals (vdW), electrostatic (ES) and Lewis acid-base interactions (AB) in dictating IONP attachment was studied under the framework of extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) analysis. Our results showed that ES interaction plays an important role in determining the net interaction between the Chlorella sp. cells and IONPs in freshwater, while the AB and vdW interactions play a more dominant role in dictating the net particle-to-cell interaction in high ionic strength media (≥100 mM NaCl), such as seawater. XDLVO predicted effective attachment between cells and surface functionalized IONPs (SF-IONPs) with an estimated secondary minimum of -3.12 kT in freshwater. This prediction is in accordance with the experimental observation in which 98.89% of cells can be magnetophoretically separated from freshwater with SF-IONPs. We have observed successful magnetophoretic separation of microalgal cells from freshwater and/or seawater for all the cases as long as XDLVO analysis predicts particle attachment. For both the conditions, no pH adjustment is required for particle-to-cell attachment.

Mechanics of membrane-cytoskeleton attachment in Paramecium

NASA Astrophysics Data System (ADS)

Campillo, C.; Jerber, J.; Fisch, C.; Simoes-Betbeder, M.; Dupuis-Williams, P.; Nassoy, P.; Sykes, C.

2012-12-01

In this paper we assess the role of the protein MKS1 (Meckel syndrome type 1) in the cortical membrane mechanics of the ciliated protist Paramecium. This protein is known to be crucial in the process of cilium formation, and we investigate its putative role in membrane-cytoskeleton attachment. Therefore, we compare cells where the gene coding for MKS1 is silenced to wild-type cells. We found that scanning electron microscopy observation of the cell surface reveals a cup-like structure in wild-type cells that is lost in silenced cells. Since this structure is based on the underlying cytoskeleton, one hypothesis to explain this observation is a disruption of membrane attachment to the cytoskeleton in the absence of MKS1 that should affect plasma membrane mechanics. We test this by probing the mechanics of wild-type and silenced cells by micropipette aspiration. Strikingly, we observe that, at the same aspiration pressure, the membrane of silenced cells is easily aspirated by the micropipette whereas that of wild-type cells enters only at a moderate velocity, an effect that suggests a detachment of the membrane from the underlying cytoskeleton in silenced cells. We quantify this detachment by measuring the deformation of the cell cortex and the rate of cell membrane entry in the micropipette. This study offers a new perspective for the characterization of membrane-cytoskeleton attachment in protists and paves the way for a better understanding of the role of membrane-cortex attachment in cilium formation.

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

PubMed

Belal, Mahmoud Helmy; Watanabe, Hisashi

2014-10-01

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

Attached and planktonic Listeria monocytogenes global proteomic responses and associated influence of strain genetics and temperature.

PubMed

Mata, Marcia M; da Silva, Wladimir P; Wilson, Richard; Lowe, Edwin; Bowman, John P

2015-02-06

Contamination of industrial and domestic food usage environments by the attachement of bacterial food-borne pathogen Listeria monocytogenes has public health and economic implications. Comprehensive proteomics experiments using label-free liquid chromatography/tandem mass spectrometry were used to compare the proteomes of two different L. monocytogenes strains (Siliken_1/2c and F2365_4b), which show very different capacities to attach to surfaces. Growth temperature and strain type were highly influential on the proteomes in both attached and planktonic cells. On the basis of the proteomic data, it is highly unlikely that specific surface proteins play a direct role in adherence to inanimate surfaces. Instead, strain-dependent responses related to cell envelope polymer biosynthesis and stress response regulation likely contribute to a different ability to attach and also to survive external stressors. Collectively, the divergent proteome-level responses observed define strain- and growth-temperature-dependent differences relevant to attachment efficacy, highlight relevant proteins involved in stress protection in attached cells, and suggest that strain differences and growth conditions are important in relation to environmental persistence.

New models of hematogenous ovarian cancer metastasis demonstrate preferential spread to the ovary and a requirement for the ovary for abdominal dissemination.

PubMed

Coffman, Lan G; Burgos-Ojeda, Daniela; Wu, Rong; Cho, Kathleen; Bai, Shoumei; Buckanovich, Ronald J

2016-09-01

Emerging evidence suggest that many high-grade serous "ovarian" cancers (HGSOC) start in the fallopian tube. Cancer cells are then recruited to the ovary and then spread diffusely through the abdomen. The mechanism of ovarian cancer spread was thought to be largely due to direct shedding of tumor cells into the peritoneal cavity with vascular spread being of limited importance. Recent work challenges this dogma, suggesting hematogenous spread of ovarian cancer may play a larger role in ovarian cancer cell metastasis than previously thought. One reason the role of vascular spread of ovarian cancer has not been fully elucidated is the lack of easily accessible models of vascular ovarian cancer metastasis. Here, we present 3 metastatic models of ovarian cancer which confirm the ability of ovarian cancer to hematogenously spread. Strikingly, we observe a high rate of metastasis to the ovary with the development of ascites in these models. Interestingly, oophorectomy resulted in a complete loss of peritoneal metastases and ascites. Taken together, our data indicate that hematogenously disseminated HGSOC cells have a unique tropism for the ovary and that hematogenous spread in ovarian cancer may be more common than appreciated. Furthermore, our studies support a critical role for the ovary in promoting HGSOC cell metastasis to the abdomen. The models developed here represent important new tools to evaluate both the mechanism of cancer cell recruitment to the ovary and understand and target key steps in ovarian cancer metastasis. Copyright © 2016 Elsevier Inc. All rights reserved.

New models of hematogenous ovarian cancer metastasis demonstrate preferential spread to the ovary and a requirement for the ovary for abdominal dissemination

PubMed Central

Coffman, Lan G; Burgos-Ojeda, Daniela; Wu, Rong; Cho, Kathleen; Bai, Shoumei; Buckanovich, Ronald J

2016-01-01

Emerging evidence suggest that many high grade serous ‘ovarian’ cancers (HGSOC) start in the fallopian tube. Cancer cells are then recruited to the ovary and then spread diffusely through the abdomen. The mechanism of ovarian cancer spread was thought to be largely due to direct shedding of tumor cells into the peritoneal cavity with vascular spread being of limited importance. Recent work challenges this dogma, suggesting hematogenous spread of ovarian cancer may play a larger role in ovarian cancer cell metastasis than previously thought. One reason the role of vascular spread of ovarian cancer has not been fully elucidated is the lack of easily accessible models of vascular ovarian cancer metastasis. Here we present three metastatic models of ovarian cancer which confirm the ability of ovarian cancer to hematogenously spread. Strikingly, we observe a high rate of metastasis to the ovary with the development of ascites in these models. Interestingly, oophorectomy resulted in a complete loss of peritoneal metastases and ascites. Taken together our data indicates that hematogenously disseminated HGSOC cells have a unique tropism for the ovary and that hematogenous spread in ovarian cancer may be more common than appreciated. Furthermore our studies support a critical role for the ovary in promoting HGSOC cell metastasis to the abdomen. The models developed here represent important new tools to evaluate both the mechanism of cancer cell recruitment to the ovary and to understand and target key steps in ovarian cancer metastasis. PMID:27083386

A new method using insert-based systems (IBS) to improve cell behavior study on flexible and rigid biomaterials.

PubMed

Grenade, Charlotte; Moniotte, Nicolas; Rompen, Eric; Vanheusden, Alain; Mainjot, Amélie; De Pauw-Gillet, Marie-Claire

2016-12-01

In vitro studies about biomaterials biological properties are essential screening tests. Yet cell cultures encounter difficulties related to cell retention on material surface or to the observation of both faces of permeable materials. The objective of the present study was to develop a reliable in vitro method to study cell behavior on rigid and flexible/permeable biomaterials elaborating two specific insert-based systems (IBS-R and IBS-F respectively). IBS-R was designed as a specific cylindrical polytetrafluoroethylene (PTFE) system to evaluate attachment, proliferation and morphology of human gingival fibroblasts (HGFs) on grade V titanium and lithium disilicate glass-ceramic discs characteristics of dental prostheses. The number of cells, their covering on discs and their morphology were determined from MTS assays and microscopic fluorescent images after 24, 48 and 72 h. IBS-F was developed as a two components system to study HGFs behavior on guided bone regeneration polyester membranes. The viability and the membrane barrier effect were evaluated by metabolic MTS assays and by scanning electron microscopy. IBS-R and IBS-F were shown to promote (1) easy and rapid handling; (2) cell retention on biomaterial surface; (3) accurate evaluation of the cellular proliferation, spreading and viability; (4) use of non-toxic material. Moreover IBS-F allowed the study of the cell migration through degradable membranes, with an access to both faces of the biomaterial and to the bottom of culture wells for medium changing.

In vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate.

PubMed

Beloti, Márcio M; de Oliveira, Paulo T; Gimenes, Rossano; Zaghete, Maria A; Bertolini, Márcio J; Rosa, Adalberto L

2006-11-01

This study was aimed at investigating the in vitro biocompatibility of a novel membrane of the composite poly(vinylidene-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT). Osteoblastic cells were obtained from human alveolar bone fragments and cultured under standard osteogenic condition until subconfluence. First passaged cells were cultured on P(VDF-TrFE)/BT and expanded polytetrafluoroethylene (e-PTFE--control) membranes in 24-well plates. Cell adhesion and spreading were evaluated at 30 min, and 4 and 24 h. For proliferation assay, cells were cultured for 1, 7, and 10 days. Cell viability was detected by trypan blue at 7 and 10 days. Total protein content and alkaline phosphatase (ALP) activity were measured at 7, 14, and 21 days. Cultures were stained with Alizarin red at 21 days, for detection of mineralized matrix. Data were compared by ANOVA and Student t test. Cell attachment (p = 0.001), cell number (p = 0.001), and ALP activity (p = 0.0001) were greater on P(VDF-TrFE)/BT. Additionally, doubling time was greater on P(VDF-TrFE)/BT (p = 0.03), indicating a decreased proliferation rate. Bone-like nodule formation took place only on P(VDF-TrFE)/BT. The present results showed that both membranes are biocompatible. However, P(VDF-TrFE)/BT presented a better in vitro biocompatibility and allowed bone-like nodule formation. Therefore, P(VDF-TrFE)/BT could be an alternative membrane to be used in guided tissue regeneration.

Behavioural properties of chick somitic mesoderm and lateral plate when explanted in vitro.

PubMed

Bellairs, R; Sanders, E J; Portch, P A

1980-04-01

Tissue culture, time-lapse cinematographic and electron microscopic techniques have been used to study the properties of chick mesoderm at several stages of differentiation. Lateral plate, unsegmented mesoderm (segmental plate), and newly formed somites were dissected from stage-12 embryos, whilst dermo-myotomes and sclerotomes were dissected from stage-18 embryos. Each type of mesoderm was found to exhibit a characteristic pattern of behaviour. The explants from the unsegmented mesoderm from the newly formed somites and from the older embryos could be placed in a developmental sequence; with increasing differentiation they settled and spread on the substrate more readily, whether explanted as pieces of tissue or as individual cells, and it was concluded that this implied an increased adhesion to the substrate. Similarly, with increasing differentiation, the cells segmented at a faster rate. No significant differences could be discerned in the internal structure of the different types of cells, although differences in the general shape were apparent. The lateral plate mesoderm cells, which bear some resemblances to the unsegmented mesoderm cells in the embryo, also show some morphological resemblances to them in vitro. However, the lateral plate cells had a much greater success in attaching to glass or platic substrates. They were also found to have the highest speed of locomotion of all the tissues studied, whereas the unsegmented had the lowest. It is concluded therefore, that although cells may look similar to one another morphologically, their behaviour may differ greatly, probably because they are already partially determined.

Contact enhancement of locomotion in spreading cell colonies

NASA Astrophysics Data System (ADS)

D'Alessandro, Joseph; Solon, Alexandre P.; Hayakawa, Yoshinori; Anjard, Christophe; Detcheverry, François; Rieu, Jean-Paul; Rivière, Charlotte

2017-10-01

The dispersal of cells from an initially constrained location is a crucial aspect of many physiological phenomena, ranging from morphogenesis to tumour spreading. In such processes, cell-cell interactions may deeply alter the motion of single cells, and in turn the collective dynamics. While contact phenomena like contact inhibition of locomotion are known to come into play at high densities, here we focus on the little explored case of non-cohesive cells at moderate densities. We fully characterize the spreading of micropatterned colonies of Dictyostelium discoideum cells from the complete set of individual trajectories. From data analysis and simulation of an elementary model, we demonstrate that contact interactions act to speed up the early population spreading by promoting individual cells to a state of higher persistence, which constitutes an as-yet unreported contact enhancement of locomotion. Our findings also suggest that the current modelling paradigm of memoryless active particles may need to be extended to account for the history-dependent internal state of motile cells.

Multipolar Spindle Pole Coalescence Is a Major Source of Kinetochore Mis-Attachment and Chromosome Mis-Segregation in Cancer Cells

PubMed Central

Silkworth, William T.; Nardi, Isaac K.; Scholl, Lindsey M.; Cimini, Daniela

2009-01-01

Many cancer cells display a CIN (Chromosome Instability) phenotype, by which they exhibit high rates of chromosome loss or gain at each cell cycle. Over the years, a number of different mechanisms, including mitotic spindle multipolarity, cytokinesis failure, and merotelic kinetochore orientation, have been proposed as causes of CIN. However, a comprehensive theory of how CIN is perpetuated is still lacking. We used CIN colorectal cancer cells as a model system to investigate the possible cellular mechanism(s) underlying CIN. We found that CIN cells frequently assembled multipolar spindles in early mitosis. However, multipolar anaphase cells were very rare, and live-cell experiments showed that almost all CIN cells divided in a bipolar fashion. Moreover, fixed-cell analysis showed high frequencies of merotelically attached lagging chromosomes in bipolar anaphase CIN cells, and higher frequencies of merotelic attachments in multipolar vs. bipolar prometaphases. Finally, we found that multipolar CIN prometaphases typically possessed γ-tubulin at all spindle poles, and that a significant fraction of bipolar metaphase/early anaphase CIN cells possessed more than one centrosome at a single spindle pole. Taken together, our data suggest a model by which merotelic kinetochore attachments can easily be established in multipolar prometaphases. Most of these multipolar prometaphase cells would then bi-polarize before anaphase onset, and the residual merotelic attachments would produce chromosome mis-segregation due to anaphase lagging chromosomes. We propose this spindle pole coalescence mechanism as a major contributor to chromosome instability in cancer cells. PMID:19668340

Selective Infection of Antigen-Specific B Lymphocytes by Salmonella Mediates Bacterial Survival and Systemic Spreading of Infection

PubMed Central

de Wit, Jelle; Martinoli, Chiara; Zagato, Elena; Janssen, Hans; Jorritsma, Tineke; Bar-Ephraïm, Yotam E.; Rescigno, Maria; Neefjes, Jacques; van Ham, S. Marieke

2012-01-01

Background The bacterial pathogen Salmonella causes worldwide disease. A major route of intestinal entry involves M cells, providing access to B cell-rich Peyer’s Patches. Primary human B cells phagocytose Salmonella typhimurium upon recognition by the specific surface Ig receptor (BCR). As it is unclear how Salmonella disseminates systemically, we studied whether Salmonella can use B cells as a transport device for spreading. Methodology/Principal Findings Human primary B cells or Ramos cell line were incubated with GFP-expressing Salmonella. Intracellular survival and escape was studied in vitro by live cell imaging, flow cytometry and flow imaging. HEL-specific B cells were transferred into C57BL/6 mice and HEL-expressing Salmonella spreading in vivo was analyzed investigating mesenteric lymph nodes, spleen and blood. After phagocytosis by B cells, Salmonella survives intracellularly in a non-replicative state which is actively maintained by the B cell. Salmonella is later excreted followed by reproductive infection of other cell types. Salmonella-specific B cells thus act both as a survival niche and a reservoir for reinfection. Adoptive transfer of antigen-specific B cells before oral infection of mice showed that these B cells mediate in vivo systemic spreading of Salmonella to spleen and blood. Conclusions/Significance This is a first example of a pathogenic bacterium that abuses the antigen-specific cells of the adaptive immune system for systemic spreading for dissemination of infection. PMID:23209805

Bacterial spread from cell to cell: beyond actin-based motility.

PubMed

Kuehl, Carole J; Dragoi, Ana-Maria; Talman, Arthur; Agaisse, Hervé

2015-09-01

Several intracellular pathogens display the ability to propagate within host tissues by displaying actin-based motility in the cytosol of infected cells. As motile bacteria reach cell-cell contacts they form plasma membrane protrusions that project into adjacent cells and resolve into vacuoles from which the pathogen escapes, thereby achieving spread from cell to cell. Seminal studies have defined the bacterial and cellular factors that support actin-based motility. By contrast, the mechanisms supporting the formation of protrusions and their resolution into vacuoles have remained elusive. Here, we review recent advances in the field showing that Listeria monocytogenes and Shigella flexneri have evolved pathogen-specific mechanisms of bacterial spread from cell to cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

Morphoregulatory functions of the RNA-binding motif protein 3 in cell spreading, polarity and migration.

PubMed

Pilotte, J; Kiosses, W; Chan, S W; Makarenkova, H P; Dupont-Versteegden, E; Vanderklish, P W

2018-05-09

RNA-binding proteins are emerging as key regulators of transitions in cell morphology. The RNA-binding motif protein 3 (RBM3) is a cold-inducible RNA-binding protein with broadly relevant roles in cellular protection, and putative functions in cancer and development. Several findings suggest that RBM3 has morphoregulatory functions germane to its roles in these contexts. For example, RBM3 helps maintain the morphological integrity of cell protrusions during cell stress and disease. Moreover, it is highly expressed in migrating neurons of the developing brain and in cancer invadopodia, suggesting roles in migration. We here show that RBM3 regulates cell polarity, spreading and migration. RBM3 was present in spreading initiation centers, filopodia and blebs that formed during cell spreading in cell lines and primary myoblasts. Reducing RBM3 triggered exaggerated spreading, increased RhoA expression, and a loss of polarity that was rescued by Rho kinase inhibition and overexpression of CRMP2. High RBM3 expression enhanced the motility of cells migrating by a mesenchymal mode involving extension of long protrusions, whereas RBM3 knockdown slowed migration, greatly reducing the ability of cells to extend protrusions and impairing multiple processes that require directional migration. These data establish novel functions of RBM3 of potential significance to tissue repair, metastasis and development.

Fundamentals in Microalgae Harvesting: From Flocculation to Self-attachment

NASA Astrophysics Data System (ADS)

Cui, Yan

Microalgae are a very promising source of biodiesel and other renewable energy due to their fast grow rates, high lipid contents and tremendous potential for water conservation and CO2 biofixation. However, a bottleneck issue with algae biofuel manufacturing is the lack of cost-effective harvesting methods. This research focuses on the technologies for improved microalgae harvesting to enable commercially viable and environmentally friendly biodiesel production. The first objective of this study was to optimize flocculation of marine microalga Nannochloropsis oculata with metal salts, aluminum sulfate (A.S.) and ferric chloride (F.C.) via response surface methodology. It was found that there existed a positive stoichiometric relationship between the flocculant dose (FD) and the initial biomass concentration (IABC). Optimum flocculation conditions were predicted at IABC of 1.7 g/l, pH 8.3, and FD of 383.5 microM for A.S., and IABC of 2.2 g/l, pH 7.9, and FD of 438.1 microM for F.C., under which the predicted maximum harvested solid concentration of algae were 32.98 and 30.10 g/l by using A.S. and F.C., respectively. The second objective was to investigate the mechanism of microalgae flocculation with metal salts. The theory of Derjaguin, Landau, Verwey and Overbeek (DLVO) was applied to understand the flocculation mechanism of a freshwater alga Scenedesmus dimorphus and a marine alga Nannochloropsis oculata under various pH and aluminum sulphate ionic strengths. Effective flocculation was achieved as a result of charge neutralization and sweep flocculation. When low flocculant dosage (<0.1 mM) was applied, charge neutralization seemed to be predominating and the DLVO predicted flocculation trends were found quantitatively accurate in matching the experimental results. In the case of high flocculant dosage, the DLVO theory failed to explain the results since sweep flocculation was found to predominate at alum dose above 1 mM. Understanding of cell-to-cell interactions of microalgae offered possibilities in design of a novel semi-immobilized algal production and harvesting method, which exploited cell to substrata interactions instead of cell-to-cell interactions. In such method, a solid carrier was used to grow and accumulate algal cells and the cost of harvesting and drying can be simply reduced by easy algae-water separation. In order to enable the envisioned algal attachment, the third objective was to investigate the cell to substrata attachment by a thermodynamic model. Based on the theoretical analysis, when the polar surface energy of the cell is greater than that of water, cellular attachment would be more favorable on materials with higher dispersive surface energy but lower polar surface energy. If the polar surface energy of the cell is smaller than that of water, more cell attachment would be expected on materials that are higher in both dispersive and polar surface energies. The model was also validated its capability in designing, selecting, and matching algal strains and solid carrier materials to enhance cell attachment. The forth objective was to investigate the effect of surface texturing on algal attachment. It was found that surface texturing had a greater effect than surface free energy, by changing the liquid wetting behavior and real contact area. The attachment is preferred when the feature size is close to the diameter of the cell attempting to settle. Larger or smaller feature dimensions have the potential to reduce cellular attachment. The fifth objective was to study the role of carrier materials and their surface roughness in attachment. If the surface chemical composition was similar, the attachment and orientation of algal cells was found to depend on the surface average roughness, wherein rougher surfaces resulted in increased attachment. Whereas, the attachment was strongly related to surface free energy as described by the thermodynamic model if materials were chemically different.

HIV-1 Activates T Cell Signaling Independently of Antigen to Drive Viral Spread.

PubMed

Len, Alice C L; Starling, Shimona; Shivkumar, Maitreyi; Jolly, Clare

2017-01-24

HIV-1 spreads between CD4 T cells most efficiently through virus-induced cell-cell contacts. To test whether this process potentiates viral spread by activating signaling pathways, we developed an approach to analyze the phosphoproteome in infected and uninfected mixed-population T cells using differential metabolic labeling and mass spectrometry. We discovered HIV-1-induced activation of signaling networks during viral spread encompassing over 200 cellular proteins. Strikingly, pathways downstream of the T cell receptor were the most significantly activated, despite the absence of canonical antigen-dependent stimulation. The importance of this pathway was demonstrated by the depletion of proteins, and we show that HIV-1 Env-mediated cell-cell contact, the T cell receptor, and the Src kinase Lck were essential for signaling-dependent enhancement of viral dissemination. This study demonstrates that manipulation of signaling at immune cell contacts by HIV-1 is essential for promoting virus replication and defines a paradigm for antigen-independent T cell signaling. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Microscopic Behavior Of Colloidal Particles Under The Effect Of Acoustic Stimulations In The Ultrasonic To Megasonic Range

NASA Astrophysics Data System (ADS)

Abdel-Fattah, Amr I.; Roberts, Peter M.

2006-05-01

It is well known that colloid attachment and detachment at solid surfaces are influenced strongly by physico-chemical conditions controlling electric double layer (EDL) and solvation-layer effects. We present experimental observations demonstrating that, in addition, acoustic waves can produce strong effects on colloid/surface interactions that can alter the behavior of colloid and fluid transport in porous media. Microscopic colloid visualization experiments were performed with polystyrene micro-spheres suspended in water in a parallel-plate glass flow cell. When acoustic energy was applied to the cell at frequencies from 500 kHz to 5 MHz, changes in colloid attachment to and detachment from the glass cell surfaces were observed. Quantitative measurements of acoustically-induced detachment of 300-nm microspheres in 0.1M NaCl solution demonstrated that roughly 30% of the colloids that were attached to the glass cell wall during flow alone could be detached rapidly by applying acoustics at frequencies in the range of 0.7 to 1.2 MHz. The remaining attached colloids could not be detached by acoustics. This implies the existence of both "strong" and "weak" attachment sites at the cell surface. Subsequent re-attachment of colloids with acoustics turned off occurred only at new, previously unoccupied sites. Thus, acoustics appears to accelerate simultaneously both the deactivation of existing weak sites where colloids are already attached, and the activation of new weak sites where future attachments can occur. Our observations indicate that acoustics (and, in general, dynamic stress) can influence colloid-colloid and colloid-surface interactions in ways that could cause significant changes in porous-media permeability and mass transport. This would occur due to either buildup or release of colloids present in the porous matrix.

The memory of the accreting plate boundary and the continuity of fracture zones

USGS Publications Warehouse

Schouten, Hans; Klitgord, Kim D.

1982-01-01

A detailed aeromagnetic anomaly map of the Mesozoic seafloor-spreading lineations southwest of Bermuda reveals the dominant magnetic grain of the oceanic crust and the character of the accreting boundary at the time of crustal formation. The magnetic anomaly pattern is that of a series of elongate lobes perpendicular to the fracture zone (flowline) trends. The linear sets of magnetic anomaly peaks and troughs have narrow regions of reduced amplitude anomalies associated with the fracture zones. During the period of Mesozoic geomagnetic polarity reversals (when 1200 km of central North Atlantic seafloor formed), the Atlantic accreting boundary consisted of stationary, elongate, spreading center cells that maintained their independence even though sometimes only minor spatial offsets existed between cells. Normal oceanic crustal structure was formed in the spreading center cells, but structural anomalies and discontinuities characteristic of fracture zones were formed at their boundaries, which parallel flowlines of Mesozoic relative plate motion in the central North Atlantic. We suggest that the memory for a stationary pattern of independent spreading center cells resides in the young brittle lithosphere at the accreting boundary where the lithosphere is weakest; here, each spreading center cell independently goes through its cylce of stress buildup, stress release, and crustal accretion, after which its memory is refreshed. The temporal offset between the peaks of the accretionary activity that takes place within each cell may provide the mechanism for maintaining the independence of adjacent spreading center cells through times when no spatial offset between the cells exists.

The Role of Cell Volume in the Dynamics of Seizure, Spreading Depression, and Anoxic Depolarization

PubMed Central

Ullah, Ghanim; Wei, Yina; Dahlem, Markus A; Wechselberger, Martin; Schiff, Steven J

2015-01-01

Cell volume changes are ubiquitous in normal and pathological activity of the brain. Nevertheless, we know little of how cell volume affects neuronal dynamics. We here performed the first detailed study of the effects of cell volume on neuronal dynamics. By incorporating cell swelling together with dynamic ion concentrations and oxygen supply into Hodgkin-Huxley type spiking dynamics, we demonstrate the spontaneous transition between epileptic seizure and spreading depression states as the cell swells and contracts in response to changes in osmotic pressure. Our use of volume as an order parameter further revealed a dynamical definition for the experimentally described physiological ceiling that separates seizure from spreading depression, as well as predicted a second ceiling that demarcates spreading depression from anoxic depolarization. Our model highlights the neuroprotective role of glial K buffering against seizures and spreading depression, and provides novel insights into anoxic depolarization and the relevant cell swelling during ischemia. We argue that the dynamics of seizures, spreading depression, and anoxic depolarization lie along a continuum of the repertoire of the neuron membrane that can be understood only when the dynamic ion concentrations, oxygen homeostasis,and cell swelling in response to osmotic pressure are taken into consideration. Our results demonstrate the feasibility of a unified framework for a wide range of neuronal behaviors that may be of substantial importance in the understanding of and potentially developing universal intervention strategies for these pathological states. PMID:26273829

Characterization of a highly efficient chevron-shaped anti-contamination device

NASA Astrophysics Data System (ADS)

Fiore, M.; Vermeersch, O.; Forte, M.; Casalis, G.; François, C.

2016-04-01

This paper is devoted to the characterization of an optimized chevron-shaped anti-contamination device (ACD). This device can prevent efficiently the propagation of turbulence from the fuselage along the attachment line (hypothetical streamline that spreads the flow going to suction side and the one going to pressure side) of swept wings and enables the development of a new laminar boundary layer downstream. More specifically, the aim is to prevent boundary-layer transition along the attachment line by a contamination process. This process is characterized by the typical Reynolds number overline{R} and the associated Poll's criterion. Thus, ACD efficiency will be expressed in terms of overline{R} values. Some experiments performed on a new numerically optimized ACD have shown its ability to prevent leading-edge contamination up to overline{R} values close to the natural transition process of the laminar boundary layer along the attachment line. The corresponding stability analysis of the laminar boundary layer is made using the Görtler-Hämmerlin stability approach. The study is completed with the different transition processes that can occur downstream the attachment line, around the airfoil, especially with crossflow analysis.

The HSV-1 mechanisms of cell-to-cell spread and fusion are critically dependent on host PTP1B.

PubMed

Carmichael, Jillian C; Yokota, Hiroki; Craven, Rebecca C; Schmitt, Anthony; Wills, John W

2018-05-01

All herpesviruses have mechanisms for passing through cell junctions, which exclude neutralizing antibodies and offer a clear path to neighboring, uninfected cells. In the case of herpes simplex virus type 1 (HSV-1), direct cell-to-cell transmission takes place between epithelial cells and sensory neurons, where latency is established. The spreading mechanism is poorly understood, but mutations in four different HSV-1 genes can dysregulate it, causing neighboring cells to fuse to produce syncytia. Because the host proteins involved are largely unknown (other than the virus entry receptor), we were intrigued by an earlier discovery that cells infected with wild-type HSV-1 will form syncytia when treated with salubrinal. A biotinylated derivative of this drug was used to pull down cellular complexes, which were analyzed by mass spectrometry. One candidate was a protein tyrosine phosphatase (PTP1B), and although it ultimately proved not to be the target of salubrinal, it was found to be critical for the mechanism of cell-to-cell spread. In particular, a highly specific inhibitor of PTP1B (CAS 765317-72-4) blocked salubrinal-induced fusion, and by itself resulted in a dramatic reduction in the ability of HSV-1 to spread in the presence of neutralizing antibodies. The importance of this phosphatase was confirmed in the absence of drugs by using PTP1B-/- cells. Importantly, replication assays showed that virus titers were unaffected when PTP1B was inhibited or absent. Only cell-to-cell spread was altered. We also examined the effects of salubrinal and the PTP1B inhibitor on the four Syn mutants of HSV-1, and strikingly different responses were found. That is, both drugs individually enhanced fusion for some mutants and reduced fusion for others. PTP1B is the first host factor identified to be specifically required for cell-to-cell spread, and it may be a therapeutic target for preventing HSV-1 reactivation disease.

Electric impedance sensing in cell-substrates for rapid and selective multipotential differentiation capacity monitoring of human mesenchymal stem cells.

PubMed

Reitinger, Stephan; Wissenwasser, Jürgen; Kapferer, Werner; Heer, Rudolf; Lepperdinger, Günter

2012-04-15

Biosensor systems which enable impedance measurements on adherent cell layers under label-free conditions are considered powerful tools for monitoring specific biological characteristics. A radio frequency identification-based sensor platform was adopted to characterize cultivation and differentiation of human bone marrow-derived multipotent stem cells (bmMSC) over periods of up to several days and weeks. Electric cell-substrate impedance sensing was achieved through fabrication of sensitive elements onto glass substrates which comprised two comb-shaped interdigitated gold electrodes covering an area of 1.8 mm×2 mm. The sensing systems were placed into the wells of a 6-well tissue culture plate, stacked onto a reader unit and could thus be handled and operated under sterile conditions. Continuous measurements were carried out with a sinusoidal voltage of 35 mV at a frequency of 10 kHz. After seeding of human bmMSC, this sensor was able to trace significant impedance changes contingent upon cell spreading and adhesion. The re-usable system was further proven suitable for live examination of cell-substrate attachment or continuous cell monitoring up to several weeks. Induction of either osteogenic or adipogenic differentiation could be validated in bmMSC cultures within a few days, in contrast to state-of-the-art protocols, which require several weeks of cultivation time. In the context of medical cell production in a GMP-compliant process, the here presented interdigitated electric microsensor technology allows the documentation of MSC quality in a fast, efficient and reliable fashion. Copyright © 2012 Elsevier B.V. All rights reserved.

Diverse roles of integrin receptors in articular cartilage.

PubMed

Shakibaei, M; Csaki, C; Mobasheri, A

2008-01-01

Integrins are heterodimeric integral membrane proteins made up of alpha and beta subunits. At least eighteen alpha and eight beta subunit genes have been described in mammals. Integrin family members are plasma membrane receptors involved in cell adhesion and active as intra- and extracellular signalling molecules in a variety of processes including embryogenesis, hemostasis, tissue repair, immune response and metastatic spread of tumour cells. Integrin beta 1 (beta1-integrin), the protein encoded by the ITGB1 gene (also known as CD29 and VLAB), is a multi-functional protein involved in cell-matrix adhesion, cell signalling, cellular defense, cell adhesion, protein binding, protein heterodimerisation and receptor-mediated activity. It is highly expressed in the human body (17.4 times higher than the average gene in the last updated revision of the human genome). The extracellular matrix (ECM) of articular cartilage is a unique environment. Interactions between chondrocytes and the ECM regulate many biological processes important to homeostasis and repair of articular cartilage, including cell attachment, growth, differentiation and survival. The beta1-integrin family of cell surface receptors appears to play a major role in mediating cell-matrix interactions that are important in regulating these fundamental processes. Chondrocyte mechanoreceptors have been proposed to incorporate beta1-integrins and mechanosensitive ion channels which link with key ECM, cytoskeletal and signalling proteins to maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte-specific gene expression. This review focuses on the expression and function of beta1-integrins in articular chondrocytes, its role in the unique biology of these cells and its distribution in cartilage.

Hair Follicle-Associated Pluripotent (HAP) Stem Cells in Gelfoam® Histoculture for Use in Spinal Cord Repair.

PubMed

Liu, Fang; Hoffman, Robert M

2018-01-01

The stem cell marker, nestin, is expressed in the hair follicle, both in cells in the bulge area (BA) and the dermal papilla (DP). Nestin-expressing hair follicle-associated-pluripotent (HAP) stem cells of both the BA and DP have been previously shown to be able to form neurons, heart muscle cells, and other non-follicle cell types. The ability of the nestin-expressing HAP stem cells from the BA and DP to repair spinal cord injury was compared. Nestin-expressing HAP stem cells from both the BA and DP grew very well on Gelfoam ® . The HAP stem cells attached to the Gelfoam ® within 1 h. They grew along the grids of the Gelfoam ® during the first 2 or 3 days. Later they spread into the Gelfoam ® . After transplantation of Gelfoam ® cultures of nestin-expressing BA or DP HAP stem cells into the injured spinal cord (including the Gelfoam ® ) nestin-expressing BA and DP cells were observed to be viable over 100 days post-surgery. Hematoxylin and eosin (H&E) staining showed connections between the transplanted cells and the host spine tissue. Immunohistochemistry showed many Tuj1-, Isl 1/2, and EN1-positive cells and nerve fibers in the transplanted area of the spinal cord after BA Gelfoam ® or DP Gelfoam ® cultures were transplanted to the spine. The spinal cord of mice was injured to effect hind-limb paralysis. Twenty-eight days after transplantation with BA or DP HAP stem cells on Gelfoam ® to the injured area of the spine, the mice recovered normal locomotion.

Methods for Maintaining Insect Cell Cultures

PubMed Central

Lynn, Dwight E.

2002-01-01

Insect cell cultures are now commonly used in insect physiology, developmental biology, pathology, and molecular biology. As the field has advanced from methods development to a standard procedure, so has the diversity of scientists using the technique. This paper describes methods that are effective for maintaining various insect cell lines. The procedures are differentiated between loosely or non-attached cell strains, attached cell strains, and strongly adherent cell strains. PMID:15455043

Nitric acid passivation does not affect in vitro biocompatibility of titanium.

PubMed

Faria, Adriana C L; Beloti, Márcio M; Rosa, Adalberto L

2003-01-01

In general, both chemical composition and surface features of implants affect cell response. The aim of this study was to evaluate the effect of titanium (Ti) passivation on the response of rat bone marrow cells, considering cell attachment, cell morphology, cell proliferation, total protein content, alkaline phosphatase (ALP) activity, and bonelike nodule formation. Cells were cultured on both commercially pure titanium (cpTi) and titanium-aluminium-vanadium alloy (Ti-6Al-4V) discs, either passivated or not. For attachment evaluation, cells were cultured for 4 and 24 hours. Cell morphology was evaluated after 4 days. After 7, 14, and 21 days, cell proliferation, total protein content, and ALP activity were evaluated. Bonelike nodule formation was evaluated after 21 days. Data were compared by analysis of variance and the Duncan multiple range test. Cell attachment, cell morphology, cell proliferation, total protein content, ALP activity, and bonelike nodule formation all were unaffected by Ti composition or passivation. Although the protocol for passivation used here could interfere with the pattern of ions released from Ti-6Al-4V and cpTi surfaces, the present study did not show any effect of this surface treatment on in vitro biocompatibility of Ti as evaluated by osteoblast attachment, proliferation, and differentiation.

Electrochemical Characteristics of Cell Cultured Ti-Nb-Zr Alloys After Nano-Crystallized Si-HA Coating.

PubMed

Jeong, Yong-Hoon; Choe, Han-Cheol

2015-01-01

The aim of this study was to investigate the electrochemical characteristics of nano crystallized Si-HA coating on Ti-Nb-Zr alloy after human osteoblast like (HOB) cell attachment. The Ti-Nb-Zr alloy was manufactured with 35 wt.% of Nb and 10 wt.% of Zr by arc melting furnace to appropriate physical properties as biomaterials. The HA and Si-substituted coatings were prepared by electron-beam physical vapor deposition method with 0.5, 0.8 and 1.2 wt.% of Si contents, and nano aging treatment was performed 500 degrees C for 1 h. The characteristics of coating surface were analyzed by field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, and X-ray diffraction, respectively. To evaluate of cell attachment on cell cultured surface, the potentiodynamic test was performed on the surface using HOB cells. The results showed that the Si-HA coating surface showed higher tendency of cell attachment than that of single HA coating, corrosion resistance value was increased by dense of cell attachment.

Inhibition of experimental ascending urinary tract infection by an epithelial cell-surface receptor analogue

NASA Astrophysics Data System (ADS)

Edén, C. Svanborg; Freter, R.; Hagberg, L.; Hull, R.; Hull, S.; Leffler, H.; Schoolnik, G.

1982-08-01

It has been shown that the establishment of urinary tract infection by Escherichia coli is dependent on attachment of the bacteria to epithelial cells1-4. The attachment involves specific epithelial cell receptors, which have been characterized as glycolipids5-10. Reversible binding to cell-surface mannosides may also be important4,11-13. This suggests an approach to the treatment of infections-that of blocking bacterial attachment with cell membrane receptor analogues. Using E. coli mutants lacking one or other of the two binding specificities (glycolipid and mannose), we show here that glycolipid analogues can block in vitro adhesion and in vivo urinary tract infection.

A new epidemic modeling approach: Multi-regions discrete-time model with travel-blocking vicinity optimal control strategy.

PubMed

Zakary, Omar; Rachik, Mostafa; Elmouki, Ilias

2017-08-01

First, we devise in this paper, a multi-regions discrete-time model which describes the spatial-temporal spread of an epidemic which starts from one region and enters to regions which are connected with their neighbors by any kind of anthropological movement. We suppose homogeneous Susceptible-Infected-Removed (SIR) populations, and we consider in our simulations, a grid of colored cells, which represents the whole domain affected by the epidemic while each cell can represent a sub-domain or region. Second, in order to minimize the number of infected individuals in one region, we propose an optimal control approach based on a travel-blocking vicinity strategy which aims to control only one cell by restricting movements of infected people coming from all neighboring cells. Thus, we show the influence of the optimal control approach on the controlled cell. We should also note that the cellular modeling approach we propose here, can also describes infection dynamics of regions which are not necessarily attached one to an other, even if no empty space can be viewed between cells. The theoretical method we follow for the characterization of the travel-locking optimal controls, is based on a discrete version of Pontryagin's maximum principle while the numerical approach applied to the multi-points boundary value problems we obtain here, is based on discrete progressive-regressive iterative schemes. We illustrate our modeling and control approaches by giving an example of 100 regions.

The synthesis and coupling of photoreactive collagen-based peptides to restore integrin reactivity to an inert substrate, chemically-crosslinked collagen

PubMed Central

Malcor, Jean-Daniel; Bax, Daniel; Hamaia, Samir W.; Davidenko, Natalia; Best, Serena M.; Cameron, Ruth E.; Farndale, Richard W.; Bihan, Dominique

2016-01-01

Collagen is frequently advocated as a scaffold for use in regenerative medicine. Increasing the mechanical stability of a collagen scaffold is widely achieved by cross-linking using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). However, this treatment consumes the carboxylate-containing amino acid sidechains that are crucial for recognition by the cell-surface integrins, abolishing cell adhesion. Here, we restore cell reactivity to a cross-linked type I collagen film by covalently linking synthetic triple-helical peptides (THPs), mimicking the structure of collagen. These THPs are ligands containing an active cell-recognition motif, GFOGER, a high-affinity binding site for the collagen-binding integrins. We end-stapled peptide strands containing GFOGER by coupling a short diglutamate-containing peptide to their N-terminus, improving the thermal stability of the resulting THP. A photoreactive Diazirine group was grafted onto the end-stapled THP to allow covalent linkage to the collagen film upon UV activation. Such GFOGER-derivatized collagen films showed restored affinity for the ligand-binding I domain of integrin α2β1, and increased integrin-dependent cell attachment and spreading of HT1080 and Rugli cell lines, expressing integrins α2β1 and α1β1, respectively. The method we describe has wide application, beyond collagen films or scaffolds, since the photoreactive diazirine will react with many organic carbon skeletons. PMID:26854392

Integration of multiple cell-matrix interactions into alginate scaffolds for promoting cardiac tissue regeneration.

PubMed

Sapir, Yulia; Kryukov, Olga; Cohen, Smadar

2011-03-01

Cardiac tissue engineering aims to repair damaged myocardial tissues by applying heart patches created in vitro. Herein, we explored the possible role of a combination of two matrix-attached peptides, the adhesion peptide G(4)RGDY and heparin-binding peptide G(4)SPPRRARVTY (HBP) in cardiac tissue regeneration. Neonatal rat cardiac cells were seeded into unmodified, single peptide or double peptide-attached alginate scaffolds, all having the same physical features of porosity, hydrogel forming and matrix stiffness. The cardiac tissue developed in the HBP/RGD-attached scaffolds revealed the best features of a functional muscle tissue, as judged by all studied parameters, i.e., immunostaining of cardiac cell markers, histology, western blot of protein expressions and metabolic activity. By day 7, well-developed myocardial fibers were observed in these cell constructs. At 14 days the HBP/RGD-attached constructs presented an isotropic myofiber arrangement, while no such arrangement was seen in the other constructs. The expression levels of α-actinin, N-cadherin and Connexin-43, showing preservation and an increase in Connexin-43 expression (Cx-43) with time, further supported the formation a contractile muscle tissue in the HBP/RGD-attached scaffolds. Collectively, the attachment of combinatorial peptides representing different signaling in ECM-cell interactions proved to play a key role, contributing to the formation of a functional cardiac muscle tissue, in vitro. Copyright © 2010 Elsevier Ltd. All rights reserved.

Understanding Product Attachment and Expected Product Lifetime by Extending Technology Acceptance Model (TAM) with Product Personalization and Innovation Diffusion Theory (IDT)

ERIC Educational Resources Information Center

Yun, Younghwa

2013-01-01

Identifying and understanding consumers is fundamental, especially for quickly spreading new products. In recent years, the popularity of digital gadgets has sky rocketed; however, there has also been a growing tendency of relative obsolescence--replacement of a product regardless of the demise of its perfect functioning. Therefore, a question is…

Accounting for Space—Quantification of Cell-To-Cell Transmission Kinetics Using Virus Dynamics Models.

PubMed

Kumberger, Peter; Durso-Cain, Karina; Uprichard, Susan L; Dahari, Harel; Graw, Frederik

2018-04-17

Mathematical models based on ordinary differential equations (ODE) that describe the population dynamics of viruses and infected cells have been an essential tool to characterize and quantify viral infection dynamics. Although an important aspect of viral infection is the dynamics of viral spread, which includes transmission by cell-free virions and direct cell-to-cell transmission, models used so far ignored cell-to-cell transmission completely, or accounted for this process by simple mass-action kinetics between infected and uninfected cells. In this study, we show that the simple mass-action approach falls short when describing viral spread in a spatially-defined environment. Using simulated data, we present a model extension that allows correct quantification of cell-to-cell transmission dynamics within a monolayer of cells. By considering the decreasing proportion of cells that can contribute to cell-to-cell spread with progressing infection, our extension accounts for the transmission dynamics on a single cell level while still remaining applicable to standard population-based experimental measurements. While the ability to infer the proportion of cells infected by either of the transmission modes depends on the viral diffusion rate, the improved estimates obtained using our novel approach emphasize the need to correctly account for spatial aspects when analyzing viral spread.

Xylella fastidiosa Differentially Accumulates Mineral Elements in Biofilm and Planktonic Cells

PubMed Central

Cobine, Paul A.; Cruz, Luisa F.; Navarrete, Fernando; Duncan, Daniel; Tygart, Melissa; De La Fuente, Leonardo

2013-01-01

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen. PMID:23349991

Xylella fastidiosa differentially accumulates mineral elements in biofilm and planktonic cells.

PubMed

Cobine, Paul A; Cruz, Luisa F; Navarrete, Fernando; Duncan, Daniel; Tygart, Melissa; De La Fuente, Leonardo

2013-01-01

Xylella fastidiosa is a bacterial plant pathogen that infects numerous plant hosts. Disease develops when the bacterium colonizes the xylem vessels and forms a biofilm. Inductively coupled plasma optical emission spectroscopy was used to examine the mineral element content of this pathogen in biofilm and planktonic states. Significant accumulations of copper (30-fold), manganese (6-fold), zinc (5-fold), calcium (2-fold) and potassium (2-fold) in the biofilm compared to planktonic cells were observed. Other mineral elements such as sodium, magnesium and iron did not significantly differ between biofilm and planktonic cells. The distribution of mineral elements in the planktonic cells loosely mirrors the media composition; however the unique mineral element distribution in biofilm suggests specific mechanisms of accumulation from the media. A cell-to-surface attachment assay shows that addition of 50 to 100 µM Cu to standard X. fastidiosa media increases biofilm, while higher concentrations (>200 µM) slow cell growth and prevent biofilm formation. Moreover cell-to-surface attachment was blocked by specific chelation of copper. Growth of X. fastidiosa in microfluidic chambers under flow conditions showed that addition of 50 µM Cu to the media accelerated attachment and aggregation, while 400 µM prevented this process. Supplementation of standard media with Mn showed increased biofilm formation and cell-to-cell attachment. In contrast, while the biofilm accumulated Zn, supplementation to the media with this element caused inhibited growth of planktonic cells and impaired biofilm formation. Collectively these data suggest roles for these minerals in attachment and biofilm formation and therefore the virulence of this pathogen.

Tobacco mosaic virus Movement Protein Enhances the Spread of RNA Silencing

PubMed Central

Vogler, Hannes; Kwon, Myoung-Ok; Dang, Vy; Sambade, Adrian; Fasler, Monika; Ashby, Jamie; Heinlein, Manfred

2008-01-01

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA. PMID:18389061

Modeling and optimization of gelatin-chitosan micro-carriers preparation for soft tissue engineering: Using Response Surface Methodology.

PubMed

Radaei, Payam; Mashayekhan, Shohreh; Vakilian, Saeid

2017-06-01

Electrospray ionization is a wide spread technique for producing polymeric microcarriers (MCs) by applying electrostatic force and ionic cross-linker, simultaneously. In this study, fabrication process of gelatin-chitosan MCs and its optimization using the Response Surface Methodology (RSM) is reported. Gelatin/chitosan (G/C) blend ratio, applied voltage and feeding flow rate, their individual and interaction effects on the diameter and mechanical strength of the MCs were investigated. The obtained models for diameter and mechanical strength of MCs have a quadratic relationship with G/C blend ratio, applied voltage and feeding flow rate. Using the desirability curve, optimized G/C blend ratios that are introduced, include the desirable quantities for MCs diameter and mechanical strength. MCs of the same desirable diameter (350μm) and different G/C blend ratio (1, 2, and 3) were fabricated and their elasticity was investigated via Atomic Force Microscopy (AFM). The biocompatibility of the MCs was evaluated using MTT assay. The results showed that human Umbilical Cord Mesenchymal Stem Cells (hUCMSCs) could attach and proliferate on fabricated MCs during 7days of culturing especially on those prepared with G/C blend ratios of 1 and 2. Such gelatin-chitosan MCs may be considered as a promising candidate for injectable tissue engineering scaffolds, supporting attachment and proliferation of hUCMSCs. Copyright © 2017 Elsevier B.V. All rights reserved.

Strong attachment of circadian pacemaker neurons on modified ultrananocrystalline diamond surfaces.

PubMed

Voss, Alexandra; Wei, HongYing; Zhang, Yi; Turner, Stuart; Ceccone, Giacomo; Reithmaier, Johann Peter; Stengl, Monika; Popov, Cyril

2016-07-01

Diamond is a promising material for a number of bio-applications, including the fabrication of platforms for attachment and investigation of neurons and of neuroprostheses, such as retinal implants. In the current work ultrananocrystalline diamond (UNCD) films were deposited by microwave plasma chemical vapor deposition, modified by UV/O3 treatment or NH3 plasma, and comprehensively characterized with respect to their bulk and surface properties, such as crystallinity, topography, composition and chemical bonding nature. The interactions of insect circadian pacemaker neurons with UNCD surfaces with H-, O- and NH2-terminations were investigated with respect to cell density and viability. The fast and strong attachment achieved without application of adhesion proteins allowed for advantageous modification of dispersion protocols for the preparation of primary cell cultures. Centrifugation steps, which are employed for pelletizing dispersed cells to separate them from dispersing enzymes, easily damage neurons. Now centrifugation can be avoided since dispersed neurons quickly and strongly attach to the UNCD surfaces. Enzyme solutions can be easily washed off without losing many of the dispersed cells. No adverse effects on the cell viability and physiological responses were observed as revealed by calcium imaging. Furthermore, the enhanced attachment of the neurons, especially on the modified UNCD surfaces, was especially advantageous for the immunocytochemical procedures with the cell cultures. The cell losses during washing steps were significantly reduced by one order of magnitude in comparison to controls. In addition, the integration of a titanium grid structure under the UNCD films allowed for individual assignment of physiologically characterized neurons to immunocytochemically stained cells. Thus, employing UNCD surfaces free of foreign proteins improves cell culture protocols and immunocytochemistry with cultured cells. The fast and strong attachment of neurons was attributed to a favorable combination of topography, surface chemistry and wettability. Copyright © 2016 Elsevier B.V. All rights reserved.

A New Biphasic Dicalcium Silicate Bone Cement Implant.

PubMed

Zuleta, Fausto; Murciano, Angel; Gehrke, Sergio A; Maté-Sánchez de Val, José E; Calvo-Guirado, José L; De Aza, Piedad N

2017-07-06

This study aimed to investigate the processing parameters and biocompatibility of a novel biphasic dicalcium silicate (C₂S) cement. Biphasic α´ L + β-C₂S ss was synthesized by solid-state processing, and was used as a raw material to prepare the cement. In vitro bioactivity and biocompatibility studies were assessed by soaking the cement samples in simulated body fluid (SBF) and human adipose stem cell cultures. Two critical-sized defects of 6 mm Ø were created in 15 NZ tibias. A porous cement made of the high temperature forms of C₂S, with a low phosphorous substitution level, was produced. An apatite-like layer covered the cement's surface after soaking in SBF. The cell attachment test showed that α´ L + β-C₂S ss supported cells sticking and spreading after 24 h of culture. The cement paste (55.86 ± 0.23) obtained higher bone-to-implant contact (BIC) percentage values (better quality, closer contact) in the histomorphometric analysis, and defect closure was significant compared to the control group (plastic). The residual material volume of the porous cement was 35.42 ± 2.08% of the initial value. The highest BIC and bone formation percentages were obtained on day 60. These results suggest that the cement paste is advantageous for initial bone regeneration.

A New Biphasic Dicalcium Silicate Bone Cement Implant

PubMed Central

Murciano, Angel; Maté-Sánchez de Val, José E.

2017-01-01

This study aimed to investigate the processing parameters and biocompatibility of a novel biphasic dicalcium silicate (C2S) cement. Biphasic α´L + β-C2Sss was synthesized by solid-state processing, and was used as a raw material to prepare the cement. In vitro bioactivity and biocompatibility studies were assessed by soaking the cement samples in simulated body fluid (SBF) and human adipose stem cell cultures. Two critical-sized defects of 6 mm Ø were created in 15 NZ tibias. A porous cement made of the high temperature forms of C2S, with a low phosphorous substitution level, was produced. An apatite-like layer covered the cement’s surface after soaking in SBF. The cell attachment test showed that α´L + β-C2Sss supported cells sticking and spreading after 24 h of culture. The cement paste (55.86 ± 0.23) obtained higher bone-to-implant contact (BIC) percentage values (better quality, closer contact) in the histomorphometric analysis, and defect closure was significant compared to the control group (plastic). The residual material volume of the porous cement was 35.42 ± 2.08% of the initial value. The highest BIC and bone formation percentages were obtained on day 60. These results suggest that the cement paste is advantageous for initial bone regeneration. PMID:28773119

Differential Attachment of Salmonella enterica and Enterohemorrhagic Escherichia coli to Alfalfa, Fenugreek, Lettuce, and Tomato Seeds

PubMed Central

Cui, Yue; Walcott, Ronald

2017-01-01

ABSTRACT Vegetable seeds have the potential to disseminate and transmit foodborne bacterial pathogens. This study was undertaken to assess the abilities of selected Salmonella and enterohemorrhagic Escherichia coli (EHEC) strains to attach to fungicide-treated versus untreated, and intact versus mechanically damaged, seeds of alfalfa, fenugreek, lettuce, and tomato. Surface-sanitized seeds (2 g) were exposed to four individual strains of Salmonella or EHEC at 20°C for 5 h. Contaminated seeds were rinsed twice, each with 10 ml of sterilized water, before being soaked overnight in 5 ml of phosphate-buffered saline at 4°C. The seeds were then vortexed vigorously for 1 min, and pathogen populations in seed rinse water and soaking buffer were determined using a standard plate count assay. In general, the Salmonella cells had higher attachment ratios than the EHEC cells. Lettuce seeds by unit weight had the highest numbers of attached Salmonella or EHEC cells, followed by tomato, alfalfa, and fenugreek seeds. In contrast, individual fenugreek seeds had more attached pathogen cells, followed by lettuce, alfalfa, and tomato seeds. Significantly more Salmonella and EHEC cells attached to mechanically damaged seeds than to intact seeds (P < 0.05). Although, on average, significantly more Salmonella and EHEC cells were recovered from untreated than fungicide-treated seeds (P < 0.05), fungicide treatment did not significantly affect the attachment of individual bacterial strains to vegetable seeds (P > 0.05), with a few exceptions. This study fills gaps in the current body of literature and helps explain bacterial interactions with vegetable seeds with differing surface characteristics. IMPORTANCE Vegetable seeds, specifically sprout seeds, have the potential to disseminate and transmit foodborne bacterial pathogens. This study investigated the interaction between two important bacterial pathogens, i.e., Salmonella and EHEC, and vegetable seeds with differing surface characteristics. This research helps understand whether seed surface structure, integrity, and fungicide treatment affect the interaction between bacterial cells and vegetable seeds. PMID:28130295

Differential Attachment of Salmonella enterica and Enterohemorrhagic Escherichia coli to Alfalfa, Fenugreek, Lettuce, and Tomato Seeds.

PubMed

Cui, Yue; Walcott, Ronald; Chen, Jinru

2017-04-01

Vegetable seeds have the potential to disseminate and transmit foodborne bacterial pathogens. This study was undertaken to assess the abilities of selected Salmonella and enterohemorrhagic Escherichia coli (EHEC) strains to attach to fungicide-treated versus untreated, and intact versus mechanically damaged, seeds of alfalfa, fenugreek, lettuce, and tomato. Surface-sanitized seeds (2 g) were exposed to four individual strains of Salmonella or EHEC at 20°C for 5 h. Contaminated seeds were rinsed twice, each with 10 ml of sterilized water, before being soaked overnight in 5 ml of phosphate-buffered saline at 4°C. The seeds were then vortexed vigorously for 1 min, and pathogen populations in seed rinse water and soaking buffer were determined using a standard plate count assay. In general, the Salmonella cells had higher attachment ratios than the EHEC cells. Lettuce seeds by unit weight had the highest numbers of attached Salmonella or EHEC cells, followed by tomato, alfalfa, and fenugreek seeds. In contrast, individual fenugreek seeds had more attached pathogen cells, followed by lettuce, alfalfa, and tomato seeds. Significantly more Salmonella and EHEC cells attached to mechanically damaged seeds than to intact seeds ( P < 0.05). Although, on average, significantly more Salmonella and EHEC cells were recovered from untreated than fungicide-treated seeds ( P < 0.05), fungicide treatment did not significantly affect the attachment of individual bacterial strains to vegetable seeds ( P > 0.05), with a few exceptions. This study fills gaps in the current body of literature and helps explain bacterial interactions with vegetable seeds with differing surface characteristics. IMPORTANCE Vegetable seeds, specifically sprout seeds, have the potential to disseminate and transmit foodborne bacterial pathogens. This study investigated the interaction between two important bacterial pathogens, i.e., Salmonella and EHEC, and vegetable seeds with differing surface characteristics. This research helps understand whether seed surface structure, integrity, and fungicide treatment affect the interaction between bacterial cells and vegetable seeds. Copyright © 2017 American Society for Microbiology.

Protein and cell micropatterning and its integration with micro/nanoparticles assembly.

PubMed

Yap, F L; Zhang, Y

2007-01-15

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

PubMed

Thomas, Richard J; Brooks, Tim J

2004-02-01

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

Cationic Surface Charge Combined with Either Vitronectin or Laminin Dictates the Evolution of Human Embryonic Stem Cells/Microcarrier Aggregates and Cell Growth in Agitated Cultures

PubMed Central

Lam, Alan Tin-Lun; Li, Jian; Chen, Allen Kuan-Liang; Reuveny, Shaul

2014-01-01

The expansion of human pluripotent stem cells (hPSC) for biomedical applications generally compels a defined, reliable, and scalable platform. Bioreactors offer a three-dimensional culture environment that relies on the implementation of microcarriers (MC), as supports for cell anchorage and their subsequent growth. Polystyrene microspheres/MC coated with adhesion-promoting extracellular matrix (ECM) protein, vitronectin (VN), or laminin (LN) have been shown to support hPSC expansion in a static environment. However, they are insufficient to promote human embryonic stem cells (hESC) seeding and their expansion in an agitated environment. The present study describes an innovative technology, consisting of a cationic charge that underlies the ECM coatings. By combining poly-L-lysine (PLL) with a coating of ECM protein, cell attachment efficiency and cell spreading are improved, thus enabling seeding under agitation in a serum-free medium. This coating combination also critically enables the subsequent formation and evolution of hPSC/MC aggregates, which ensure cell viability and generate high yields. Aggregate dimensions of at least 300 μm during early cell growth give rise to ≈15-fold expansion at 7 days' culture. Increasing aggregate numbers at a quasi-constant size of ≈300 μm indicates hESC growth within a self-regulating microenvironment. PLL+LN enables cell seeding and aggregate evolution under constant agitation, whereas PLL+VN requires an intermediate 2-day static pause to attain comparable aggregate sizes and correspondingly high expansion yields. The cells' highly reproducible bioresponse to these defined and characterized MC surface properties is universal across multiple cell lines, thus confirming the robustness of this scalable expansion process in a defined environment. PMID:24641164

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

NASA Astrophysics Data System (ADS)

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

2010-03-01

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

Degradation of extracellular matrix by mouse trophoblast outgrowths: a model for implantation

PubMed Central

Glass, RH; Aggeler, J; Spindle, A; Pederson, RA; Werb, Z

1983-01-01

During implantation the embryo attaches to the endometrial surface and trophoblast traverses the uterine epithelium, anchoring in the uterine connective tissue. To determine whether trophoblast can facilitate invasion of the uterus by degrading components of normal uterine extracellular matrix, mouse blastocysts were cultured on a radio-labeled extracellular matrix that contained glycoproteins, elastin, and collagen. The embryos attached to the matrix, and trophoblast spread over the surface. Starting on day 5 of culture there was a release of labeled peptides into the medium. The radioactive peptides released from the matrix by the embryos had molecular weights ranging from more than 25,000 to more than 200. By day 7 there were areas where individual trophoblast cells had separated from one another, revealing the underlying substratum that was cleared of matrix. When trophoblast cells were lysed with NH(4)OH on day 8, it was apparent that the area underneath the trophoblast outgrowth had been cleared of matrix. Scanning electron microscopy and time-lapse cinemicrography confirmed that the digestion of matrix was highly localized, taking place only underneath the trophoblast, with no evidence of digestion of the matrix beyond the periphery of the trophoblast outgrowth. The sharp boundaries of degredation observed may be due to localized proteinase secretion by trophoblast, to membrane proteinases on the surface of trophoblast, or to endocytosis. Digestion of the matrix was not dependent on plasminogen, thus ruling out a role for plasminogen activator. Digestion was not inhibited by a variety of hormones and inhibitors, including progesterone, 17β-estradiol, leupeptin, EDTA, colchicine, NH(4)Cl, or ε-aminocaproic acid. This system of culturing embryos on extracellular matrix may be useful in determining the processes that regulate trophoblast migration and invasion into the maternal tissues during implantation.0 PMID:6339525

Change in surface properties of zirconia and initial attachment of osteoblastlike cells with hydrophilic treatment.

PubMed

Watanabe, Hiroaki; Saito, Kensuke; Kokubun, Katsutoshi; Sasaki, Hodaka; Yoshinari, Masao

2012-01-01

The objectives of this study were to characterize change in surface properties of tetragonal zirconia polycrystals (TZP) after hydrophilic treatment, and to determine the effect of such changes on initial attachment of osteoblast-like cells. Roughened surfaces were produced by alumina-blasting and acid-etching. Hydrophilic treatment comprised application of immediately after blasting and acid-etching (Blast/Etch), oxygen plasma (O2-Plasma), ultraviolet light (UV). Specimens stored in air were used as a control. The water contact angle was determined and surface analysis was performed using an X-ray photoelectron spectroscopy. Blast/Etch, O2-Plasma and UV specimens showed superhydrophilicity, and these hydrophilic treatments to TZP elicited a marked decrease in carbon content and an increase in hydroxyl groups. Hydrophilic treatments enhanced initial attachment of osteoblast-like cells and a change in cell morphologies. These results indicate that Blast/Etch, O2-Plasma, or UV treatment has potential in the creation and maintenance of superhydrophilic surfaces and enhancing initial attachment of osteoblast-like cells.

In vitro biocompatibility tests of glass ionomer cements impregnated with collagen or bioactive glass to fibroblasts.

PubMed

Subbarao, C; Neelakantan, P; Subbarao, C V

2012-01-01

To evaluate the biocompatibility of glass ionomer cement (GIC) impregnated with collagen or bioactive glass to BHK-21 fibroblasts in vitro. Mineral Trioxide Aggregate was used as the standard for comparison. Human maxillary central incisors (n = 70) were instrumented with a rotary NiTi system and filled. Following resection of the apical 3mm, root end cavities were prepared and restored with conventional GIC (group 1) or GIC with 0.01%, 0.1% or 1% collagen (groups 2, 3, 4 respectively) or, 10%, 30% or 50% bioactive glass (groups 5, 6, 7 respectively), or Mineral Trioxide Aggregate (group 8). The root slices were incubated in tissue culture plates with BHK-21 fibroblast cell line. Phase contrast and scanning electron microscopes were used to score cell quantity, morphology and cell attachment. The data were statistically analyzed by one way ANOVA with Post Hoc Tukey HSD test (p = 0.05). Group 5 showed the highest scores which was significantly higher than all other groups (p < 0.05) except group 8, with which there was no significant difference (p > 0.05). Glass ionomer cement with 10% bioactive glass showed better adhesion and spreading of cells than glass ionomer cement with 0.01% collagen. The biocompatibility of collagen and bioactive glass was concentration dependent. The addition of bioactive glass improved the biocompatibility of glass ionomer cement to fibroblasts better than addition of collagen.

Computational prediction of strain-dependent diffusion of transcription factors through the cell nucleus.

PubMed

Nava, Michele M; Fedele, Roberto; Raimondi, Manuela T

2016-08-01

Nuclear spreading plays a crucial role in stem cell fate determination. In previous works, we reported evidence of multipotency maintenance for mesenchymal stromal cells cultured on three-dimensional engineered niche substrates, fabricated via two-photon laser polymerization. We correlated maintenance of multipotency to a more roundish morphology of these cells with respect to those cultured on conventional flat substrates. To interpret these findings, here we present a multiphysics model coupling nuclear strains induced by cell adhesion to passive diffusion across the cell nucleus. Fully three-dimensional reconstructions of cultured cells were developed on the basis of confocal images: in particular, the level of nuclear spreading resulted significantly dependent on the cell localization within the niche architecture. We assumed that the cell diffusivity varies as a function of the local volumetric strain. The model predictions indicate that the higher the level of spreading of the cell, the higher the flux across the nucleus of small solutes such as transcription factors. Our results point toward nuclear spreading as a primary mechanism by which the stem cell translates its shape into a fate decision, i.e., by amplifying the diffusive flow of transcriptional activators into the nucleus.

SNARE-mediated trafficking of {alpha}{sub 5}{beta}{sub 1} integrin is required for spreading in CHO cells

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

Skalski, Michael; Coppolino, Marc G.

2005-10-07

In this study, the role of SNARE-mediated membrane traffic in regulating integrin localization was examined and the requirement for SNARE function in cellular spreading was quantitatively assessed. Membrane traffic was inhibited with the VAMP-specific catalytic light chain from tetanus toxin (TeTx-LC), a dominant-negative form (E329Q) of N-ethylmaleimide-sensitive fusion protein (NSF), and brefeldin A (BfA). Inhibition of membrane traffic with either E329Q-NSF or TeTx-LC, but not BfA, significantly inhibited spreading of CHO cells on fibronectin. Spreading was rescued in TeTx-LC-expressing cells by co-transfection with a TeTx-resistant cellubrevin/VAMP3. E329Q-NSF, a general inhibitor of SNARE function, was a more potent inhibitor of cellmore » spreading than TeTx-LC, suggesting that tetanus toxin-insensitive SNAREs contribute to adhesion. It was found that E329Q-NSF prevented trafficking of {alpha}{sub 5}{beta}{sub 1} integrins from a central Rab11-containing compartment to sites of protrusion during cell adhesion, while TeTx-LC delayed this trafficking. These results are consistent with a model of cellular adhesion that implicates SNARE function as an important component of integrin trafficking during the process of cell spreading.« less

Pigmented basal cell carcinoma mimicking a superficial spreading melanoma.

PubMed

Hasbún Acuña, Paula; Cullen Aravena, Roberto; Maturana Donaire, César; Ares Mora, Raúl; Porras Kusmanic, Ninoska

2016-12-20

Basal cell carcinoma is the most common form of skin cancer, especially in elderly people. Pigmented basal cell carcinoma is a rare subtype and has been described in the literature as a nodular and hyperpigmented lesion; rarely, it can appear as an extensive pigmented plate, which may be clinically indistinguishable from superficial spreading melanoma and Bowen disease. Dermatoscopy has a high sensitivity in the diagnosis of basal cell carcinoma. When Menzies criteria are used; however, the final diagnosis is made by histopathology. The objective of the present report is to analyze the case of a patient with pigmented basal cell carcinoma simulating a superficial spreading melanoma.

Watch Out for the "Living Dead": Cell-Free Enzymes and Their Fate.

PubMed

Baltar, Federico

2017-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the "gatekeepers" of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell's fate. In contrast, cell-free enzymes belong to a kind of "living dead" realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go "beyond the living things," studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles.

Non-cell autonomous cell death caused by transmission of Huntingtin aggregates in Drosophila.

PubMed

Babcock, Daniel T; Ganetzky, Barry

2015-01-01

Recent evidence indicates that protein aggregates can spread between neurons in several neurodegenerative diseases but much remains unknown regarding the underlying mechanisms responsible for this spreading and its role in disease progression. We recently demonstrated that mutant Huntingtin aggregates spread between cells within the Drosophila brain resulting in non-cell autonomous loss of a pair of large neurons in the posterior protocerebrum. However, the full extent of neuronal loss throughout the brain was not determined. Here we examine the effects of driving expression of mutant Huntingtin in Olfactory Receptor Neurons (ORNs) by using a marker for cleaved caspase activity to monitor neuronal apoptosis as a function of age. We find widespread caspase activity in various brain regions over time, demonstrating that non-cell autonomous damage is widespread. Improved understanding of which neurons are most vulnerable and why should be useful in developing treatment strategies for neurodegenerative diseases that involve transcellular spreading of aggregates.

Cell attachment functionality of bioactive conducting polymers for neural interfaces.

PubMed

Green, Rylie A; Lovell, Nigel H; Poole-Warren, Laura A

2009-08-01

Bioactive coatings for neural electrodes that are tailored for cell interactions have the potential to produce superior implants with improved charge transfer capabilities. In this study synthetically produced anionically modified laminin peptides DEDEDYFQRYLI and DCDPGYIGSR were used to dope poly(3,4-ethylenedioxythiophene) (PEDOT) electrodeposited on platinum (Pt) electrodes. Performance of peptide doped films was compared to conventional polymer PEDOT/paratoluene sulfonate (pTS) films using SEM, XPS, cyclic voltammetry, impedance spectroscopy, mechanical hardness and adherence. Bioactivity of incorporated peptides and their affect on cell growth was assessed using a PC12 neurite outgrowth assay. It was demonstrated that large peptide dopants produced softer PEDOT films with a minimal decrease in electrochemical stability, compared to the conventional dopant, pTS. Cell studies revealed that the YFQRYLI ligand retained neurite outgrowth bioactivity when DEDEDYFQRYLI was used as a dopant, but the effect was strongly dependant on initial cell attachment. Alternate peptide dopant, DCDPGYIGSR was found to impart superior cell attachment properties when compared to DEDEDYFQRYLI, but attachment on both peptide doped polymers could be enhanced by coating with whole native laminin.

Attachment Capability of Antagonistic Yeast Rhodotorula glutinis to Botrytis cinerea Contributes to Biocontrol Efficacy.

PubMed

Li, Boqiang; Peng, Huaimin; Tian, Shiping

2016-01-01

Rhodotorula glutinis as an antagonism show good biocontrol performance against various post-harvest diseases in fruits. In the present study, strong attachment capability of R. glutinis to spores and hyphae of Botrytis cinerea was observed. Further analysis showed that certain protein components on the yeast cell surface played critical role during the interaction between R. glutinis and B. cinerea. The components mainly distributed at the poles of yeast cells and might contain glycosylation modification, as tunicamycin treated yeast cells lost attachment capability to B. cinerea. To investigate contributions of attachment capability of R. glutinis to its biocontrol efficacy, yeast cells were mutagenized with 3% methane-sulfonic acid ethyl ester (EMS), and a mutant CE4 with stable non-attaching phenotype was obtained. No significant difference was found on colony, cell morphology, reproductive ability, and capsule formation between the mutant and wild-type. However, there was a distinct difference in India ink positive staining patterns between the two strains. Moreover, wild-type strain of R. glutinis showed better performance on inhibiting spore germination and mycelial growth of B. cinerea than CE4 strain when yeast cells and B. cinerea were co-cultured in vitro. In biocontrol assay, both wild-type and CE4 strains showed significant biocontrol efficacy against gray mold caused by B. cinerea in apple fruit, whereas, control effect of CE4 strain was lower than that of wild-type. Our findings provided new evidences that attachment capability of R. glutinis to B. cinerea contributed to its biocontrol efficacy.

Attachment Capability of Antagonistic Yeast Rhodotorula glutinis to Botrytis cinerea Contributes to Biocontrol Efficacy

PubMed Central

Li, Boqiang; Peng, Huaimin; Tian, Shiping

2016-01-01

Rhodotorula glutinis as an antagonism show good biocontrol performance against various post-harvest diseases in fruits. In the present study, strong attachment capability of R. glutinis to spores and hyphae of Botrytis cinerea was observed. Further analysis showed that certain protein components on the yeast cell surface played critical role during the interaction between R. glutinis and B. cinerea. The components mainly distributed at the poles of yeast cells and might contain glycosylation modification, as tunicamycin treated yeast cells lost attachment capability to B. cinerea. To investigate contributions of attachment capability of R. glutinis to its biocontrol efficacy, yeast cells were mutagenized with 3% methane-sulfonic acid ethyl ester (EMS), and a mutant CE4 with stable non-attaching phenotype was obtained. No significant difference was found on colony, cell morphology, reproductive ability, and capsule formation between the mutant and wild-type. However, there was a distinct difference in India ink positive staining patterns between the two strains. Moreover, wild-type strain of R. glutinis showed better performance on inhibiting spore germination and mycelial growth of B. cinerea than CE4 strain when yeast cells and B. cinerea were co-cultured in vitro. In biocontrol assay, both wild-type and CE4 strains showed significant biocontrol efficacy against gray mold caused by B. cinerea in apple fruit, whereas, control effect of CE4 strain was lower than that of wild-type. Our findings provided new evidences that attachment capability of R. glutinis to B. cinerea contributed to its biocontrol efficacy. PMID:27199931

Evaluation of Blood Cell Attachment on Er:Yag Laser Applied Root Surface Using Scanning Electron Microscopy

PubMed Central

CEKICI, Ali; MADEN, Ilay; YILDIZ, Sercan; SAN, Tangul; ISIK, Gulden

2013-01-01

Background: Periodontal regeneration is dependent on the uninterrupted adhesion, maturation and absorption of fibrin clots to a periodontally compromised root surface. The modification of the root surface with different agents has been used for better fibrin clot formation and blood cell attachment. It is known that Er:YAG laser application on dentin removes the smear layer succesfully. Aim: The aim of this study is to observe blood cell attachment and fibrin network formation following ER:YAG laser irradiation on periodontally compromised root surfaces in comparison to chemical root conditioning techniques in vitro. Materials and methods: 40 dentin blocks prepared from freshly extracted periodontally compromised hopeless teeth. Specimens were divided in 5 groups; those applied with PBS, EDTA, Citric acid and Er:YAG. They were further divided into two groups: those which had received these applications, and the control group. The specimens were evaluated with scanning electron microscope and micrographs were taken. Smear layer and blood cell attachment scoring was performed. Results: In the Er:YAG laser applied group, smear layer were totally removed. In the blood applied specimens, better fibrin clot formation and blood cell attachment were observed in the Er:YAG group. In the group that had been applied with citric acid, the smear layer was also removed. The smear layer could not be fully removed in the EDTA group. Conclusion: Er:YAG laser application on the root dentin seems to form a suitable surface for fibrin clot formation and blood cell attachment. Further clinical studies to support these results are necessitated. PMID:23533017

Antioxidant ameliorating effects against H2O2-induced cytotoxicity in primary endometrial cells.

PubMed

Zal, F; Khademi, F; Taheri, R; Mostafavi-Pour, Z

2018-02-01

Oxidative stress and a disrupted antioxidant system are involved in a variety of pregnancy complications. In the present study, the role of vitamin E (Vit E) and folate as radical scavengers on the GSH homeostasis in stress oxidative induced in rat endometrial cells was investigated. Primary endometrial stromal cell cultures treated with 50 and 200 µM of H 2 O 2 and evaluated the cytoprotective effects of Vit E (5 µM) and folate (0.01 µM) in H 2 O 2 -treated cells for 24 h. Following the exposure of endometrial cells to H 2 O 2 alone and in the presence of Vit E and/or folate, cell survival, glutathione peroxidase (GPx) and glutathione reductase activities and the level of reduced glutathione (GSH) were measured. Cell adhesions comprise of cell attachment and spreading on collagen were determined. Flow cytometric analysis using annexin V was used to measure apoptosis. H 2 O 2 treatment showed a marked decrease in cell viability, GPx and GR activities and the level of GSH. Although Vit E or folate had some protective effect, combination therapy with Vit E and folate attenuated all the changes due to H 2 O 2 toxicity. An increasing number of alive cells was showed in the cells exposed to H 2 O 2 (50 µM) accompanied by co-treatment with Vit E and folic acid. The present findings indicate that co-administration of Vit E and folate before and during pregnancy may maintain a viable pregnancy and contribute to its clinical efficacy for the treatment of some idiopathic infertility.

A miniaturized bioreactor system for the evaluation of cell interaction with designed substrates in perfusion culture.

PubMed

Sun, T; Donoghue, P S; Higginson, J R; Gadegaard, N; Barnett, S C; Riehle, M O

2012-12-01

In tissue engineering, chemical and topographical cues are normally developed using static cell cultures but then applied directly to tissue cultures in three dimensions (3D) and under perfusion. As human cells are very sensitive to changes in the culture environment, it is essential to evaluate the performance of any such cues in a perfused environment before they are applied to tissue engineering. Thus, the aim of this research was to bridge the gap between static and perfusion cultures by addressing the effect of perfusion on cell cultures within 3D scaffolds. For this we developed a scaled-down bioreactor system, which allows evaluation of the effectiveness of various chemical and topographical cues incorporated into our previously developed tubular ε-polycaprolactone scaffold under perfused conditions. Investigation of two exemplary cell types (fibroblasts and cortical astrocytes) using the miniaturized bioreactor indicated that: (a) quick and firm cell adhesion in the 3D scaffold was critical for cell survival in perfusion culture compared with static culture; thus, cell-seeding procedures for static cultures might not be applicable, therefore it was necessary to re-evaluate cell attachment on different surfaces under perfused conditions before a 3D scaffold was applied for tissue cultures; (b) continuous medium perfusion adversely influenced cell spread and survival, which could be balanced by intermittent perfusion; (c) micro-grooves still maintained their influences on cell alignment under perfused conditions, while medium perfusion demonstrated additional influence on fibroblast alignment but not on astrocyte alignment on grooved substrates. This research demonstrated that the mini-bioreactor system is crucial for the development of functional scaffolds with suitable chemical and topographical cues by bridging the gap between static culture and perfusion culture. Copyright © 2011 John Wiley & Sons, Ltd.

Neuronal and Astrocytic Monoacylglycerol Lipase Limit the Spread of Endocannabinoid Signaling in the Cerebellum.

PubMed

Chen, Yao; Liu, Xiaojie; Vickstrom, Casey R; Liu, Michelle J; Zhao, Li; Viader, Andreu; Cravatt, Benjamin F; Liu, Qing-Song

2016-01-01

Endocannabinoids are diffusible lipophilic molecules that may spread to neighboring synapses. Monoacylglycerol lipase (MAGL) is the principal enzyme that degrades the endocannabinoid 2-arachidonoylglycerol (2-AG). Using knock-out mice in which MAGL is deleted globally or selectively in neurons and astrocytes, we investigated the extent to which neuronal and astrocytic MAGL limit the spread of 2-AG-mediated retrograde synaptic depression in cerebellar slices. A brief tetanic stimulation of parallel fibers in the molecular layer induced synaptically evoked suppression of excitation (SSE) in Purkinje cells, and both neuronal and astrocytic MAGL contribute to the termination of this form of endocannabinoid-mediated synaptic depression. The spread of SSE among Purkinje cells occurred only after global knock-out of MAGL or pharmacological blockade of either MAGL or glutamate uptake, but no spread was detected following neuron- or astrocyte-specific deletion of MAGL. The spread of endocannabinoid signaling was also influenced by the spatial pattern of synaptic stimulation, because it did not occur at spatially dispersed parallel fiber synapses induced by stimulating the granular layer. The tetanic stimulation of parallel fibers did not induce endocannabinoid-mediated synaptic suppression in Golgi cells even after disruption of MAGL and glutamate uptake, suggesting that heightened release of 2-AG by Purkinje cells does not spread the retrograde signal to parallel fibers that innervate Golgi cells. These results suggest that both neuronal and astrocytic MAGL limit the spatial diffusion of 2-AG and confer synapse-specificity of endocannabinoid signaling.

Information hiding technique

NASA Astrophysics Data System (ADS)

Younger, Michael; Budulas, Peter P.; Young, Stuart H.

2002-08-01

Spread spectrum communication techniques have been recognized as a viable method to gain an advantage in interference environments. Many military-oriented systems have been initiated, and some civil systems have been attempted. Spread spectrum allows the ability to hide the signal of interest below or in the noise floor, so as not to be detected. A spread spectrum system is one in which the transmitted signal is spread over a wide frequency band, much wider, in fact, than the minimum bandwidth required to transmit the information being sent. We at Army Research Lab (ARL) are proposing using the same technique on the Internet with port hopping. The information would be transmitted in data packets over multiple ports. The port used would vary per packet or per session basis. This port hopping gives you and the recipients the ability to take datagram's and spread them out over a multitude of ports. This will hide information among the Internet noise. This will allow trusted communications between the transmitter and receiver because of the port coding sequence. There are 64K possible ports to span datagram. Jamming of transmission would be limiting the ability of the sniffer/listener. Also, the listener will find it difficult to use a man in the middle attach, since the data will be spread over multiple ports and only the receiver and transmitter will know the specific port sequencing for the datagram.

Radiological detection of extracapsular spread in head and neck squamous cell carcinoma (HNSCC) cervical metastases.

PubMed

Url, C; Schartinger, V H; Riechelmann, H; Glückert, R; Maier, H; Trumpp, M; Widmann, G

2013-10-01

Extracapsular spread of cervical lymph nodes deteriorates the prognosis of patients with head and neck squamous cell carcinoma. Postoperative radiochemotherapy is superior to postoperative radiotherapy alone in patients with histologically proven extracapsular spread. If extracapsular spread can be detected preoperatively, patients may favor primary radiochemotherapy instead of primary surgery plus postoperative radiochemotherapy. Computed tomography (CT) scans of nodal positive head and neck squamous cell carcinoma patients treated between 2008 and 2010 with comprehensive neck dissection as part of first line surgical treatment were retrospectively scanned for extracapsular spread by two blinded radiologists. If a positive lymph node was identified by the pathologist, CT scans were assessed for extracapsular spread retrospectively. CT criteria for Extracapsular spread were apparent fat and soft tissue infiltration or infiltration of sternocleidomastoid muscle, internal jugular vein or carotid artery. Radiologic judgment was compared with histological evidence of extracapsular spread and specificity and sensitivity of CT detection was calculated. Forty-nine patients with histologically proven positive lymph nodes (pN+) were included. Extracapsular spread was histologically proven in 17 cases; the number of all affected lymph nodes was not listed. Radiologist 1 found extracapsular spread in CT scans of 15/49 patients and radiologist 2 in 16/49 patients (Cohen's kappa=0.86; p<0.01). Sensitivity of radiologic extracapsular spread detection was 73% (95% confidential index (CI): 44.0-89.7%) and specificity 91% (75.0-98.0%). Extracapsular spread depicted on computed tomography using strict criteria has high specificity. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Toxoplasma gondii-infected natural killer cells display a hypermotility phenotype in vivo.

PubMed

Ueno, Norikiyo; Lodoen, Melissa B; Hickey, Graeme L; Robey, Ellen A; Coombes, Janine L

2015-01-01

Toxoplasma gondii is a highly prevalent intracellular protozoan parasite that causes severe disease in congenitally infected or immunocompromised hosts. T. gondii is capable of invading immune cells and it has been suggested that the parasite harnesses the migratory pathways of these cells to spread through the body. Although in vitro evidence suggests that the parasite further enhances its spread by inducing a hypermotility phenotype in parasitized immune cells, in vivo evidence for this phenomenon is scarce. Here we use a physiologically relevant oral model of T. gondii infection, in conjunction with two-photon laser scanning microscopy, to address this issue. We found that a small proportion of natural killer (NK) cells in mesenteric lymph nodes contained parasites. Compared with uninfected 'bystander' NK cells, these infected NK cells showed faster, more directed and more persistent migratory behavior. Consistent with this, infected NK cells showed impaired spreading and clustering of the integrin, LFA-1, when exposed to plated ligands. Our results provide the first evidence for a hypermigratory phenotype in T. gondii-infected NK cells in vivo, providing an anatomical context for understanding how the parasite manipulates immune cell motility to spread through the host.

The role of basal cells in adhesion of columnar epithelium to airway basement membrane.

PubMed

Evans, M J; Plopper, C G

1988-08-01

In this report, we present a new concept of the role of the basal cell in airway epithelium. Previously, the basal cell was thought to be the progenitor cell for the columnar epithelium. However, several studies have shown that this concept may not be correct. The morphologic aspects of the basal cell suggest that it could play a role in adhesion of the columnar epithelium to the basement membrane. Basal cells form attachments with columnar cells (desmosomes) and with the basement membrane (hemidesmosomes). Columnar cells do not form hemidesmosome attachments with the basement membrane. Basal cells could strengthen the adhesion of columnar cells to the basement membrane by forming hemidesmosome attachments to the basement membrane and desmosome attachments with adjacent columnar cells. Incidental evidence from 2 existing publications concerning airway microanatomy support this concept. As columnar cells grow taller, the proportion of the cell surface in contact with the basement membrane becomes progressively smaller, and thus the cell surface area related to adhesion also becomes smaller. It was found that the number of basal cells per millimeter of basement membrane was closely related to the height of the columnar cell epithelium (r = 0.98), but not to the number of columnar cells (r = 0.42). The consistency of the relationship between increased columnar cell height (and thus decreased surface area for adhesion) and the number of basal cells present (r = 0.98) supports the concept that the basal cell plays a role in adhesion of columnar cells to the basement membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Extra-embryonic tissue spreading directs early embryo morphogenesis in killifish

PubMed Central

Reig, Germán; Cerda, Mauricio; Sepúlveda, Néstor; Flores, Daniela; Castañeda, Victor; Tada, Masazumi; Härtel, Steffen; Concha, Miguel L.

2017-01-01

The spreading of mesenchymal-like cell layers is critical for embryo morphogenesis and tissue repair, yet we know little of this process in vivo. Here we take advantage of unique developmental features of the non-conventional annual killifish embryo to study the principles underlying tissue spreading in a simple cellular environment, devoid of patterning signals and major morphogenetic cell movements. Using in vivo experimentation and physical modelling we reveal that the extra-embryonic epithelial enveloping cell layer, thought mainly to provide protection to the embryo, directs cell migration and the spreading of embryonic tissue during early development. This function relies on the ability of embryonic cells to couple their autonomous random motility to non-autonomous signals arising from the expansion of the extra-embryonic epithelium, mediated by cell membrane adhesion and tension. Thus, we present a mechanism of extra-embryonic control of embryo morphogenesis that couples the mechanical properties of adjacent tissues in the early killifish embryo. PMID:28580937

Vinculin promotes cell spreading by mechanically coupling integrins to the cytoskeleton

NASA Technical Reports Server (NTRS)

Ezzell, R. M.; Goldmann, W. H.; Wang, N.; Parasharama, N.; Ingber, D. E.

1997-01-01

Mouse F9 embryonic carcinoma 5.51 cells that lack the cytoskeletal protein vinculin spread poorly on extracellular matrix compared with wild-type F9 cells or two vinculin-transfected clones (5.51Vin3 and Vin4; Samuels et al., 1993, J. Cell Biol. 121, 909-921). In the present study, we used this model system to determine how the presence of vinculin promotes cytoskeletal alterations and associated changes in cell shape. Microscopic analysis of cell spreading at early times, revealed that 5.51 cells retained the ability to form filopodia; however, they could not form lamellipodia, assemble stress fibers, or efficiently spread over the culture substrate. Detergent (Triton X-100) studies revealed that these major differences in cell morphology and cytoskeletal organization did not result from differences in levels of total polymerized or cross-linked actin. Biochemical studies showed that 5.51 cells, in addition to lacking vinculin, exhibited slightly reduced levels of alpha-actinin and paxillin in their detergent-insoluble cytoskeleton. The absence of vinculin correlated with a decrease in the mechanical stiffness of the integrin-cytoskeleton linkage, as measured using cell magnetometry. Furthermore, when vinculin was replaced by transfection in 5.51Vin3 and 5.51Vin4 cells, the levels of cytoskeletal-associated alpha-actinin and paxillin, the efficiency of transmembrane mechanical coupling, and the formation of actin stress fibers were all restored to near wild-type levels. These findings suggest that vinculin may promote cell spreading by stabilizing focal adhesions and transferring mechanical stresses that drive cytoskeletal remodeling, rather than by altering the total level of actin polymerization or cross-linking.

Dabrafenib

MedlinePlus

... to treat a certain type of non-small cell lung cancer (NSCLC) that has spread to nearby tissues or ... the action of an abnormal protein that signals cancer cells to multiply. This helps stop the spread of ...

Trametinib

MedlinePlus

... to treat a certain type of non-small-cell lung cancer (NSCLC) that has spread to nearby tissues or ... the action of an abnormal protein that signals cancer cells to multiply. This helps stop the spread of ...

Attachment and internalization of a Chlamydia trachomatis lymphogranuloma venereum strain by McCoy cells: kinetics of infectivity and effect of lectins and carbohydrates.

PubMed Central

Söderlund, G; Kihlström, E

1983-01-01

The kinetics of attachment and ingestion of Chlamydia trachomatis serotype L1 by monolayers of McCoy cells were studied by using a method that discriminated between attachment and uptake. When about 1% of the McCoy cells was infected, the proteinase K-resistant chlamydial fraction, regarded as ingested chlamydiae, reached a constant value after about 3 h of incubation at 37 degrees C. Uptake of chlamydiae at 4 degrees C could not be demonstrated. The attached and ingested chlamydial fractions were constant over an eightfold increase in chlamydial inoculum. Chitobiose and chitotriose, the di- and trisaccharides of N-acetyl-D-glucosamine, reduced the association of C. trachomatis serotype L1 with McCoy cells. Higher concentrations of chitobiose also selectively inhibited ingestion of chlamydiae. A corresponding effect of chitobiose was also observed on the number of chlamydial inclusions. Wheat germ agglutinin, specific for N-acetyl-D-glucosamine residues, reduced the association of chlamydiae when incubated at 4 degrees C, but not at 37 degrees C. A small inhibiting effect of concanavalin A on association of chlamydiae, but no effect of the corresponding carbohydrates, indicates a nonspecific effect on chlamydial attachment of this lectin. These results suggest that beta 1 leads to 4-linked oligomers of N-acetyl-D-glucosamine are important in the specificity of attachment of C. trachomatis to McCoy cells. PMID:6642670

Enhancement of Cell Attachment to a Substrate Coated with Oral Bacterial Endotoxin by Plasma Fibronectin

DTIC Science & Technology

1983-01-01

root surfaces is unpredict- human gingival fibroblasts (Aleo. De Renzis able (World Workshop in PeriodonticN & Farber 1975). Clearly, if new attachment...1966). of periodontal tissues to a tooth is to be A preliminary characterization of the made possible, therapeutic measures must FIBRONECTIN ENHANCES...CELL ATTACHMENT 155 list he developed it remove. alter. or other- population of bacteria wsithin the gingival wise inactivate the toxic principle of

Traction in smooth muscle cells varies with cell spreading

NASA Technical Reports Server (NTRS)

Tolic-Norrelykke, Iva Marija; Wang, Ning

2005-01-01

Changes in cell shape regulate cell growth, differentiation, and apoptosis. It has been suggested that the regulation of cell function by the cell shape is a result of the tension in the cytoskeleton and the distortion of the cell. Here we explore the association between cell-generated mechanical forces and the cell morphology. We hypothesized that the cell contractile force is associated with the degree of cell spreading, in particular with the cell length. We measured traction fields of single human airway smooth muscle cells plated on a polyacrylamide gel, in which fluorescent microbeads were embedded to serve as markers of gel deformation. The traction exerted by the cells at the cell-substrate interface was determined from the measured deformation of the gel. The traction was measured before and after treatment with the contractile agonist histamine, or the relaxing agonist isoproterenol. The relative increase in traction induced by histamine was negatively correlated with the baseline traction. On the contrary, the relative decrease in traction due to isoproterenol was independent of the baseline traction, but it was associated with cell shape: traction decreased more in elongated than in round cells. Maximum cell width, mean cell width, and projected area of the cell were the parameters most tightly coupled to both baseline and histamine-induced traction in this study. Wide and well-spread cells exerted larger traction than slim cells. These results suggest that cell contractility is controlled by cell spreading.

Use of high concentrations of dimethyl sulfoxide for cryopreservation of HepG2 cells adhered to glass and polydimethylsiloxane matrices.

PubMed

Nagahara, Yukitoshi; Sekine, Hiroaki; Otaki, Mari; Hayashi, Masakazu; Murase, Norio

2016-02-01

Animal cells are generally cryopreserved in cryovials in a cell suspension state containing 5%-10% v/v dimethyl sulfoxide (DMSO) used as a cryoprotective agent. However, cryopreservation of cells in an attached state has not been intensively studied, and the effective freezing solution remains unknown. Here we determined the suitable DMSO concentration for the cryopreservation of human hepatoma HepG2 cells attached to glass and polydimethylsiloxane (PDMS) matrices coated with poly-l-lysine. With the use of the glass matrix, the rate of cell adhesion increased with the DMSO concentration up to 30% v/v in the freezing solution. In contrast, the cell-adhesion rate remained constant in the case of the PDMS matrix irrespective of the DMSO concentration between 10% v/v and 30% v/v. The viability of post-thawed cells attached to glass or PDMS matrix was also investigated. The viability was highest at the DMSO concentration of 20% v/v in the freezing solution. The DMSO concentration of 30% v/v, however, had a cytotoxic effect on the cell viability. Thus, the 20% v/v DMSO concentration was found to be most suitable for the cryopreservation of HepG2 cells in the attached state. This dose is high compared to the DMSO concentration used for the cryopreservation of cells in the suspended state. Copyright © 2015 Elsevier Inc. All rights reserved.

Earth observation taken by the Expedition 43 crew

NASA Image and Video Library

2015-04-18

ISS043E125469 (04/18/2015) --- A dying sun turns the Earth gold with cities sparkling to match the stars that spread out from the International Space Station. NASA astronaut Terry Verts tweeted this wondrous image with the attached comment about the stars: " See those light clusters? (center sky left and right)They are Magellanic clouds, a duo of dwarf galaxies seen from the southern hemisphere".

INTEGRIN-MEDIATED CELL ATTACHMENT SHOWS TIME-DEPENDENT UPREGULATION OF GAP JUNCTION COMMUNICATION.

EPA Science Inventory

Integrin-mediated Cell Attachment Shows Time-Dependent Upregulation of Gap Junction
Communication

Rachel Grindstaff and Carl Blackman, National Health & Environmental Effects Research
Laboratory, Office of Research and Development, US EPA, Research Triang...

High Concentration of Red Clay as an Alternative for Antibiotics in Aquaculture.

PubMed

Jung, Jaejoon; Jee, Seung Cheol; Sung, Jung-Suk; Park, Woojun

2016-01-01

The use of antibiotics in aquaculture raises environmental and food safety concerns because chronic exposure of an aquatic ecosystem to antibiotics can result in the spread of antibiotic resistance, bioaccumulation of antibiotics in the organisms, and transfer of antibiotics to humans. In an attempt to overcome these problems, high-concentration red clay was applied as an alternative antibiotic against the following common fish pathogens: Aeromonas salmonicida, Vibrio alginolyticus, and Streptococcus equinus. The growth of A. salmonicida and V. alginolyticus was retarded by red clay, whereas that of S. equinus was promoted. Phase contrast and scanning electron microscopy analyses confirmed the attachment of red clay on cell surfaces, resulting in rapid gravitational removal and cell surface damage in both A. salmonicida and V. alginolyticus, but not in S. equinus. Different cell wall properties of grampositive species may explain the unharmed cell surface of S. equinus. Significant levels of oxidative stress were generated in only the former two species, whereas significant changes in membrane permeability were found only in S. equinus, probably because of its physiological adaptation. The bacterial communities in water samples from Oncorhynchus mykiss aquacultures supplemented with red clay showed similar structure and diversity as those from oxytetracycline-treated water. Taken together, the antibiotic effects of high concentrations of red clay in aquaculture can be attributed to gravitational removal, cell surface damage, and oxidative stress production, and suggest that red clay may be used as an alternative for antibiotics in aquaculture.

Adipose tissue-derived stem cell response to the differently processed 316L stainless steel substrates.

PubMed

Faghihi, Shahab; Zia, Sonia; Taha, Masoumeh Fakhr

2012-12-01

Stainless steel (SS) is one of the most applicable materials in fabrication of cardiac implants. The aim of this study is to investigate the effect of atomic structure of polycrystalline stainless steel on the response of adipose tissue-derived stem cells (ADSCs). Samples are prepared from differently processed extruded rod and rolled sheet of 316L SS having different crystallographic structure. X-ray diffraction analysis indicated (200) and (111) orientations with distinct volume fractions in the specimens. Morphology and ADSCs behavior including adhesion, proliferation and differentiation are assessed. The expression of cardiac specific protein (cardiac troponin I) and genes of differentiating cardiomyocytes is analyzed by immunofluorescence and RT-PCR. The number of attached and grown cells on the rod sample is higher than the sheet sample also the scanning electron microscopy (SEM) analysis of ADSCs grown on the samples demonstrates higher cell density and spreading pattern on the surface of rod sample. In differentiated ADSCs on the rod sample the expression of all genes except ANF are detectable, while on the sheet sample only the MEF2C and β-MHC are expressed. This study shows that the cellular response is influenced by the crystal structure of the substrate therefore; the skill to alter the structure of substrate may lend itself to engineer a biomaterial which could be suitable for differentiation of stem cells into a definite lineage. Copyright © 2012 Elsevier Ltd. All rights reserved.

MicroRNA miR-30 family regulates non-attachment growth of breast cancer cells

PubMed Central

2013-01-01

Background A subset of breast cancer cells displays increased ability to self-renew and reproduce breast cancer heterogeneity. The characterization of these so-called putative breast tumor-initiating cells (BT-ICs) may open the road for novel therapeutic strategies. As microRNAs (miRNAs) control developmental programs in stem cells, BT-ICs may also rely on specific miRNA profiles for their sustained activity. To explore the notion that miRNAs may have a role in sustaining BT-ICs, we performed a comprehensive profiling of miRNA expression in a model of putative BT-ICs enriched by non-attachment growth conditions. Results We found breast cancer cells grown under non-attachment conditions display a unique pattern of miRNA expression, highlighted by a marked low expression of miR-30 family members relative to parental cells. We further show that miR-30a regulates non-attachment growth. A target screening revealed that miR-30 family redundantly modulates the expression of apoptosis and proliferation-related genes. At least one of these targets, the anti-apoptotic protein AVEN, was able to partially revert the effect of miR-30a overexpression. Finally, overexpression of miR-30a in vivo was associated with reduced breast tumor progression. Conclusions miR30-family regulates the growth of breast cancer cells in non-attachment conditions. This is the first analysis of target prediction in a whole family of microRNAs potentially involved in survival of putative BT-ICs. PMID:23445407

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

PubMed Central

Zhao, Nan; Zhu, Donghui

2014-01-01

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

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

PubMed

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

2018-06-07

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

Extracellular Polymeric Substance Production and Aggregated Bacteria Colonization Influence the Competition of Microbes in Biofilms.

PubMed

Jayathilake, Pahala G; Jana, Saikat; Rushton, Steve; Swailes, David; Bridgens, Ben; Curtis, Tom; Chen, Jinju

2017-01-01

The production of extracellular polymeric substance (EPS) is important for the survival of biofilms. However, EPS production is costly for bacteria and the bacterial strains that produce EPS (EPS+) grow in the same environment as non-producers (EPS-) leading to competition between these strains for nutrients and space. The outcome of this competition is likely to be dependent on factors such as initial attachment, EPS production rate, ambient nutrient levels and quorum sensing. We use an Individual-based Model (IbM) to study the competition between EPS+ and EPS- strains by varying the nature of initial colonizers which can either be in the form of single cells or multicellular aggregates. The microbes with EPS+ characteristics obtain a competitive advantage if they initially colonize the surface as smaller aggregates and are widely spread-out between the cells of EPS-, when both are deposited on the substratum. Furthermore, the results show that quorum sensing-regulated EPS production may significantly reduce the fitness of EPS producers when they initially deposit as aggregates. The results provide insights into how the distribution of bacterial aggregates during initial colonization could be a deciding factor in the competition among different strains in biofilms.

Low modulus and bioactive Ti/α-TCP/Ti-mesh composite prepared by spark plasma sintering.

PubMed

Guo, Yu; Tan, Yanni; Liu, Yong; Liu, Shifeng; Zhou, Rui; Tang, Hanchun

2017-11-01

A titanium mesh scaffold composite filled with Ti/α-TCP particles was prepared by spark plasma sintering (SPS). The microstructures and interfacial reactions of the composites were investigated by scanning electron microscopy (SEM), Energy Dispersive Spectroscopy (EDS) and X-ray diffraction (XRD) analyses. The compressive strength and elastic modulus were also measured. In vitro bioactivity and biocompatibility was evaluated by using simulated body fluid and cells culture, respectively. After high temperature sintering, Ti oxides, Ti x P y and CaTiO 3 were formed. The formation of Ti oxides and Ti x P y were resulted from the diffusion of O and P elements from α-TCP to Ti. CaTiO 3 was the reaction product of Ti and α-TCP. The composite of 70Ti/α-TCP incorporated with Ti mesh showed a high compressive strength of 589MPa and a low compressive modulus of 30GPa. The bioactivity test showed the formation of a thick apatite layer on the composite and well-spread cells attachment. A good combination of mechanical properties and bioactivity indicated a high potential application of Ti/α-TCP/Ti-mesh composite for orthopedic implants. Copyright © 2017. Published by Elsevier B.V.

Increasing Mechanical Strength of Gelatin Hydrogels by Divalent Metal Ion Removal

PubMed Central

Xing, Qi; Yates, Keegan; Vogt, Caleb; Qian, Zichen; Frost, Megan C.; Zhao, Feng

2014-01-01

The usage of gelatin hydrogel is limited due to its instability and poor mechanical properties, especially under physiological conditions. Divalent metal ions present in gelatin such as Ca2+ and Fe2+ play important roles in the gelatin molecule interactions. The objective of this study was to determine the impact of divalent ion removal on the stability and mechanical properties of gelatin gels with and without chemical crosslinking. The gelatin solution was purified by Chelex resin to replace divalent metal ions with sodium ions. The gel was then chemically crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Results showed that the removal of divalent metal ions significantly impacted the formation of the gelatin network. The purified gelatin hydrogels had less interactions between gelatin molecules and form larger-pore network which enabled EDC to penetrate and crosslink the gel more efficiently. The crosslinked purified gels showed small swelling ratio, higher crosslinking density and dramatically increased storage and loss moduli. The removal of divalent ions is a simple yet effective method that can significantly improve the stability and strength of gelatin hydrogels. The in vitro cell culture demonstrated that the purified gelatin maintained its ability to support cell attachment and spreading. PMID:24736500

Controlling the mechanics and nanotopography of biocompatible scaffolds through dielectrophoresis with carbon nanotubes.

PubMed

Lu, Yu-Lun; Cheng, Chao-Min; LeDuc, Philip R; Ho, Mon-Shu

2008-08-01

Creating a biocompatible carbon-nanotube polymer scaffold is an area that has a number of potential applications. Herein, a dielectrophoretic approach was pursued to integrate carbon nanotubes into a polymeric material for fabricating a nanoscale composite scaffold with increased and controllable mechanical strength. The adhesion force, which combines the surface energy of the sample and the interfacial energy between the tip and sample, was estimated to be 55.39 +/- 6.72 nN away from the center of the protrusions at a distance of 0.5 microm while being 24.01 +/- 4.45 nN at the center. The adhesion force for the center of the cavities was 42.47 +/- 6.91 and 88.21 +/- 15.05 nN at 0.5 microm away from the center. NIH 3T3 fibroblast cells were then utilized to test the cellular biocompatibility of this multiwalled carbon nanotubes (MWCNTs) film. Cells were cultured on the surface and then their attachment, spreading, and proliferation behaviors were observed. This nanotube-polymer scaffolding approach has a wide range of potential applications including in complex device fabrication as well as in developing biomimetic and tissue engineering scaffolds, and artificial organs.

Induction of virulence factors in Giardia duodenalis independent of host attachment

PubMed Central

Emery, Samantha J.; Mirzaei, Mehdi; Vuong, Daniel; Pascovici, Dana; Chick, Joel M.; Lacey, Ernest; Haynes, Paul A.

2016-01-01

Giardia duodenalis is responsible for the majority of parasitic gastroenteritis in humans worldwide. Host-parasite interaction models in vitro provide insights into disease and virulence and help us to understand pathogenesis. Using HT-29 intestinal epithelial cells (IEC) as a model we have demonstrated that initial sensitisation by host secretions reduces proclivity for trophozoite attachment, while inducing virulence factors. Host soluble factors triggered up-regulation of membrane and secreted proteins, including Tenascins, Cathepsin-B precursor, cystatin, and numerous Variant-specific Surface Proteins (VSPs). By comparison, host-cell attached trophozoites up-regulated intracellular pathways for ubiquitination, reactive oxygen species (ROS) detoxification and production of pyridoxal phosphate (PLP). We reason that these results demonstrate early pathogenesis in Giardia involves two independent host-parasite interactions. Motile trophozoites respond to soluble secreted signals, which deter attachment and induce expression of virulence factors. Trophozoites attached to host cells, in contrast, respond by up-regulating intracellular pathways involved in clearance of ROS, thus anticipating the host defence response. PMID:26867958

Models of collective cell spreading with variable cell aspect ratio: a motivation for degenerate diffusion models.

PubMed

Simpson, Matthew J; Baker, Ruth E; McCue, Scott W

2011-02-01

Continuum diffusion models are often used to represent the collective motion of cell populations. Most previous studies have simply used linear diffusion to represent collective cell spreading, while others found that degenerate nonlinear diffusion provides a better match to experimental cell density profiles. In the cell modeling literature there is no guidance available with regard to which approach is more appropriate for representing the spreading of cell populations. Furthermore, there is no knowledge of particular experimental measurements that can be made to distinguish between situations where these two models are appropriate. Here we provide a link between individual-based and continuum models using a multiscale approach in which we analyze the collective motion of a population of interacting agents in a generalized lattice-based exclusion process. For round agents that occupy a single lattice site, we find that the relevant continuum description of the system is a linear diffusion equation, whereas for elongated rod-shaped agents that occupy L adjacent lattice sites we find that the relevant continuum description is connected to the porous media equation (PME). The exponent in the nonlinear diffusivity function is related to the aspect ratio of the agents. Our work provides a physical connection between modeling collective cell spreading and the use of either the linear diffusion equation or the PME to represent cell density profiles. Results suggest that when using continuum models to represent cell population spreading, we should take care to account for variations in the cell aspect ratio because different aspect ratios lead to different continuum models.

Porcine Reproductive and Respiratory Syndrome Virus Utilizes Nanotubes for Intercellular Spread

PubMed Central

Guo, Rui; Katz, Benjamin B.; Tomich, John M.; Gallagher, Tom

2016-01-01

ABSTRACT Intercellular nanotube connections have been identified as an alternative pathway for cellular spreading of certain viruses. In cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), nanotubes were observed connecting two distant cells with contiguous membranes, with the core infectious viral machinery (viral RNA, certain replicases, and certain structural proteins) present in/on the intercellular nanotubes. Live-cell movies tracked the intercellular transport of a recombinant PRRSV that expressed green fluorescent protein (GFP)-tagged nsp2. In MARC-145 cells expressing PRRSV receptors, GFP-nsp2 moved from one cell to another through nanotubes in the presence of virus-neutralizing antibodies. Intercellular transport of viral proteins did not require the PRRSV receptor as it was observed in receptor-negative HEK-293T cells after transfection with an infectious clone of GFP-PRRSV. In addition, GFP-nsp2 was detected in HEK-293T cells cocultured with recombinant PRRSV-infected MARC-145 cells. The intercellular nanotubes contained filamentous actin (F-actin) with myosin-associated motor proteins. The F-actin and myosin IIA were identified as coprecipitates with PRRSV nsp1β, nsp2, nsp2TF, nsp4, nsp7-nsp8, GP5, and N proteins. Drugs inhibiting actin polymerization or myosin IIA activation prevented nanotube formation and viral clusters in virus-infected cells. These data lead us to propose that PRRSV utilizes the host cell cytoskeletal machinery inside nanotubes for efficient cell-to-cell spread. This form of virus transport represents an alternative pathway for virus spread, which is resistant to the host humoral immune response. IMPORTANCE Extracellular virus particles transmit infection between organisms, but within infected hosts intercellular infection can be spread by additional mechanisms. In this study, we describe an alternative pathway for intercellular transmission of PRRSV in which the virus uses nanotube connections to transport infectious viral RNA, certain replicases, and certain structural proteins to neighboring cells. This process involves interaction of viral proteins with cytoskeletal proteins that form the nanotube connections. Intercellular viral spread through nanotubes allows the virus to escape the neutralizing antibody response and may contribute to the pathogenesis of viral infections. The development of strategies that interfere with this process could be critical in preventing the spread of viral infection. PMID:26984724

Listeria membrane protrusion collapse: Requirement of Cyclophilin A for Listeria cell-to-cell spreading.

PubMed

Dhanda, Aaron S; Lulic, Katarina T; Vogl, A Wayne; Mc Gee, Margaret M; Chiu, Robert H; Guttman, Julian A

2018-05-04

Listeria generate actin-rich tubular protrusions at the plasma membrane that propel the bacteria into neighbouring cells. The precise molecular mechanisms governing the formation of these protrusions remain poorly defined. Here we demonstrate that the PPIase Cyclophilin A (CypA) is hijacked by Listeria at membrane protrusions used for cell-to-cell spreading. CypA localizes within the F-actin of these structures and is crucial for their proper formation, as in cells depleted of CypA, these extended actin-rich structures are mis-shaped and collapsed due to changes within the F-actin network. The lack of structural integrity within the Listeria membrane protrusions hampers the microbes from spreading from CypA null cells. Our results demonstrate a crucial role for CypA during Listeria infections.

Cell-to-cell transmission of retroviruses: Innate immunity and interferon-induced restriction factors

PubMed Central

Jolly, Clare

2011-01-01

It has been known for some time that retroviruses can disseminate between immune cells either by conventional cell-free transmission or by directed cell-to-cell spread. Over the past few years there has been increasing interest in how retroviruses may use cell-to-cell spread to promote more rapid infection kinetics and circumvent humoral immunity. Effective humoral immune responses are intimately linked with innate immunity and the interplay between retroviruses and innate immunity is a rapidly expanding area of research that has been advanced considerably by the identification of cellular restriction factors that provide barriers to retroviral infection. The effect of innate immunity and restriction factors on retroviral cell-to-cell spread has been comparatively little studied; however recent work suggests this maybe changing. Here I will review some recent advances in what is a budding area of retroviral research. PMID:21247613

Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)-Streptavidin and Biotinylated Fibronectin

PubMed Central

Anamelechi, Charles C.; Clermont, Edward C.; Novak, Matthew T.; Reichert, William M.

2014-01-01

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately. PMID:19348476

Shape-shifting trypanosomes: Flagellar shortening followed by asymmetric division in Trypanosoma congolense from the tsetse proventriculus.

PubMed

Peacock, Lori; Kay, Christopher; Bailey, Mick; Gibson, Wendy

2018-05-01

Trypanosomatids such as Leishmania and Trypanosoma are digenetic, single-celled, parasitic flagellates that undergo complex life cycles involving morphological and metabolic changes to fit them for survival in different environments within their mammalian and insect hosts. According to current consensus, asymmetric division enables trypanosomatids to achieve the major morphological rearrangements associated with transition between developmental stages. Contrary to this view, here we show that the African trypanosome Trypanosoma congolense, an important livestock pathogen, undergoes extensive cell remodelling, involving shortening of the cell body and flagellum, during its transition from free-swimming proventricular forms to attached epimastigotes in vitro. Shortening of the flagellum was associated with accumulation of PFR1, a major constituent of the paraflagellar rod, in the mid-region of the flagellum where it was attached to the substrate. However, the PFR1 depot was not essential for attachment, as it accumulated several hours after initial attachment of proventricular trypanosomes. Detergent and CaCl2 treatment failed to dislodge attached parasites, demonstrating the robust nature of flagellar attachment to the substrate; the PFR1 depot was also unaffected by these treatments. Division of the remodelled proventricular trypanosome was asymmetric, producing a small daughter cell. Each mother cell went on to produce at least one more daughter cell, while the daughter trypanosomes also proliferated, eventually resulting in a dense culture of epimastigotes. Here, by observing the synchronous development of the homogeneous population of trypanosomes in the tsetse proventriculus, we have been able to examine the transition from proventricular forms to attached epimastigotes in detail in T. congolense. This transition is difficult to observe in vivo as it happens inside the mouthparts of the tsetse fly. In T. brucei, this transition is achieved by asymmetric division of long trypomastigotes in the proventriculus, yielding short epimastigotes, which go on to colonise the salivary glands. Thus, despite their close evolutionary relationship and shared developmental route within the vector, T. brucei and T. congolense have evolved different ways of accomplishing the same developmental transition from proventricular form to attached epimastigote.

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

PubMed

Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

2013-10-25

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Single-Crystalline, Nanoporous Gallium Nitride Films With Fine Tuning of Pore Size for Stem Cell Engineering.

PubMed

Han, Lin; Zhou, Jing; Sun, Yubing; Zhang, Yu; Han, Jung; Fu, Jianping; Fan, Rong

2014-11-01

Single-crystalline nanoporous gallium nitride (GaN) thin films were fabricated with the pore size readily tunable in 20-100 nm. Uniform adhesion and spreading of human mesenchymal stem cells (hMSCs) seeded on these thin films peak on the surface with pore size of 30 nm. Substantial cell elongation emerges as pore size increases to ∼80 nm. The osteogenic differentiation of hMSCs occurs preferentially on the films with 30 nm sized nanopores, which is correlated with the optimum condition for cell spreading, which suggests that adhesion, spreading, and stem cell differentiation are interlinked and might be coregulated by nanotopography.

Quantification of Agrobacterium tumefaciens C58 attachment to Arabidopsis thaliana roots.

PubMed

Petrovicheva, Anna; Joyner, Jessica; Muth, Theodore R

2017-10-02

Agrobacterium tumefaciens is the causal agent of crown gall disease and is a vector for DNA transfer in transgenic plants. The transformation process by A. tumefaciens has been widely studied, but the attachment stage has not been well characterized. Most measurements of attachment have used microscopy and colony counting, both of which are labor and time intensive. To reduce the time and effort required to analyze bacteria attaching to plant tissues, we developed a quantitative real-time PCR (qPCR) assay to quantify attached A. tumefaciens using the chvE gene as marker for the presence of the bacteria. The qPCR detection threshold of A. tumefaciens from pure culture was 104 cell equivalents/ml. The A. tumefaciens minimum threshold concentration from root-bound populations was determined to be 105 cell equivalents/ml inoculum to detect attachment above background. The qPCR assay can be used for measuring A. tumefaciens attachment in applications such as testing the effects of mutations on bacterial adhesion molecules or biofilm formation, comparing attachment across various plant species and ecotypes, and detecting mutations in putative attachment receptors expressed in plant roots. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Substratum interfacial energetic effects on the attachment of marine bacteria

NASA Astrophysics Data System (ADS)

Ista, Linnea Kathryn

Biofilms represent an ancient, ubiquitous and influential form of life on earth. Biofilm formation is initiated by attachment of bacterial cells from an aqueous suspension onto a suitable attachment substratum. While in certain, well studied cases initial attachment and subsequent biofilm formation is mediated by specific ligand-receptor pairs on the bacteria and attachment substratum, in the open environment, including the ocean, it is assumed to be non-specific and mediated by processes similar to those that drive adsorption of colloids at the water-solid interface. Colloidal principles are studied to determine the molecular and physicochemical interactions involved in the attachment of the model marine bacterium, Cobetia marina to model self-assembled monolayer surfaces. In the simplest application of colloidal principles the wettability of attachment substrata, as measured by the advancing contact angle of water (theta AW) on the surface, is frequently used as an approximation for the surface tension. We demonstrate the applicability of this approach for attachment of C. marina and algal zoospores and extend it to the development of a means to control attachment and release of microorganisms by altering and tuning surface thetaAW. In many cases, however, thetaAW does not capture all the information necessary to model attachment of bacteria to attachment substrata; SAMs with similar thetaAW attach different number of bacteria. More advanced colloidal models of initial bacterial attachment have evolved over the last several decades, with the emergence of the model proposed by van Oss, Chaudhury and Good (VCG) as preeminent. The VCG model enables calculation of interfacial tensions by dividing these into two major interactions thought to be important at biointerfaces: apolar, Lifshitz-van der Waals and polar, Lewis acid-base (including hydrogen bonding) interactions. These interfacial tensions are combined to yield DeltaGadh, the free energy associated with attachment of bacteria to a substratum. We use VCG to model DeltaGadh and interfacial tensions as they relate to model bacterial attachment on SAMs that accumulate cells to different degrees. Even with the more complex interactions measured by VCG, surface energy of the attachment substratum alone was insufficient to predict attachment. VCG was then employed to model attachment of C. marina to a series of SAMs varying systematically in the number of ethylene glycol residues present in the molecule; an identical series has been previously shown to vary dramatically in the number of cells attached as a function of ethylene glycols present. Our results indicate that while VCG adequately models the interfacial tension between water and ethylene glycol SAMs in a manner that predicts bacterial attachment, DeltaGadh as calculated by VCG neither qualitatively nor quantitatively reflects the attachment data. The VCG model, thus, fails to capture specific information regarding the interactions between the attaching bacteria, water, and the SAM. We show that while hydrogen-bond accepting interactions are very well captured by this model, the ability for SAMs and bacteria to donate hydrogen bonds is not adequately described as the VCG model is currently applied. We also describe ways in which VCG fails to capture two specific biological aspects that may be important in bacterial attachment to surfaces:1.) specific interactions between molecules on the surface and bacteria and 2.) bacterial cell surface heterogeneities that may be important in differential attachment to different substrata.

Adhesion of glucosyltransferase phase variants to Streptococcus gordonii bacterium-glucan substrata may involve lipoteichoic acid.

PubMed

Vickerman, M M; Jones, G W

1992-10-01

Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3H]thymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp+ and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria.

Adhesion of glucosyltransferase phase variants to Streptococcus gordonii bacterium-glucan substrata may involve lipoteichoic acid.

PubMed Central

Vickerman, M M; Jones, G W

1992-01-01

Growing Streptococcus gordonii Spp+ phase variants, which have normal levels of glucosyltransferase (GTF) activity, use sucrose to promote their accumulation on surfaces by forming a cohesive bacterium-insoluble glucan polymer mass (BPM). Spp- phase variants, which have lower levels of GTF activity, do not form BPMs and do not remain in BPMs formed by Spp+ cells when grown in mixed cultures. To test the hypothesis that segregation of attached Spp+ and unattached Spp- cells was due to differences in adhesiveness, adhesion between washed, [3H]thymidine-labeled cells and preformed BPM substrata was measured. Unexpectedly, the results showed that cells of both phenotypes, as well as GTF-negative cells, attached equally well to preformed BPMs, indicating that attachment to BPMs was independent of cell surface GTF activity. Initial characterization of this binding interaction suggested that a protease-sensitive component on the washed cells may be binding to lipoteichoic acids sequestered in the BPM, since exogenous lipoteichoic acid inhibited adhesion. Surprisingly, the adhesion of both Spp+ and Spp- cells was markedly inhibited in the presence of sucrose, which also released lipoteichoic acid from the BPM. These in vitro findings suggest that, in vivo, sucrose and lipoteichoic acid may modify dental plaque development by enhancing or inhibiting the attachment of additional bacteria. PMID:1398940

Integrin Expression Regulates Neuroblastoma Attachment and Migration1

PubMed Central

Meyer, Amy; van Golen, Cynthia M.; Kim, Bhumsoo; van Golen, Kenneth L.; Feldman, Eva L.

2004-01-01

Abstract Neuroblastoma (NBL) is the most common malignant disease of infancy, and children with bone metastasis have a mortality rate greater than 90%. Two major classes of proteins, integrins and growth factors, regulate the metastatic process. We have previously shown that tumorigenic NBL cells express higher levels of the type I insulin-like growth factor receptor (IGF-IR) and that β1 integrin expression is inversely proportional to tumorigenic potential in NBL. In the current study, we analyze the effect of β1 integrin and IGF-IR on NBL cell attachment and migration. Nontumorigenic S-cells express high levels of β1 integrin, whereas tumorigenic N-cells express little β1 integrin. Alterations in β1 integrin are due to regulation at the protein level, as translation is decreased in N-type cells. Moreover, inhibition of protein synthesis shows that β1 integrin is degraded more slowly in S-type cells (SHEP) than in N-type cells (SH-SY5Y and IMR32). Inhibition of α5β1 integrin prevents SHEP (but not SH-SY5Y or IMR32) cell attachment to fibronectin and increases SHEP cell migration. Increases in IGF-IR decrease β1 integrin expression, and enhance SHEP cell migration, potentially through increased expression of αvβ3. These data suggest that specific classes of integrins in concert with IGF-IR regulate NBL attachment and migration. PMID:15256055

Molecular mechanisms of cell-cell spread of intracellular bacterial pathogens.

PubMed

Ireton, Keith

2013-07-17

Several bacterial pathogens, including Listeria monocytogenes, Shigella flexneri and Rickettsia spp., have evolved mechanisms to actively spread within human tissues. Spreading is initiated by the pathogen-induced recruitment of host filamentous (F)-actin. F-actin forms a tail behind the microbe, propelling it through the cytoplasm. The motile pathogen then encounters the host plasma membrane, forming a bacterium-containing protrusion that is engulfed by an adjacent cell. Over the past two decades, much progress has been made in elucidating mechanisms of F-actin tail formation. Listeria and Shigella produce tails of branched actin filaments by subverting the host Arp2/3 complex. By contrast, Rickettsia forms tails with linear actin filaments through a bacterial mimic of eukaryotic formins. Compared with F-actin tail formation, mechanisms controlling bacterial protrusions are less well understood. However, recent findings have highlighted the importance of pathogen manipulation of host cell-cell junctions in spread. Listeria produces a soluble protein that enhances bacterial protrusions by perturbing tight junctions. Shigella protrusions are engulfed through a clathrin-mediated pathway at 'tricellular junctions'--specialized membrane regions at the intersection of three epithelial cells. This review summarizes key past findings in pathogen spread, and focuses on recent developments in actin-based motility and the formation and internalization of bacterial protrusions.

Investigating the Influence of the Initial Biomass Distribution and Injection Strategies on Biofilm-Mediated Calcite Precipitation in Porous Media

DOE PAGES

Hommel, Johannes; Lauchnor, Ellen; Gerlach, Robin; ...

2015-12-16

Attachment of bacteria in porous media is a complex mixture of processes resulting in the transfer and immobilization of suspended cells onto a solid surface within the porous medium. However, quantifying the rate of attachment is difficult due to the many simultaneous processes possibly involved in attachment, including straining, sorption, and sedimentation, and the difficulties in measuring metabolically active cells attached to porous media. Preliminary experiments confirmed the difficulty associated with measuring active Sporosarcina pasteurii cells attached to porous media. However, attachment is a key process in applications of biofilm-mediated reactions in the subsurface such as microbially induced calcite precipitation.more » Independent of the exact processes involved, attachment determines both the distribution and the initial amount of attached biomass and as such the initial reaction rate. As direct experimental investigations are difficult, this study is limited to a numerical investigation of the effect of various initial biomass distributions and initial amounts of attached biomass. This is performed for various injection strategies, changing the injection rate as well as alternating between continuous and pulsed injections. The results of this study indicate that, for the selected scenarios, both the initial amount and the distribution of attached biomass have minor influence on the Ca 2+ precipitation efficiency as well as the distribution of the precipitates compared to the influence of the injection strategy. The influence of the initial biomass distribution on the resulting final distribution of the precipitated calcite is limited, except for the continuous injection at intermediate injection rate. But even for this injection strategy, the Ca 2+ precipitation efficiency shows no significant dependence on the initial biomass distribution.« less

Investigating the Influence of the Initial Biomass Distribution and Injection Strategies on Biofilm-Mediated Calcite Precipitation in Porous Media

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

Hommel, Johannes; Lauchnor, Ellen; Gerlach, Robin

Attachment of bacteria in porous media is a complex mixture of processes resulting in the transfer and immobilization of suspended cells onto a solid surface within the porous medium. However, quantifying the rate of attachment is difficult due to the many simultaneous processes possibly involved in attachment, including straining, sorption, and sedimentation, and the difficulties in measuring metabolically active cells attached to porous media. Preliminary experiments confirmed the difficulty associated with measuring active Sporosarcina pasteurii cells attached to porous media. However, attachment is a key process in applications of biofilm-mediated reactions in the subsurface such as microbially induced calcite precipitation.more » Independent of the exact processes involved, attachment determines both the distribution and the initial amount of attached biomass and as such the initial reaction rate. As direct experimental investigations are difficult, this study is limited to a numerical investigation of the effect of various initial biomass distributions and initial amounts of attached biomass. This is performed for various injection strategies, changing the injection rate as well as alternating between continuous and pulsed injections. The results of this study indicate that, for the selected scenarios, both the initial amount and the distribution of attached biomass have minor influence on the Ca 2+ precipitation efficiency as well as the distribution of the precipitates compared to the influence of the injection strategy. The influence of the initial biomass distribution on the resulting final distribution of the precipitated calcite is limited, except for the continuous injection at intermediate injection rate. But even for this injection strategy, the Ca 2+ precipitation efficiency shows no significant dependence on the initial biomass distribution.« less

Social climber attachment in forming networks produces a phase transition in a measure of connectivity

NASA Astrophysics Data System (ADS)

Taylor, Dane; Larremore, Daniel B.

2012-09-01

The formation and fragmentation of networks are typically studied using percolation theory, but most previous research has been restricted to studying a phase transition in cluster size, examining the emergence of a giant component. This approach does not study the effects of evolving network structure on dynamics that occur at the nodes, such as the synchronization of oscillators and the spread of information, epidemics, and neuronal excitations. We introduce and analyze an alternative link-formation rule, called social climber (SC) attachment, that may be combined with arbitrary percolation models to produce a phase transition using the largest eigenvalue of the network adjacency matrix as the order parameter. This eigenvalue is significant in the analyses of many network-coupled dynamical systems in which it measures the quality of global coupling and is hence a natural measure of connectivity. We highlight the important self-organized properties of SC attachment and discuss implications for controlling dynamics on networks.

Potential mechanisms for the effects of tea extracts on the attachment, biofilm formation and cell size of Streptococcus mutans.

PubMed

Wang, Yi; Lee, Sui M; Dykes, Gary A

2013-01-01

Tea can inhibit the attachment of Streptococcus mutans to surfaces and subsequent biofilm formation. Five commercial tea extracts were screened for their ability to inhibit attachment and biofilm formation by two strains of S. mutans on glass and hydroxyapatite surfaces. The mechanisms of these effects were investigated using scanning electron microscopy (SEM) and phytochemical screening. The results indicated that extracts of oolong tea most effectively inhibited attachment and extracts of pu-erh tea most effectively inhibited biofilm formation. SEM images showed that the S. mutans cells treated with extracts of oolong tea, or grown in medium containing extracts of pu-erh tea, were coated with tea components and were larger with more rounded shapes. The coatings on the cells consisted of flavonoids, tannins and indolic compounds. The ratio of tannins to simple phenolics in each of the coating samples was ∼3:1. This study suggests potential mechanisms by which tea components may inhibit the attachment and subsequent biofilm formation of S. mutans on tooth surfaces, such as modification of cell surface properties and blocking of the activity of proteins and the structures used by the bacteria to interact with surfaces.

Effects of atmospheric air plasma treatment of graphite and carbon felt electrodes on the anodic current from Shewanella attached cells.

PubMed

Epifanio, Monica; Inguva, Saikumar; Kitching, Michael; Mosnier, Jean-Paul; Marsili, Enrico

2015-12-01

The attachment of electrochemically active microorganisms (EAM) on an electrode is determined by both the chemistry and topography of the electrode surface. Pre-treatment of the electrode surface by atmospheric air plasma introduces hydrophilic functional groups, thereby increasing cell attachment and electroactivity in short-term experiments. In this study, we use graphite and carbon felt electrodes to grow the model EAM Shewanella loihica PV-4 at oxidative potential (0.2 V vs. Ag/AgCl). Cell attachment and electroactivity are measured through electrodynamic methods. Atmospheric air plasma pre-treatment increases cell attachment and current output at graphite electrodes by 25%, while it improves the electroactivity of the carbon felt electrodes by 450%. Air plasma pre-treatment decreased the coulombic efficiency on both carbon felt and graphite electrodes by 60% and 80%, respectively. Microbially produced flavins adsorb preferentially at the graphite electrode, and air plasma pre-treatment results in lower flavin adsorption at both graphite and carbon felt electrodes. Results show that air plasma pre-treatment is a feasible option to increase current output in bioelectrochemical systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Cable attachment for a radioactive brachytherapy source capsule

DOEpatents

Gross, Ian G; Pierce, Larry A

2006-07-18

In cancer brachytherapy treatment, a small californium-252 neutron source capsule is attached to a guide cable using a modified crimping technique. The guide cable has a solid cylindrical end, and the attachment employs circumferential grooves micromachined in the solid cable end. The attachment was designed and tested, and hardware fabricated for use inside a radioactive hot cell. A welding step typically required in other cable attachments is avoided.

T cell virological synapses and HIV-1 pathogenesis.

PubMed

Chen, Benjamin K

2012-12-01

Human immunodeficiency virus type 1 is the cause of a modern global pandemic associated with progressive acquired immune deficiency. The infection is characterized by the loss of the primary target of viral infection, the CD4+ T cell. The measurement of plasma viremia in patients can predict the rate of CD4+ cell decline; however, it is not clear whether this cell-free plasma virus represents the engine that drives viral spread. Active viral replication is mainly observed within lymphoid tissues that are hotbeds of cell-cell interactions that initiate and organize immune responses. It is well established that cell-cell interactions enhance viral spread in vitro. Dendritic cell-T cell interactions, which lie at the heart of adaptive immune responses, enhance viral infection in vitro. Interactions between infected and uninfected CD4+ T cells are a dominant route of viral spread in vitro and are likely to play a central role in viral dissemination in vivo. Future studies will test existing paradigms of HIV-1 dissemination to determine whether virus-transmitting contacts between infected and uninfected T cells called virological synapses are the dominant mode of viral spread in vivo. Here, we review the status of our understanding of this mode of infection with a focus on T cell-T cell interactions and examine how it may explain resistance to neutralizing antibodies and or the generation of genetic diversity of HIV.

Watch Out for the “Living Dead”: Cell-Free Enzymes and Their Fate

PubMed Central

Baltar, Federico

2018-01-01

Microbes are the engines driving biogeochemical cycles. Microbial extracellular enzymatic activities (EEAs) are the “gatekeepers” of the carbon cycle. The total EEA is the sum of cell-bound (i.e., cell-attached), and dissolved (i.e., cell-free) enzyme activities. Cell-free enzymes make up a substantial proportion (up to 100%) of the total marine EEA. Although we are learning more about how microbial diversity and function (including total EEA) will be affected by environmental changes, little is known about what factors control the importance of the abundant cell-free enzymes. Since cell-attached EEAs are linked to the cell, their fate will likely be linked to the factors controlling the cell’s fate. In contrast, cell-free enzymes belong to a kind of “living dead” realm because they are not attached to a living cell but still are able to perform their function away from the cell; and as such, the factors controlling their activity and fate might differ from those affecting cell-attached enzymes. This article aims to place cell-free EEA into the wider context of hydrolysis of organic matter, deal with recent studies assessing what controls the production, activity and lifetime of cell-free EEA, and what their fate might be in response to environmental stressors. This perspective article advocates the need to go “beyond the living things,” studying the response of cells/organisms to different stressors, but also to study cell-free enzymes, in order to fully constrain the future and evolution of marine biogeochemical cycles. PMID:29354095

Chk1 and Mps1 jointly regulate correction of merotelic kinetochore attachments.

PubMed

Petsalaki, Eleni; Zachos, George

2013-03-01

If uncorrected, merotelic kinetochore attachments can induce mis-segregated chromosomes in anaphase. We show that checkpoint kinase 1 (Chk1) protects vertebrate cells against merotelic attachments and lagging chromosomes and is required for correction of merotelic attachments during a prolonged metaphase. Decreased Chk1 activity leads to hyper-stable kinetochore microtubules, unstable binding of MCAK, Kif2b and Mps1 to centromeres or kinetochores and reduced phosphorylation of Hec1 by Aurora-B. Phosphorylation of Aurora-B at serine 331 (Ser331) by Chk1 is high in prometaphase and decreases significantly in metaphase cells. We propose that Ser331 phosphorylation is required for optimal localization of MCAK, Kif2b and Mps1 to centromeres or kinetochores and for Hec1 phosphorylation. Furthermore, inhibition of Mps1 activity diminishes initial recruitment of MCAK and Kif2b to centromeres or kinetochores, impairs Hec1 phosphorylation and exacerbates merotelic attachments in Chk1-deficient cells. We propose that Chk1 and Mps1 jointly regulate Aurora-B, MCAK, Kif2b and Hec1 to correct merotelic attachments. These results suggest a role for Chk1 and Mps1 in error correction.

Attached and Unattached Bacterial Communities in a 120-Meter Corehole in an Acidic, Crystalline Rock Aquifer

PubMed Central

Lehman, R. Michael; Roberto, Francisco F.; Earley, Drummond; Bruhn, Debby F.; Brink, Susan E.; O'Connell, Sean P.; Delwiche, Mark E.; Colwell, Frederick S.

2001-01-01

The bacteria colonizing geologic core sections (attached) were contrasted with those found suspended in the groundwater (unattached) by examining the microbiology of 16 depth-paired core and groundwater samples using a suite of culture-independent and culture-dependent analyses. One hundred twenty-two meters was continuously cored from a buried chalcopyrite ore hosted in a biotite-quartz-monzonite porphyry at the Mineral Park Mine near Kingman, Ariz. Every fourth 1.5-m core was acquired using microbiologically defensible methods, and these core sections were aseptically processed for characterization of the attached bacteria. Groundwater samples containing unattached bacteria were collected from the uncased corehole at depth intervals corresponding to the individual cores using an inflatable straddle packer sampler. The groundwater was acidic (pH 2.8 to 5.0), with low levels of dissolved oxygen and high concentrations of sulfate and metals, including ferrous iron. Total numbers of attached cells were less than 105 cells g of core material−1 while unattached cells numbered about 105 cells ml of groundwater−1. Attached and unattached acidophilic heterotrophs were observed throughout the depth profile. In contrast, acidophilic chemolithotrophs were not found attached to the rock but were commonly observed in the groundwater. Attached communities were composed of low numbers (<40 CFU g−1) of neutrophilic heterotrophs that exhibited a high degree of morphologic diversity, while unattached communities contained higher numbers (ca. 103 CFU ml−1) of neutrophilic heterotrophs of limited diversity. Sulfate-reducing bacteria were restricted to the deepest samples of both core and groundwater. 16S ribosomal DNA sequence analysis of attached, acidophilic isolates indicated that organisms closely related to heterotrophic, acidophilic mesophiles such as Acidiphilium organovorum and, surprisingly, to the moderately thermophilic Alicyclobacillus acidocaldarius were present. The results indicate that viable (but possibly inactive) microorganisms were present in the buried ore and that there was substantial distinction in biomass and physiological capabilities between attached and unattached populations. PMID:11319087

PEG attachment to osteoblasts enhances mechanosensitivity.

PubMed

Hamamura, Kazunori; Weng, Yiming; Zhao, Jun; Yokota, Hiroki; Xie, Dong

2008-06-01

Fluid flow induces proliferation and differentiation of osteoblasts, and fibrous structure like a primary cilium on a cell surface contributes to flow sensing and flow-driven gene regulation. We address a question: Does attachment of synthetic polymers on a cell surface enhance mechanosensitivity of osteoblasts? Using MC3T3 osteoblast cells (C4 clone) and a PEG polymer, one of whose termini was covalently linked to a succinimidyl succinate group (functionalized PEG-PEGSS), we examined attachment of PEGSS to osteoblasts and evaluated its effects on the mRNA expression of stress-responsive genes. AFM images exhibited globular PEGSS conformation of approximately 100 nm in size, and SEM images confirmed the attachment of a cluster of pancake-like PEGSS molecules on the osteoblast surface. Compared to control cells incubated with unfunctionalized PEG, real-time PCR revealed that RNA upregulation of c-fos, egr1, ATF3 and Cox2 genes was magnified in the cells incubated with PEGSS. These results support a PEG-induced increase in mechanosensitivity of osteoblasts and indicate that the described approach would be useful to accelerate growth and development of osteoblasts for bone repair and tissue engineering.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro.

PubMed

Zhang, Justina Su; Guri, Anilda; Corredig, Milena; Morales-Rayas, Rocio; Hassan, Ashraf; Griffiths, Mansel; LaPointe, Gisèle

2016-12-01

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Effect of Ag and Ag+N Ion Implantation on Cell Attachment Properties

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

Urkac, Emel Sokullu; Oztarhan, Ahmet; Gurhan, Ismet Deliloglu

2009-03-10

Implanted biomedical prosthetic devices are intended to perform safely, reliably and effectively in the human body thus the materials used for orthopedic devices should have good biocompatibility. Ultra High Molecular Weight Poly Ethylene (UHMWPE) has been commonly used for total hip joint replacement because of its very good properties. In this work, UHMWPE samples were Ag and Ag+N ion implanted by using the Metal-Vapor Vacuum Arc (MEVVA) ion implantation technique. Samples were implanted with a fluency of 1017 ion/cm2 and extraction voltage of 30 kV. Rutherford Backscattering Spectrometry (RBS) was used for surface studies. RBS showed the presence of Agmore » and N on the surface. Cell attachment properties investigated with model cell lines (L929 mouse fibroblasts) to demonstrate that the effect of Ag and Ag+N ion implantation can favorably influence the surface of UHMWPE for biomedical applications. Scanning electron microscopy (SEM) was used to demonstrate the cell attachment on the surface. Study has shown that Ag+N ion implantation represents more effective cell attachment properties on the UHMWPE surfaces.« less

Correcting bulk in-plane motion artifacts in MRI using the point spread function.

PubMed

Lin, Wei; Wehrli, Felix W; Song, Hee Kwon

2005-09-01

A technique is proposed for correcting both translational and rotational motion artifacts in magnetic resonance imaging without the need to collect additional navigator data or to perform intensive postprocessing. The method is based on measuring the point spread function (PSF) by attaching one or two point-sized markers to the main imaging object. Following the isolation of a PSF marker from the acquired image, translational motion could be corrected directly from the modulation transfer function, without the need to determine the object's positions during the scan, although the shifts could be extracted if desired. Rotation is detected by analyzing the relative displacements of two such markers. The technique was evaluated with simulations, phantom and in vivo experiments.

Allogeneic Transplantation of Müller-Derived Retinal Ganglion Cells Improves Retinal Function in a Feline Model of Ganglion Cell Depletion

PubMed Central

Becker, Silke; Eastlake, Karen; Jayaram, Hari; Jones, Megan F.; Brown, Robert A.; McLellan, Gillian J.; Charteris, David G.; Khaw, Peng T.

2016-01-01

Human Müller glia with stem cell characteristics (hMGSCs) have been shown to improve retinal function upon transplantation into rat models of retinal ganglion cell (RGC) depletion. However, their translational potential may depend upon successful engraftment and improvement of retinal function in experimental models with anatomical and functional features resembling those of the human eye. We investigated the effect of allogeneic transplantation of feline Müller glia with the ability to differentiate into cells expressing RGC markers, following ablation of RGCs by N-methyl-d-aspartate (NMDA). Unlike previous observations in the rat, transplantation of hMGSC-derived RGCs into the feline vitreous formed aggregates and elicited a severe inflammatory response without improving visual function. In contrast, allogeneic transplantation of feline MGSC (fMGSC)-derived RGCs into the vitrectomized eye improved the scotopic threshold response (STR) of the electroretinogram (ERG). Despite causing functional improvement, the cells did not attach onto the retina and formed aggregates on peripheral vitreous remnants, suggesting that vitreous may constitute a barrier for cell attachment onto the retina. This was confirmed by observations that cellular scaffolds of compressed collagen and enriched preparations of fMGSC-derived RGCs facilitated cell attachment. Although cells did not migrate into the RGC layer or the optic nerve, they significantly improved the STR and the photopic negative response of the ERG, indicative of increased RGC function. These results suggest that MGSCs have a neuroprotective ability that promotes partial recovery of impaired RGC function and indicate that cell attachment onto the retina may be necessary for transplanted cells to confer neuroprotection to the retina. Significance Müller glia with stem cell characteristics are present in the adult human retina, but they do not have regenerative ability. These cells, however, have potential for development of cell therapies to treat retinal disease. Using a feline model of retinal ganglion cell (RGC) depletion, cell grafting methods to improve RGC function have been developed. Using cellular scaffolds, allogeneic transplantation of Müller glia-derived RGC promoted cell attachment onto the retina and enhanced retinal function, as judged by improvement of the photopic negative and scotopic threshold responses of the electroretinogram. The results suggest that the improvement of RGC function observed may be ascribed to the neuroprotective ability of these cells and indicate that attachment of the transplanted cells onto the retina is required to promote effective neuroprotection. PMID:26718648

Child Maltreatment Is Associated with a Reduction of the Oxytocin Receptor in Peripheral Blood Mononuclear Cells

PubMed Central

Krause, Sabrina; Boeck, Christina; Gumpp, Anja M.; Rottler, Edit; Schury, Katharina; Karabatsiakis, Alexander; Buchheim, Anna; Gündel, Harald; Kolassa, Iris-Tatjana; Waller, Christiane

2018-01-01

Background: Child maltreatment (CM) and attachment experiences are closely linked to alterations in the human oxytocin (OXT) system. However, human data about oxytocin receptor (OXTR) protein levels are lacking. Therefore, we investigated oxytocin receptor (OXTR) protein levels in circulating immune cells and related them to circulating levels of OXT in peripheral blood. We hypothesized reduced OXTR protein levels, associated with both, experiences of CM and an insecure attachment representation. Methods: OXTR protein expressions were analyzed by western blot analyses in peripheral blood mononuclear cells (PBMC) and plasma OXT levels were determined by radioimmunoassay (RIA) in 49 mothers. We used the Childhood Trauma Questionnaire (CTQ) to assess adverse childhood experiences. Attachment representations (secure vs. insecure) were classified using the Adult Attachment Projective Picture System (AAP) and levels of anxiety and depression were assessed with the German version of the Hospital Depression and Anxiety scale (HADS-D). Results: CM-affected women showed significantly lower OXTR protein expression with significantly negative correlations between the OXTR protein expression and the CTQ sum score, whereas plasma OXT levels showed no significant differences in association with CM. Lower OXTR protein expression in PBMC were particularly pronounced in the group of insecurely attached mothers compared to the securely attached group. Anxiety levels were significantly higher in CM-affected women. Conclusion: This study demonstrated a significant association between CM and an alteration of OXTR protein expression in human blood cells as a sign for chronic, long-lasting alterations in this attachment-related neurobiological system. PMID:29535656

β1 Integrins Mediate Attachment of Mesenchymal Stem Cells to Cartilage Lesions

PubMed Central

Zwolanek, Daniela; Flicker, Magdalena; Kirstätter, Elisabeth; Zaucke, Frank; van Osch, Gerjo J.V.M.; Erben, Reinhold G.

2015-01-01

Abstract Mesenchymal stem cells (MSC) may have great potential for cell-based therapies of osteoarthritis. However, after injection in the joint, only few cells adhere to defective articular cartilage and contribute to cartilage regeneration. Little is known about the molecular mechanisms of MSC attachment to defective articular cartilage. Here, we developed an ex vivo attachment system, using rat osteochondral explants with artificially created full-thickness cartilage defects in combination with genetically labeled MSC isolated from bone marrow of human placental alkaline phosphatase transgenic rats. Binding of MSC to full-thickness cartilage lesions was improved by serum, but not hyaluronic acid, and was dependent on the presence of divalent cations. Additional in vitro tests showed that rat MSC attach, in a divalent cation-dependent manner, to collagen I, collagen II, and fibronectin, but not to collagen XXII or cartilage oligomeric matrix protein (COMP). RGD peptides partially blocked the adhesion of MSC to fibronectin in vitro and to cartilage lesions ex vivo. Furthermore, the attachment of MSC to collagen I and II in vitro and to cartilage lesions ex vivo was almost completely abolished in the presence of a β1 integrin blocking antibody. In conclusion, our data suggest that attachment of MSC to ex vivo full-thickness cartilage lesions is almost entirely β1 integrin-mediated, whereby both RGD- and collagen-binding integrins are involved. These findings suggest a key role of integrins during MSC attachment to defective cartilage and may pave the way for improved MSC-based therapies in the future. PMID:26309781

Reversals and collisions optimize protein exchange in bacterial swarms

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

Amiri, Aboutaleb; Harvey, Cameron; Buchmann, Amy

Swarming groups of bacteria coordinate their behavior by self-organizing as a population to move over surfaces in search of nutrients and optimal niches for colonization. Many open questions remain about the cues used by swarming bacteria to achieve this self-organization. While chemical cue signaling known as quorum sensing is well-described, swarming bacteria often act and coordinate on time scales that could not be achieved via these extracellular quorum sensing cues. Here, cell-cell contact-dependent protein exchange is explored as amechanism of intercellular signaling for the bacterium Myxococcus xanthus. A detailed biologically calibrated computational model is used to study how M. xanthusmore » optimizes the connection rate between cells and maximizes the spread of an extracellular protein within the population. The maximum rate of protein spreading is observed for cells that reverse direction optimally for swarming. Cells that reverse too slowly or too fast fail to spread extracellular protein efficiently. In particular, a specific range of cell reversal frequencies was observed to maximize the cell-cell connection rate and minimize the time of protein spreading. Furthermore, our findings suggest that predesigned motion reversal can be employed to enhance the collective behavior of biological synthetic active systems.« less

Monitoring change in refractive index of cytosol of animal cells on affinity surface under osmotic stimulus for label-free measurement of viability.

PubMed

Park, Jina; Jin, Sung Il; Kim, Hyung Min; Ahn, Junhyoung; Kim, Yeon-Gu; Lee, Eun Gyo; Kim, Min-Gon; Shin, Yong-Beom

2015-02-15

We demonstrated that a metal-clad waveguide (MCW)-based biosensor can be applied to label-free measurements of viability of adherent animal cells with osmotic stimulation in real time. After Chinese hamster ovary (CHO) and human embryonic kidney cell 293 (HEK293) cells were attached to a Concanavalin A (Con A)-modified sensor surface, the magnitudes of cell responses to non-isotonic stimulation were compared between live and dead cells. The live cells exhibited a change in the refractive index (RI) of the cytosol caused by a redistribution of water through the cell membrane, which was induced by the osmotic stimulus, but the dead cells did not. Moreover, the normalized change in the RI measured via the MCW sensor was linearly proportional to the viability of attached cells and the resolution in monitoring cell viability was about 0.079%. Therefore, the viability of attached animal cells can be measured without labels by observing the relative differences in the RI of cytosol in isotonic and non-isotonic buffers. Copyright © 2014 Elsevier B.V. All rights reserved.

Chlamydia trachomatis-host cell interactions: role of the chlamydial major outer membrane protein as an adhesin.

PubMed Central

Su, H; Watkins, N G; Zhang, Y X; Caldwell, H D

1990-01-01

The major outer membrane protein (MOMP) of Chlamydia trachomatis is characterized by four symmetrically spaced variable domains (VDs I to IV) whose sequences vary among serotypes. The surface-exposed portions of these VDs contain contiguous sequences that are both serotyping determinants and in vivo target sites for neutralizing antibodies. Previous studies using surface proteolysis of C. trachomatis B implicated VDs II and IV of the MOMP of this serotype in the attachment of chlamydiae to host cells. In this study, we used monoclonal antibodies (MAbs) specific to antigenic determinants located in VDs II and IV of the MOMP of serotype B to further investigate the role of the MOMP in the attachment of chlamydiae to host cells. MABs specific to serotype- and subspecies-specific epitopes located in exposed VDs II and IV, respectively, neutralized chlamydial infectivity for hamster kidney cells by blocking chlamydial attachment. We radioiodinated these MAbs and used them to determine the number and topology of the surface-exposed VDs II and IV epitopes on chlamydial elementary bodies. VDs II and IV each comprised approximately 2.86 x 10(4) negatively charged sites and were in proximity on the chlamydial cell surface. These studies suggest that the MAbs blocked chlamydial attachment by inhibiting electrostatic interactions with host cells. We examined the effects of thermal inactivation on both chlamydial attachment and conformation of the MOMP. Heat-inactivated chlamydiae failed to attach to host cells and exhibited a conformational change in an inaccessible invariant hydrophobic nonapeptide sequence located within VD IV of the MOMPs of C. trachomatis serotypes. These findings suggest that in addition to electrostatic interactions, a common hydrophobic component of the MOMP also contributes to the binding of chlamydiae to host cells. Thus, we propose that the MOMP functions as a chlamydial adhesin by promoting nonspecific (electrostatic and hydrophobic) interactions with host cells. Surface-accessible negatively charged VDs appear to be important in electrostatic binding, while the invariant region of VD IV may provide a subsurface hydrophobic depression which further promotes binding of chlamydiae to host cells through hydrophobic interactions. Images PMID:2318528

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane.

PubMed

Pedersen, Gitte A; Jensen, Helene H; Schelde, Anne-Sofie B; Toft, Charlotte; Pedersen, Hans N; Ulrichsen, Maj; Login, Frédéric H; Amieva, Manuel R; Nejsum, Lene N

2017-01-01

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth.

The basolateral vesicle sorting machinery and basolateral proteins are recruited to the site of enteropathogenic E. coli microcolony growth at the apical membrane

PubMed Central

Pedersen, Gitte A.; Jensen, Helene H.; Schelde, Anne-Sofie B.; Toft, Charlotte; Pedersen, Hans N.; Ulrichsen, Maj; Login, Frédéric H.; Amieva, Manuel R.

2017-01-01

Foodborne Enteropathogenic Escherichia coli (EPEC) infections of the small intestine cause diarrhea especially in children and are a major cause of childhood death in developing countries. EPEC infects the apical membrane of the epithelium of the small intestine by attaching, effacing the microvilli under the bacteria and then forming microcolonies on the cell surface. We first asked the question where on epithelial cells EPEC attaches and grows. Using models of polarized epithelial monolayers, we evaluated the sites of initial EPEC attachment to the apical membrane and found that EPEC preferentially attached over the cell-cell junctions and formed microcolonies preferentially where three cells come together at tricellular tight junctions. The ability of EPEC to adhere increased when host cell polarity was compromised yielding EPEC access to basolateral proteins. EPEC pedestals contain basolateral cytoskeletal proteins. Thus, we asked if attached EPEC causes reorganization the protein composition of the host cell plasma membrane at sites of microcolony formation. We found that EPEC microcolony growth at the apical membrane resulted in a local accumulation of basolateral plasma membrane proteins surrounding the microcolony. Basolateral marker protein aquaporin-3 localized to forming EPEC microcolonies. Components of the basolateral vesicle targeting machinery were re-routed. The Exocyst (Exo70) was recruited to individual EPEC as was the basolateral vesicle SNARE VAMP-3. Moreover, several Rab variants were also recruited to the infection site, and their dominant-negative equivalents were not. To quantitatively study the recruitment of basolateral proteins, we created a pulse of the temperature sensitive basolateral VSVG, VSVG3-SP-GFP, from the trans-Golgi Network. We found that after release from the TGN, significantly more VSVG3-SP-GFP accumulated at the site of microcolony growth than on equivalent membrane regions of uninfected cells. This suggests that trafficking of vesicles destined for the basolateral membrane are redirected to the apical site of microcolony growth. Thus, in addition to disrupting host cell fence function, local host cell plasma membrane protein composition is changed by altered protein trafficking and recruitment of basolateral proteins to the apical microcolony. This may aid EPEC attachment and subsequent microcolony growth. PMID:28636623

Possession attachment predicts cell phone use while driving.

PubMed

Weller, Joshua A; Shackleford, Crystal; Dieckmann, Nathan; Slovic, Paul

2013-04-01

Distracted driving has become an important public health concern. However, little is known about the predictors of this health-risking behavior. One overlooked risk factor for distracted driving is the perceived attachment that one feels toward his or her phone. Prior research has suggested that individuals develop bonds toward objects, and qualitative research suggests that the bond between young drivers and their phones can be strong. It follows that individuals who perceive a strong attachment to their phone would be more likely to use it, even when driving. In a nationally representative sample of young drivers (17-28 years), participants (n = 1,006) completed a survey about driving behaviors and phone use. Risk perception surrounding cell phone use while driving and perceived attachment to one's phone were assessed by administering factor-analytically derived scales that were created as part of a larger project. Attachment toward one's phone predicted the proportion of trips in which a participant reported using their cell phone while driving, beyond that accounted for by risk perception and overall phone use. Further, attachment predicted self-reported distracted driving behaviors, such as the use of social media while driving. Attachment to one's phone may be an important but overlooked risk factor for the engagement of potentially health-risking driving behaviors. Understanding that phone attachment may adversely affect driving behaviors has the potential to inform prevention and intervention efforts designed to reduce distracted driving behaviors, especially in young drivers. 2013 APA, all rights reserved

Quantitative measurements of intercellular adhesion between a macrophage and cancer cells using a cup-attached AFM chip.

PubMed

Kim, Hyonchol; Yamagishi, Ayana; Imaizumi, Miku; Onomura, Yui; Nagasaki, Akira; Miyagi, Yohei; Okada, Tomoko; Nakamura, Chikashi

2017-07-01

Intercellular adhesion between a macrophage and cancer cells was quantitatively measured using atomic force microscopy (AFM). Cup-shaped metal hemispheres were fabricated using polystyrene particles as a template, and a cup was attached to the apex of the AFM cantilever. The cup-attached AFM chip (cup-chip) approached a murine macrophage cell (J774.2), the cell was captured on the inner concave of the cup, and picked up by withdrawing the cup-chip from the substrate. The cell-attached chip was advanced towards a murine breast cancer cell (FP10SC2), and intercellular adhesion between the two cells was quantitatively measured. To compare cell adhesion strength, the work required to separate two adhered cells (separation work) was used as a parameter. Separation work was almost 2-fold larger between a J774.2 cell and FP10SC2 cell than between J774.2 cell and three additional different cancer cells (4T1E, MAT-LyLu, and U-2OS), two FP10SC2 cells, or two J774.2 cells. FP10SC2 was established from 4T1E as a highly metastatic cell line, indicates separation work increased as the malignancy of cancer cells became higher. One possible explanation of the strong adhesion of macrophages to cancer cells observed in this study is that the measurement condition mimicked the microenvironment of tumor-associated macrophages (TAMs) in vivo, and J774.2 cells strongly expressed CD204, which is a marker of TAMs. The results of the present study, which were obtained by measuring cell adhesion strength quantitatively, indicate that the fabricated cup-chip is a useful tool for measuring intercellular adhesion easily and quantitatively. Copyright © 2017 Elsevier B.V. All rights reserved.

Bone Genes in the Kidney of Stone Formers

NASA Astrophysics Data System (ADS)

Evan, Andrew P.; Bledsoe, Sharon B.

2008-09-01

Intraoperative papillary biopsies from kidneys of idiopathic-calcium oxalate stone formers (ICSF) have revealed a distinct pattern of mineral deposition in the interstitium of the renal papilla. The earliest sites of these deposits, termed Randall's plaque, are found in the basement membrane of thin loops of Henle and appear to spread into the surrounding interstitium down to the papillary epithelium. Recent studies show kidney stones of ICSF patients grow attached to the renal papilla and at sites of Randall's plaque. Together these observations suggest that plaque formation may be the critical step in stone formation. In order to control plaque formation and thereby reduce future kidney stone development, the mechanism of plaque deposition must be understood. Because the renal papilla has unique anatomical features similar to bone and the fact that the interstitial deposits of ICSF patients are formed of biological apatite, this paper tests the hypothesis that sites of interstitial plaque form as a result of cell-mediated osteoblast-like activity.

TIM-1 Promotes Hepatitis C Virus Cell Attachment and Infection.

PubMed

Wang, Jing; Qiao, Luhua; Hou, Zhouhua; Luo, Guangxiang

2017-01-15

Human TIM and TAM family proteins were recently found to serve as phosphatidylserine (PS) receptors which promote infections by many different viruses, including dengue virus, West Nile virus, Ebola virus, Marburg virus, and Zika virus. In the present study, we provide substantial evidence demonstrating that TIM-1 is important for efficient infection by hepatitis C virus (HCV). The knockdown of TIM-1 expression significantly reduced HCV infection but not HCV RNA replication. Likewise, TIM-1 knockout in Huh-7.5 cells remarkably lowered HCV cell attachment and subsequent HCV infection. More significantly, the impairment of HCV infection in the TIM-1 knockout cells could be restored completely by ectopic expression of TIM-1 but not TIM-3 or TIM-4. Additionally, HCV infection and cell attachment were inhibited by PS but not by phosphatidylcholine (PC), demonstrating that TIM-1-mediated enhancement of HCV infection is PS dependent. The exposure of PS on the HCV envelope was confirmed by immunoprecipitation of HCV particles with a PS-specific monoclonal antibody. Collectively, these findings demonstrate that TIM-1 promotes HCV infection by serving as an attachment receptor for binding to PS exposed on the HCV envelope. TIM family proteins were recently found to enhance infections by many different viruses, including several members of the Flaviviridae family. However, their importance in HCV infection has not previously been examined experimentally. The TIM family proteins include three members in humans: TIM-1, TIM-3, and TIM-4. The findings derived from our studies demonstrate that TIM-1, but not TIM-3 or TIM-4, promotes HCV infection by functioning as an HCV attachment factor. Knockout of the TIM-1 gene resulted in a remarkable reduction of HCV cell attachment and infection. PS-containing liposomes blocked HCV cell attachment and subsequent HCV infection. HCV particles could also be precipitated with a PS-specific monoclonal antibody. These findings suggest that TIM-1 and its binding ligand, PS, may serve as novel targets for antiviral intervention. Copyright © 2017 American Society for Microbiology.

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect.

PubMed

Koga, Hideyuki; Shimaya, Masayuki; Muneta, Takeshi; Nimura, Akimoto; Morito, Toshiyuki; Hayashi, Masaya; Suzuki, Shiro; Ju, Young-Jin; Mochizuki, Tomoyuki; Sekiya, Ichiro

2008-01-01

Current cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect. For ex vivo analysis in rabbits, the cartilage defect was faced upward, filled with synovial MSC suspension, and held stationary for 2.5 to 15 minutes. The number of attached cells was examined. For in vivo analysis in rabbits, an autologous synovial MSC suspension was placed on the cartilage defect, and the position was maintained for 10 minutes to adhere the cells to the defect. For the control, either the same cell suspension was injected intra-articularly or the defects were left empty. The three groups were compared macroscopically and histologically. For ex vivo analysis in humans, in addition to the similar experiment in rabbits, the expression and effects of neutralizing antibodies for adhesion molecules were examined. Ex vivo analysis in rabbits demonstrated that the number of attached cells increased in a time-dependent manner, and more than 60% of cells attached within 10 minutes. The in vivo study showed that a large number of transplanted synovial MSCs attached to the defect at 1 day, and the cartilage defect improved at 24 weeks. The histological score was consistently better than the scores of the two control groups (same cell suspension injected intra-articularly or defects left empty) at 4, 12, and 24 weeks. Ex vivo analysis in humans provided similar results to those in rabbits. Intercellular adhesion molecule 1-positive cells increased between 1 minute and 10 minutes, and neutralizing antibodies for intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and activated leukocyte-cell adhesion molecule inhibited the attachment. Placing MSC suspension on the cartilage defect for 10 minutes resulted in adherence of >60% of synovial MSCs to the defect, and promoted cartilage regeneration. This adherent method makes it possible to adhere MSCs with low invasion, without periosteal coverage, and without a scaffold.

Magnetic resonance investigation of magnetic-labeled baker's yeast cells

NASA Astrophysics Data System (ADS)

Godoy Morais, J. P. M.; Azevedo, R. B.; Silva, L. P.; Lacava, Z. G. M.; Báo, S. N.; Silva, O.; Pelegrini, F.; Gansau, C.; Buske, N.; Safarik, I.; Safarikova, M.; Morais, P. C.

2004-05-01

In this study, the interaction of DMSA-coated magnetite nanoparticles (5 and 10 nm core-size) with Saccharomyces cerevisae was investigated using magnetic resonance (MR) and transmission electron microscopy (TEM). The TEM micrographs revealed magnetite nanoparticles attached externally to the cell wall. The MR data support the strong interaction among the nanoparticles supported by the cells. A remarkable shift in the resonance field was used as signature of particle attachment to the cell wall.

Effect of modified pectin molecules on the growth of bone cells.

PubMed

Kokkonen, Hanna E; Ilvesaro, Joanna M; Morra, Marco; Schols, Henk A; Tuukkanen, Juha

2007-02-01

The aim of this study was to investigate molecular candidates for bone implant nanocoatings, which could improve biocompatibility of implant materials. Primary rat bone cells and murine preosteoblastic MC3T3-E1 cells were cultured on enzymatically modified hairy regions (MHR-A and MHR-B) of apple pectins. MHRs were covalently attached to tissue culture polystyrene (TCPS) or glass. Uncoated substrata or bone slices were used as controls. Cell attachment, proliferation, and differentiation were investigated with fluorescence and confocal microscopy. Bone cells seem to prefer MHR-B coating to MHR-A coating. On MHR-A samples, the overall numbers as well as proportions of active osteoclasts were diminished compared to those on MHR-B, TCPS, or bone. Focal adhesions indicating attachment of the osteoblastic cells were detected on MHR-B and uncoated controls but not on MHR-A. These results demonstrate the possibility to modify surfaces with pectin nanocoatings.

A role for PVRL4-driven cell–cell interactions in tumorigenesis

PubMed Central

Pavlova, Natalya N; Pallasch, Christian; Elia, Andrew EH; Braun, Christian J; Westbrook, Thomas F; Hemann, Michael; Elledge, Stephen J

2013-01-01

During all stages of tumor progression, cancer cells are subjected to inappropriate extracellular matrix environments and must undergo adaptive changes in order to evade growth constraints associated with the loss of matrix attachment. A gain of function screen for genes that enable proliferation independently of matrix anchorage identified a cell adhesion molecule PVRL4 (poliovirus-receptor-like 4), also known as Nectin-4. PVRL4 promotes anchorage-independence by driving cell-to-cell attachment and matrix-independent integrin β4/SHP-2/c-Src activation. Solid tumors frequently have copy number gains of the PVRL4 locus and some have focal amplifications. We demonstrate that the transformation of breast cancer cells is dependent on PVRL4. Furthermore, growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer. DOI: http://dx.doi.org/10.7554/eLife.00358.001 PMID:23682311

Phenotypic Heterogeneity in Expression of the K1 Polysaccharide Capsule of Uropathogenic Escherichia coli and Downregulation of the Capsule Genes during Growth in Urine.

PubMed

King, Jane E; Aal Owaif, Hasan A; Jia, Jia; Roberts, Ian S

2015-07-01

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTI). The K1 capsule on the surface of UPEC strains is a key virulence factor, and its expression may be important in the onset and progression of UTI. In order to understand capsule expression in more detail, we analyzed its expression in the UPEC strain UTI89 during growth in rich medium (LB medium) and urine and during infection of a bladder epithelial cell line. Comparison of capsule gene transcription using a chromosomal gfp reporter fusion showed a significant reduction in transcription during growth in urine compared to that during growth in LB medium. When examined at the single-cell level, following growth in both media, capsule gene expression appears to be heterogeneous, with two distinct green fluorescent protein (GFP)-expressing populations. Using anti-K1 antibody, we showed that this heterogeneity in gene expression results in two populations of encapsulated and unencapsulated cells. We demonstrated that the capsule hinders attachment to and invasion of epithelial cells and that the unencapsulated cells within the population preferentially adhere to and invade bladder epithelial cells. We found that once internalized, UTI89 starts to produce capsule to aid in its intracellular survival and spread. We propose that this observed phenotypic diversity in capsule expression is a fitness strategy used by the bacterium to deal with the constantly changing environment of the urinary tract. Copyright © 2015 King et al.

Modeling potential Emerald Ash Borer spread through GIS/cell-based/gravity models with data bolstered by web-based inputs

Treesearch

Louis R. Iverson; Anantha M. Prasad; Davis Sydnor; Jonathan Bossenbroek; Mark W. Schwartz; Mark W. Schwartz

2006-01-01

We model the susceptibility and potential spread of the organism across the eastern United States and especially through Michigan and Ohio using Forest Inventory and Analysis (FIA) data. We are also developing a cell-based model for the potential spread of the organism. We have developed a web-based tool for public agencies and private individuals to enter the...

Separation of integrin-dependent adhesion from morphological changes based on differential PLC specificities.

PubMed

Wooten, D K; Teague, T K; McIntyre, B W

1999-01-01

In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholine-specific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin alpha4beta1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC.

Cell-mediated fibre recruitment drives extracellular matrix mechanosensing in engineered fibrillar microenvironments

NASA Astrophysics Data System (ADS)

Baker, Brendon M.; Trappmann, Britta; Wang, William Y.; Sakar, Mahmut S.; Kim, Iris L.; Shenoy, Vivek B.; Burdick, Jason A.; Chen, Christopher S.

2015-12-01

To investigate how cells sense stiffness in settings structurally similar to native extracellular matrices, we designed a synthetic fibrous material with tunable mechanics and user-defined architecture. In contrast to flat hydrogel surfaces, these fibrous materials recapitulated cell-matrix interactions observed with collagen matrices including stellate cell morphologies, cell-mediated realignment of fibres, and bulk contraction of the material. Increasing the stiffness of flat hydrogel surfaces induced mesenchymal stem cell spreading and proliferation; however, increasing fibre stiffness instead suppressed spreading and proliferation for certain network architectures. Lower fibre stiffness permitted active cellular forces to recruit nearby fibres, dynamically increasing ligand density at the cell surface and promoting the formation of focal adhesions and related signalling. These studies demonstrate a departure from the well-described relationship between material stiffness and spreading established with hydrogel surfaces, and introduce fibre recruitment as a previously undescribed mechanism by which cells probe and respond to mechanics in fibrillar matrices.

Comparison of methods for quantitating Salmonella enterica Typhimurium and Heidelberg strain attachment to reusable plastic shipping container coupons and preliminary assessment of sanitizer efficacy.

PubMed

Shi, Zhaohao; Baker, Christopher A; Lee, Sang In; Park, Si Hong; Kim, Sun Ae; Ricke, Steven C

2016-09-01

Salmonella serovars, one of the leading contributors to foodborne illness and are especially problematic for foods that are not cooked before consumption, such as fresh produce. The shipping containers that are used to transport and store fresh produce may play a role in cross contamination and subsequent illnesses. However, methods for quantitatively attached cells are somewhat variable. The overall goal of this study was to compare conventional plating with molecular methods for quantitating attached representative strains for Salmonella Typhimurium and Heidelberg on reusable plastic containers (RPC) coupons, respectively. We attached Salmonella enterica serovar Typhimurium ATCC 14028 and serovar Heidelberg SL486 (parent and an antibiotic resistant marker strain) to plastic coupons (2.54 cm(2)) derived from previously used shipping containers by growing for 72 h in tryptic soy broth. The impact of the concentration of sanitizer on log reductions between unsanitized and sanitized coupons was evaluated by exposing attached S. Typhimurium cells to 200 ppm and 200,000 ppm sodium hypochlorite (NaClO). Differences in sanitizer effectiveness between serovars were also evaluated with attached S. Typhimurium compared to attached S. Heidelberg populations after being exposed to 200 ppm peracetic acid (PAA). Treatment with NaClO caused an average of 2.73 ± 0.23 log CFU of S. Typhimurium per coupon removed with treatment at 200 ppm while 3.36 ± 0.54 log CFU were removed at 200,000 ppm. Treatment with PAA caused an average of 2.62 ± 0.15 log CFU removed for S. Typhimurium and 1.41 ± 0.17 log CFU for S. Heidelberg (parent) and 1.61 ± 0.08 log CFU (marker). Lastly, scanning electron microscopy (SEM) was used to visualize cell attachment and coupon surface topography. SEM images showed that remaining attached cell populations were visible even after sanitizer application. Conventional plating and qPCR yielded similar levels of enumerated bacterial populations indicating a high concordance between the two methods. Therefore, qPCR could be used for the rapid quantification of Salmonella attached on RPC.

Combination Space Station Handrail Clamp and Pointing Device

NASA Technical Reports Server (NTRS)

Hughes, Stephen J. (Inventor)

1999-01-01

A device for attaching an experiment carrier to a space station handrail is provided. The device has two major components, a clamping mechanism for attachment to a space station handrail, and a pointing carrier on which an experiment package can be mounted and oriented. The handrail clamp uses an overcenter mechanism and the carrier mechanism uses an adjustable preload ball and socket for carrier positioning. The handrail clamp uses a stack of disk springs to provide a spring loaded button. This configuration provides consistent clamping force over a range of possible handrail thicknesses. Three load points are incorporated in the clamping mechanism thereby spreading the clamping load onto three separate points on the handrail. A four bar linkage is used to provide for a single actuation lever for all three load points. For additional safety, a secondary lock consisting of a capture plate and push lock keeps the clamp attached to the handrail in the event of main clamp failure. For the carrier positioning mechanism, a ball in a spring loaded socket uses friction to provide locking torque; however. the ball and socket are torque limited so that the ball ran slip under kick loads (125 pounds or greater). A lead screw attached to disk spring stacks is used to provide an adjustable spring force on the socket. A locking knob is attached to the lead screw to allow for hand manipulation of the lead screw.

Interaction of herpes simplex virus glycoprotein gC with mammalian cell surface molecules.

PubMed Central

Tal-Singer, R; Peng, C; Ponce De Leon, M; Abrams, W R; Banfield, B W; Tufaro, F; Cohen, G H; Eisenberg, R J

1995-01-01

The entry of herpes simplex virus (HSV) into mammalian cells is a multistep process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. We explored the interaction between gC and the cell surface by using purified proteins in the absence of detergent. Truncated forms of gC and gD, gC1(457t), gC2(426t), and gD1(306t), lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. We studied the ability of these proteins to bind to mammalian cells, to bind to immobilized heparin, to block HSV type 1 (HSV-1) attachment to cells, and to inhibit plaque formation by HSV-1. Each of these gC proteins bound to conformation-dependent monoclonal antibodies and to human complement component C3b, indicating that they maintained the same conformation of gC proteins expressed in mammalian cells. Biotinylated gC1(457t) and gC2(426t) each bind to several cell lines. Binding was inhibited by an excess of unlabeled gC but not by gD, indicating specificity. The attachment of gC to cells involves primarily heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding by 50% but had no effect on gD binding. Moreover, binding of gC to two heparan sulfate-deficient L-cell lines, gro2C and sog9, both of which are mostly resistant to HSV infection, was markedly reduced. Purified gD1 (306t), however, bound equally well to the two mutant cell lines. In contrast, saturating amounts of gC1(457t) interfered with HSV-1 attachment to cells but failed to block plaque formation, suggesting a role for gC in attachment but not penetration. A mutant form of gC lacking residues 33 to 123, gC1(delta 33-123t), expressed in the baculovirus system, bound significantly less well to cells than did gC1(457t) and competed poorly with biotinylated gC1(457t) for binding. These results suggest that residues 33 to 123 are important for gC attachment to cells. In contrast, both the mutant and wild-type forms of gC bound to immobilized heparin, indicating that binding of these proteins to the cell surface involves more than a simple interaction with heparin. To determine that the contribution of the N-terminal region of gC is important for HSV attachment, we compared several properties of a mutant HSV-1 which contains gC lacking amino acids 33 to 123 to those of its parental virus, which contains full-length gC. The mutant bound less well to cells than the parental virus but exhibited normal growth properties.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7769707

Microfluidic Fabrication of Cell Adhesive Chitosan Microtubes

PubMed Central

Oh, Jonghyun; Kim, Keekyoung; Won, Sung Wook; Cha, Chaenyung; Gaharwar, Akhilesh; Selimović, Šeila; Bae, Hojae; Lee, Kwang Ho; Lee, Dong Hwan; Lee, Sang-Hoon; Khademhosseini, Ali

2013-01-01

Chitosan has been used as a scaffolding material in tissue engineering due to its mechanical properties and biocompatibility. With increased appreciation of the effect of micro- and nanoscale environments on cellular behavior, there is increased emphasis on generating microfabricated chitosan structures. Here we employed a microfluidic coaxial flow-focusing system to generate cell adhesive chitosan microtubes of controlled sizes by modifying the flow rates of a chitosan pre-polymer solution and phosphate buffered saline (PBS). The microtubes were extruded from a glass capillary with a 300 μm inner diameter. After ionic crosslinking with sodium tripolyphosphate (TPP), fabricated microtubes had inner and outer diameter ranges of 70-150 μm and 120-185 μm. Computational simulation validated the controlled size of microtubes and cell attachment. To enhance cell adhesiveness on the microtubes, we mixed gelatin with the chitosan pre-polymer solution and adjusted the pH values of the chitosan pre-polymer solution with gelatin and TPP. During the fabrication of microtubes, fibroblasts suspended in core PBS flow adhered to the inner surface of chitosan-gelatin microtubes. To achieve physiological pH values, we adjusted pH values of chiotsan pre-polymer solution and TPP. In particular, we were able to improve cell viability to 92% with pH values of 5.8 and 7.4 for chitosan and TPP solution respectively. Cell culturing for three days showed that the addition of the gelatin enhanced cell spreading and proliferation inside the chitosan-gelatin microtubes. The microfluidic fabrication method for ionically crosslinked chitosan microtubes at physiological pH can be compatible with a variety of cells and used as a versatile platform for microengineered tissue engineering. PMID:23355068

Hydrolyzable Tannins (Chebulagic Acid and Punicalagin) Target Viral Glycoprotein-Glycosaminoglycan Interactions To Inhibit Herpes Simplex Virus 1 Entry and Cell-to-Cell Spread▿

PubMed Central

Lin, Liang-Tzung; Chen, Ting-Ying; Chung, Chueh-Yao; Noyce, Ryan S.; Grindley, T. Bruce; McCormick, Craig; Lin, Ta-Chen; Wang, Guey-Horng; Lin, Chun-Ching; Richardson, Christopher D.

2011-01-01

Herpes simplex virus 1 (HSV-1) is a common human pathogen that causes lifelong latent infection of sensory neurons. Non-nucleoside inhibitors that can limit HSV-1 recurrence are particularly useful in treating immunocompromised individuals or cases of emerging acyclovir-resistant strains of herpesvirus. We report that chebulagic acid (CHLA) and punicalagin (PUG), two hydrolyzable tannins isolated from the dried fruits of Terminalia chebula Retz. (Combretaceae), inhibit HSV-1 entry at noncytotoxic doses in A549 human lung cells. Experiments revealed that both tannins targeted and inactivated HSV-1 viral particles and could prevent binding, penetration, and cell-to-cell spread, as well as secondary infection. The antiviral effect from either of the tannins was not associated with induction of type I interferon-mediated responses, nor was pretreatment of the host cell protective against HSV-1. Their inhibitory activities targeted HSV-1 glycoproteins since both natural compounds were able to block polykaryocyte formation mediated by expression of recombinant viral glycoproteins involved in attachment and membrane fusion. Our results indicated that CHLA and PUG blocked interactions between cell surface glycosaminoglycans and HSV-1 glycoproteins. Furthermore, the antiviral activities from the two tannins were significantly diminished in mutant cell lines unable to produce heparan sulfate and chondroitin sulfate and could be rescued upon reconstitution of heparan sulfate biosynthesis. We suggest that the hydrolyzable tannins CHLA and PUG may be useful as competitors for glycosaminoglycans in the management of HSV-1 infections and that they may help reduce the risk for development of viral drug resistance during therapy with nucleoside analogues. PMID:21307190