On strain and stress in living cells

NASA Astrophysics Data System (ADS)

Cox, Brian N.; Smith, David W.

2014-11-01

Recent theoretical simulations of amelogenesis and network formation and new, simple analyses of the basic multicellular unit (BMU) allow estimation of the order of magnitude of the strain energy density in populations of living cells in their natural environment. A similar simple calculation translates recent measurements of the force-displacement relation for contacting cells (cell-cell adhesion energy) into equivalent volume energy densities, which are formed by averaging the changes in contact energy caused by a cell's migration over the cell's volume. The rates of change of these mechanical energy densities (energy density rates) are then compared to the order of magnitude of the metabolic activity of a cell, expressed as a rate of production of metabolic energy per unit volume. The mechanical energy density rates are 4-5 orders of magnitude smaller than the metabolic energy density rate in amelogenesis or bone remodeling in the BMU, which involve modest cell migration velocities, and 2-3 orders of magnitude smaller for innervation of the gut or angiogenesis, where migration rates are among the highest for all cell types. For representative cell-cell adhesion gradients, the mechanical energy density rate is 6 orders of magnitude smaller than the metabolic energy density rate. The results call into question the validity of using simple constitutive laws to represent living cells. They also imply that cells need not migrate as inanimate objects of gradients in an energy field, but are better regarded as self-powered automata that may elect to be guided by such gradients or move otherwise. Thus Ġel=d/dt 1/2 >[(C11+C12)ɛ02+2μγ02]=(C11+C12)ɛ0ɛ˙0+2μγ0γ˙0 or Ġel=ηEɛ0ɛ˙0+η‧Eγ0γ˙0 with 1.4≤η≤3.4 and 0.7≤η‧≤0.8 for Poisson's ratio in the range 0.2≤ν≤0.4 and η=1.95 and η‧=0.75 for ν=0.3. The spatial distribution of shear strains arising within an individual cell as cells slide past one another during amelogenesis is not known in detail. However, estimates can be inferred from the known relative velocities of the cells' centers of mass. When averaged over a volume comparable to the cell size, representative values of the strain are, to order of magnitude, ɛ0≈0.1 and γ0≈0.1. The shape distortions of cells seen, for example, in Fig. 1c, imply peak strains in minor segments of a cell of magnitude unity, ɛ0≈1 and γ0≈1; these values represent the upper bound of plausible values and are included for discussion of the extremes of attainable strain energy rates.Given the strain magnitudes, the strain rates follow from the fact that a cell switches from one contacting neighbor in the adjacent row to the next in approximately 0.25 d, during which motion the strains might vary from zero to their maximum values and back again. Thus the most probable shear strain rate is inferred to be γ˙0=10-6 s-1 and the most probable tensile strain rate is inferred to be ɛ˙0≈10-6 s-1, with high bounds γ˙0=10-5 s-1 and ɛ˙0=10-5 s-1.

Dual-color dual-focus line-scanning FCS for quantitative analysis of receptor-ligand interactions in living specimens.

PubMed

Dörlich, René M; Chen, Qing; Niklas Hedde, Per; Schuster, Vittoria; Hippler, Marc; Wesslowski, Janine; Davidson, Gary; Nienhaus, G Ulrich

2015-05-07

Cellular communication in multi-cellular organisms is mediated to a large extent by a multitude of cell-surface receptors that bind specific ligands. An in-depth understanding of cell signaling networks requires quantitative information on ligand-receptor interactions within living systems. In principle, fluorescence correlation spectroscopy (FCS) based methods can provide such data, but live-cell applications have proven extremely challenging. Here, we have developed an integrated dual-color dual-focus line-scanning fluorescence correlation spectroscopy (2c2f lsFCS) technique that greatly facilitates live-cell and tissue experiments. Absolute ligand and receptor concentrations and their diffusion coefficients within the cell membrane can be quantified without the need to perform additional calibration experiments. We also determine the concentration of ligands diffusing in the medium outside the cell within the same experiment by using a raster image correlation spectroscopy (RICS) based analysis. We have applied this robust technique to study the interactions of two Wnt antagonists, Dickkopf1 and Dickkopf2 (Dkk1/2), to their cognate receptor, low-density-lipoprotein-receptor related protein 6 (LRP6), in the plasma membrane of living HEK293T cells. We obtained significantly lower affinities than previously reported using in vitro studies, underscoring the need to measure such data on living cells or tissues.

Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

PubMed

Yoshie, Sachiko; Ogasawara, Yuki; Ikehata, Masateru; Ishii, Kazuyuki; Suzuki, Yukihisa; Wada, Keiji; Wake, Kanako; Nakasono, Satoshi; Taki, Masao; Ohkubo, Chiyoji

2016-01-01

The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2 , and an early developmental gene, Hba-x , in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc . in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

Stoichiometry of Nck-dependent actin polymerization in living cells

PubMed Central

Ditlev, Jonathon A.; Michalski, Paul J.; Huber, Greg; Rivera, Gonzalo M.; Mohler, William A.

2012-01-01

Regulation of actin dynamics through the Nck/N-WASp (neural Wiskott–Aldrich syndrome protein)/Arp2/3 pathway is essential for organogenesis, cell invasiveness, and pathogen infection. Although many of the proteins involved in this pathway are known, the detailed mechanism by which it functions remains undetermined. To examine the signaling mechanism, we used a two-pronged strategy involving computational modeling and quantitative experimentation. We developed predictions for Nck-dependent actin polymerization using the Virtual Cell software system. In addition, we used antibody-induced aggregation of membrane-targeted Nck SH3 domains to test these predictions and to determine how the number of molecules in Nck aggregates and the density of aggregates affected localized actin polymerization in living cells. Our results indicate that the density of Nck molecules in aggregates is a critical determinant of actin polymerization. Furthermore, results from both computational simulations and experimentation support a model in which the Nck/N-WASp/Arp2/3 stoichiometry is 4:2:1. These results provide new insight into activities involving localized actin polymerization, including tumor cell invasion, microbial pathogenesis, and T cell activation. PMID:22613834

Identification and super-resolution imaging of ligand-activated receptor dimers in live cells

NASA Astrophysics Data System (ADS)

Winckler, Pascale; Lartigue, Lydia; Giannone, Gregory; de Giorgi, Francesca; Ichas, François; Sibarita, Jean-Baptiste; Lounis, Brahim; Cognet, Laurent

2013-08-01

Molecular interactions are key to many chemical and biological processes like protein function. In many signaling processes they occur in sub-cellular areas displaying nanoscale organizations and involving molecular assemblies. The nanometric dimensions and the dynamic nature of the interactions make their investigations complex in live cells. While super-resolution fluorescence microscopies offer live-cell molecular imaging with sub-wavelength resolutions, they lack specificity for distinguishing interacting molecule populations. Here we combine super-resolution microscopy and single-molecule Förster Resonance Energy Transfer (FRET) to identify dimers of receptors induced by ligand binding and provide super-resolved images of their membrane distribution in live cells. By developing a two-color universal-Point-Accumulation-In-the-Nanoscale-Topography (uPAINT) method, dimers of epidermal growth factor receptors (EGFR) activated by EGF are studied at ultra-high densities, revealing preferential cell-edge sub-localization. This methodology which is specifically devoted to the study of molecules in interaction, may find other applications in biological systems where understanding of molecular organization is crucial.

A multi-emitter fitting algorithm for potential live cell super-resolution imaging over a wide range of molecular densities.

PubMed

Takeshima, T; Takahashi, T; Yamashita, J; Okada, Y; Watanabe, S

2018-05-25

Multi-emitter fitting algorithms have been developed to improve the temporal resolution of single-molecule switching nanoscopy, but the molecular density range they can analyse is narrow and the computation required is intensive, significantly limiting their practical application. Here, we propose a computationally fast method, wedged template matching (WTM), an algorithm that uses a template matching technique to localise molecules at any overlapping molecular density from sparse to ultrahigh density with subdiffraction resolution. WTM achieves the localization of overlapping molecules at densities up to 600 molecules μm -2 with a high detection sensitivity and fast computational speed. WTM also shows localization precision comparable with that of DAOSTORM (an algorithm for high-density super-resolution microscopy), at densities up to 20 molecules μm -2 , and better than DAOSTORM at higher molecular densities. The application of WTM to a high-density biological sample image demonstrated that it resolved protein dynamics from live cell images with subdiffraction resolution and a temporal resolution of several hundred milliseconds or less through a significant reduction in the number of camera images required for a high-density reconstruction. WTM algorithm is a computationally fast, multi-emitter fitting algorithm that can analyse over a wide range of molecular densities. The algorithm is available through the website. https://doi.org/10.17632/bf3z6xpn5j.1. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Nanopipettes as Monitoring Probes for the Single Living Cell: State of the Art and Future Directions in Molecular Biology.

PubMed

Bulbul, Gonca; Chaves, Gepoliano; Olivier, Joseph; Ozel, Rifat Emrah; Pourmand, Nader

2018-06-06

Examining the behavior of a single cell within its natural environment is valuable for understanding both the biological processes that control the function of cells and how injury or disease lead to pathological change of their function. Single-cell analysis can reveal information regarding the causes of genetic changes, and it can contribute to studies on the molecular basis of cell transformation and proliferation. By contrast, whole tissue biopsies can only yield information on a statistical average of several processes occurring in a population of different cells. Electrowetting within a nanopipette provides a nanobiopsy platform for the extraction of cellular material from single living cells. Additionally, functionalized nanopipette sensing probes can differentiate analytes based on their size, shape or charge density, making the technology uniquely suited to sensing changes in single-cell dynamics. In this review, we highlight the potential of nanopipette technology as a non-destructive analytical tool to monitor single living cells, with particular attention to integration into applications in molecular biology.

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells.

PubMed

Freeman, Cassandra A; Samuel, Premsingh S D; Kompala, Dhinakar S

2017-07-01

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017. © 2017 American Institute of Chemical Engineers.

Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

NASA Astrophysics Data System (ADS)

Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.

2017-02-01

Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging

PubMed Central

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-01-01

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. PMID:28835375

Sequential establishment of stripe patterns in an expanding cell population.

PubMed

Liu, Chenli; Fu, Xiongfei; Liu, Lizhong; Ren, Xiaojing; Chau, Carlos K L; Li, Sihong; Xiang, Lu; Zeng, Hualing; Chen, Guanhua; Tang, Lei-Han; Lenz, Peter; Cui, Xiaodong; Huang, Wei; Hwa, Terence; Huang, Jian-Dong

2011-10-14

Periodic stripe patterns are ubiquitous in living organisms, yet the underlying developmental processes are complex and difficult to disentangle. We describe a synthetic genetic circuit that couples cell density and motility. This system enabled programmed Escherichia coli cells to form periodic stripes of high and low cell densities sequentially and autonomously. Theoretical and experimental analyses reveal that the spatial structure arises from a recurrent aggregation process at the front of the continuously expanding cell population. The number of stripes formed could be tuned by modulating the basal expression of a single gene. The results establish motility control as a simple route to establishing recurrent structures without requiring an extrinsic pacemaker.

‘Living cantilever arrays’ for characterization of mass of single live cells in fluids†

PubMed Central

Park, Kidong; Jang, Jaesung; Irimia, Daniel; Sturgis, Jennifer; Lee, James; Robinson, J. Paul; Toner, Mehmet; Bashir, Rashid

2013-01-01

The size of a cell is a fundamental physiological property and is closely regulated by various environmental and genetic factors. Optical or confocal microscopy can be used to measure the dimensions of adherent cells, and Coulter counter or flow cytometry (forward scattering light intensity) can be used to estimate the volume of single cells in a flow. Although these methods could be used to obtain the mass of single live cells, no method suitable for directly measuring the mass of single adherent cells without detaching them from the surface is currently available. We report the design, fabrication, and testing of ‘living cantilever arrays’, an approach to measure the mass of single adherent live cells in fluid using silicon cantilever mass sensor. HeLa cells were injected into microfluidic channels with a linear array of functionalized silicon cantilevers and the cells were subsequently captured on the cantilevers with positive dielectrophoresis. The captured cells were then cultured on the cantilevers in a microfluidic environment and the resonant frequencies of the cantilevers were measured. The mass of a single HeLa cell was extracted from the resonance frequency shift of the cantilever and was found to be close to the mass value calculated from the cell density from the literature and the cell volume obtained from confocal microscopy. This approach can provide a new method for mass measurement of a single adherent cell in its physiological condition in a non-invasive manner, as well as optical observations of the same cell. We believe this technology would be very valuable for single cell time-course studies of adherent live cells. PMID:18584076

Cost-Effective Live Cell Density Determination of Liquid Cultured Microorganisms.

PubMed

Kutschera, Alexander; Lamb, Jacob J

2018-02-01

Live monitoring of microorganisms growth in liquid medium is a desired parameter for many research fields. A wildly used approach for determining microbial liquid growth quantification is based on light scattering as the result of the physical interaction of light with microbial cells. These measurements are generally achieved using costly table-top instruments; however, a live, reliable, and straight forward instrument constructed using parts that are inexpensive may provide opportunities for many researchers. Here, such an instrument has been constructed and tested. It consists of modular test tube holding chambers, each with a low power monochromatic light-emitting diode, and a monolithic photodiode. A microcontroller connects to all modular chambers to control the diodes, and send the live data to either an LCD screen, or a computer. This work demonstrate that this modular instrument can determine precise cell concentrations for the bacteria Escherichia coli and Pseudomonas syringae pv. tomato DC3000, as well as Saccharomyces cerevisiae yeast.

Sensitivity of Geoelectrical Measurements to the Presence of Bacteria in Porous Media

EPA Science Inventory

We investigated the sensitivity of low frequency electrical measurements (0.1-1000 Hz) to (i) microbial cell density, (ii) live and dead cells, and (iii) microbial attachment onto mineral surfaces of clean quartz sands and iron oxide coated sands. Three strains of Pseudomonas aer...

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2014-10-01

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

Diffusion of rhodamine B and bovine serum albumin in fibrin gels seeded with primary endothelial cells.

PubMed

Shkilnyy, Andriy; Proulx, Pierre; Sharp, Jamie; Lepage, Martin; Vermette, Patrick

2012-05-01

Scaffolds with adequate mass transport properties are needed in many tissue engineering applications. Fibrin is considered a good biological material to fabricate such scaffolds. However, very little is known about mass transport in fibrin. Therefore, a method based on the analysis of fluorescence intensity for measuring the apparent diffusion coefficient of rhodamine B and fluorescein-labelled bovine serum albumin (FITC-BSA) is described. The experiments are performed in fibrin gels with and without human umbilical vein endothelial cells (HUVEC). The apparent diffusion coefficients of rhodamine B and FITC-BSA in fibrin (fibrinogen concentration of 4 mg/mL) with different cell densities are reported. A LIVE/DEAD(®) assay is performed to confirm the viability of HUVEC seeded at high densities. Diffusion coefficients for rhodamine B remain more or less constant up to 5×10(5) cells/mL and correlate well with literature values measured by other methods in water systems. This indicates that the presence of HUVEC in the fibrin gels (up to 5×10(5) cells/mL) has almost no effect on the diffusion coefficients. Higher cell densities (>5×10(5) cells/mL) result in a decrease of the diffusion coefficients. Diffusion coefficients of rhodamine B and FITC-BSA obtained by this method agree with diffusion coefficients in water predicted by the Stokes-Einstein equation. The experimental design used in this study can be applied to measure diffusion coefficients in different types of gels seeded or not with living cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Design and validation of a pulsatile perfusion bioreactor for 3D high cell density cultures.

PubMed

Chouinard, Julie A; Gagnon, Serge; Couture, Marc G; Lévesque, Alain; Vermette, Patrick

2009-12-15

This study presents the design and validation of a pulsatile flow perfusion bioreactor able to provide a suitable environment for 3D high cell density cultures for tissue engineering applications. Our bioreactor system is mobile, does not require the use of traditional cell culture incubators and is easy to sterilize. It provides real-time monitoring and stable control of pH, dissolved oxygen concentration, temperature, pressure, pulsation frequency, and flow rate. In this bioreactor system, cells are cultured in a gel within a chamber perfused by a culture medium fed by hollow fibers. Human umbilical vein endothelial cells (HUVEC) suspended in fibrin were found to be living, making connections and proliferating up to five to six times their initial seeding number after a 48-h culture period. Cells were uniformly dispersed within the 14.40 mm x 17.46 mm x 6.35 mm chamber. A larger fraction of the cells suspended in 6.35-mm thick gels and cultured in a traditional CO(2) incubator were found to be round and dead [corrected]. In control experiments carried out in a traditional cell culture incubator, the scarcely found living cells were mostly on top of the gels, while cells cultured under perfusion bioreactor conditions were found to be alive and uniformly distributed across the gel. 2009 Wiley Periodicals, Inc.

Under the Microscope: Single-Domain Antibodies for Live-Cell Imaging and Super-Resolution Microscopy.

PubMed

Traenkle, Bjoern; Rothbauer, Ulrich

2017-01-01

Single-domain antibodies (sdAbs) have substantially expanded the possibilities of advanced cellular imaging such as live-cell or super-resolution microscopy to visualize cellular antigens and their dynamics. In addition to their unique properties including small size, high stability, and solubility in many environments, sdAbs can be efficiently functionalized according to the needs of the respective imaging approach. Genetically encoded intrabodies fused to fluorescent proteins (chromobodies) have become versatile tools to study dynamics of endogenous proteins in living cells. Additionally, sdAbs conjugated to organic dyes were shown to label cellular structures with high density and minimal fluorophore displacement making them highly attractive probes for super-resolution microscopy. Here, we review recent advances of the chromobody technology to visualize localization and dynamics of cellular targets and the application of chromobody-based cell models for compound screening. Acknowledging the emerging importance of super-resolution microscopy in cell biology, we further discuss advantages and challenges of sdAbs for this technology.

Application of propidium monoazide quantitative real-time PCR to quantify the viability of Lactobacillus delbrueckii ssp. bulgaricus.

PubMed

Shao, Yuyu; Wang, Zhaoxia; Bao, Qiuhua; Zhang, Heping

2016-12-01

In this study, a combination of propidium monoazide (PMA) and quantitative real-time PCR (qPCR) was used to develop a method to determine the viability of cells of Lactobacillus delbrueckii ssp. bulgaricus ND02 (L. bulgaricus) that may have entered into a viable but nonculturable state. This can happen due to its susceptibility to cold shock during lyophilization and storage. Propidium monoazide concentration, PMA incubation time, and light exposure time were optimized to fully exploit the PMA-qPCR approach to accurately assess the total number of living L. bulgaricus ND02. Although PMA has little influence on living cells, when concentrations of PMA were higher than 30μg/mL the number of PCR-positive living bacteria decreased from 10 6 to 10 5 cfu/mL in comparison with qPCR enumeration. Mixtures of living and dead cells were used as method verification samples for enumeration by PMA-qPCR, demonstrating that this method was feasible and effective for distinguishing living cells of L. bulgaricus when mixed with a known number of dead cells. We suggest that several conditions need to be studied further before PMA-qPCR methods can be accurately used to distinguish living from dead cells for enumeration under more realistic sampling situations. However, this research provides a rapid way to enumerate living cells of L. bulgaricus and could be used to optimize selection of cryoprotectants in the lyophilization process and develop technologies for high cell density cultivation and optimal freeze-drying processes. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Engineering Multifunctional Living Paints: Thin, Convectively-Assembled Biocomposite Coatings of Live Cells and Colloidal Latex Particles Deposited by Continuous Convective-Sedimentation Assembly

NASA Astrophysics Data System (ADS)

Jenkins, Jessica Shawn

Advanced composite materials could be revolutionized by the development of methods to incorporate living cells into functional materials and devices. This could be accomplished by continuously and rapidly depositing thin ordered arrays of adhesive colloidal latex particles and live cells that maintain stability and preserve microbial reactivity. Convective assembly is one method of rapidly assembling colloidal particles into thin (<10 microm thick), ordered films with engineered compositions, thicknesses, and particle packing that offer several advantages over thicker randomly ordered composites, including enhanced cell stability and increased reactivity through minimized diffusion resistance to nutrients and reduced light scattering. This method can be used to precisely deposit live bacteria, cyanobacteria, yeast, and algae into biocomposite coatings, forming reactive biosensors, photoabsorbers, or advanced biocatalysts. This dissertation developed new continuous deposition and coating characterization methods for fabricating and characterizing <10 microm thick colloid coatings---monodispersed latex particle or cell suspensions, bimodal blends of latex particles or live cells and microspheres, and trimodal formulations of biomodal latex and live cells on substrates such as aluminum foil, glass, porous Kraft paper, polyester, and polypropylene. Continuous convective-sedimentation assembly (CSA) is introduced to enable fabrication of larger surface area and long coatings by constantly feeding coating suspension to the meniscus, thus expanding the utility of convective assembly to deposit monolayer or very thin films or multi-layer coatings composed of thin layers on a large scale. Results show thin, tunable coatings can be fabricated from diverse coating suspensions and critical coating parameters that control thickness and structure. Particle size ratio and charge influence deposition, convective mixing or demixing and relative particle locations. Substrate wettability and suspension composition influence coating microstructure by controlling suspension delivery and spreading across the substrate. Microbes behave like colloidal particles during CSA, allowing for deposition of very thin stable biocomposite coatings of latex-live cell blends. CSA of particle-cell blends result in open-packed structures (15-45% mean void space), instead of tightly packed coatings attainable with single component systems, confirming the existence of significant polymer particle-cell interactions and formation of particle aggregates that disrupt coating microstructure during deposition. Tunable process parameters, such as particle concentration, fluid sonication, and fluid density, influence coating homogeneity when the meniscus is continuously supplied. Fluid density modification and fluid sonication affect particle sedimentation and distribution in the coating growth front whereas the suspended particle concentration strongly affects coating thickness, but has almost no effect on void space. Changing the suspension delivery mode (topside versus underside CCSA) yields disparate meniscus volumes and uneven particle delivery to the drying front, which enables control of the coating microstructure by varying the total number of particles available for deposition. The judicious combination of all these parameters will enable deposition of uniform, thin, latex-cell monolayers over areas on the order of tens of square centimeters or larger. To demonstrate the utility of biocomposite coatings, this dissertation investigated photoreactive coatings (artificial leaves) from suspensions of latex particles and nitrogen-limited Rps. palustris CGA009 or sulfur-limited C. reinhardtii CC-124. These coatings demonstrated stable, sustained (>90 hours) photohydrogen production under anoxygenic conditions. Nutrient reduction slows cell division, minimizing coating outgrowth, and promotes photohydrogen generation, improving coating reactivity. Scanning electron microscopy of microstructure revealed how coating reactivity can be controlled by the size and distribution of the nanopores in the biocomposite layers. Variations in colloid microsphere size and suspension composition do not affect coating reactivity, but both parameters alter coating microstructure. Porous paper coated with thin coatings of colloidal particles and cells to enable coatings to be used in a gas-phase without dehydration may offer higher volumetric productivity for hydrogen production. Future work should focus on optimization of cell density, light intensity, media cycling, and acetate concentration.

Magnetic levitation of single cells

PubMed Central

Durmus, Naside Gozde; Tekin, H. Cumhur; Guven, Sinan; Sridhar, Kaushik; Arslan Yildiz, Ahu; Calibasi, Gizem; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

2015-01-01

Several cellular events cause permanent or transient changes in inherent magnetic and density properties of cells. Characterizing these changes in cell populations is crucial to understand cellular heterogeneity in cancer, immune response, infectious diseases, drug resistance, and evolution. Although magnetic levitation has previously been used for macroscale objects, its use in life sciences has been hindered by the inability to levitate microscale objects and by the toxicity of metal salts previously applied for levitation. Here, we use magnetic levitation principles for biological characterization and monitoring of cells and cellular events. We demonstrate that each cell type (i.e., cancer, blood, bacteria, and yeast) has a characteristic levitation profile, which we distinguish at an unprecedented resolution of 1 × 10−4 g⋅mL−1. We have identified unique differences in levitation and density blueprints between breast, esophageal, colorectal, and nonsmall cell lung cancer cell lines, as well as heterogeneity within these seemingly homogenous cell populations. Furthermore, we demonstrate that changes in cellular density and levitation profiles can be monitored in real time at single-cell resolution, allowing quantification of heterogeneous temporal responses of each cell to environmental stressors. These data establish density as a powerful biomarker for investigating living systems and their responses. Thereby, our method enables rapid, density-based imaging and profiling of single cells with intriguing applications, such as label-free identification and monitoring of heterogeneous biological changes under various physiological conditions, including antibiotic or cancer treatment in personalized medicine. PMID:26124131

Assessing resolution in live cell structured illumination microscopy

NASA Astrophysics Data System (ADS)

Pospíšil, Jakub; Fliegel, Karel; Klíma, Miloš

2017-12-01

Structured Illumination Microscopy (SIM) is a powerful super-resolution technique, which is able to enhance the resolution of optical microscope beyond the Abbe diffraction limit. In the last decade, numerous SIM methods that achieve the resolution of 100 nm in the lateral dimension have been developed. The SIM setups with new high-speed cameras and illumination pattern generators allow rapid acquisition of the live specimen. Therefore, SIM is widely used for investigation of the live structures in molecular and live cell biology. Quantitative evaluation of resolution enhancement in a real sample is essential to describe the efficiency of super-resolution microscopy technique. However, measuring the resolution of a live cell sample is a challenging task. Based on our experimental findings, the widely used Fourier ring correlation (FRC) method does not seem to be well suited for measuring the resolution of SIM live cell video sequences. Therefore, the resolution assessing methods based on Fourier spectrum analysis are often used. We introduce a measure based on circular average power spectral density (PSDca) estimated from a single SIM image (one video frame). PSDca describes the distribution of the power of a signal with respect to its spatial frequency. Spatial resolution corresponds to the cut-off frequency in Fourier space. In order to estimate the cut-off frequency from a noisy signal, we use a spectral subtraction method for noise suppression. In the future, this resolution assessment approach might prove useful also for single-molecule localization microscopy (SMLM) live cell imaging.

Development of an electro-responsive platform for the controlled transfection of mammalian cells

NASA Astrophysics Data System (ADS)

Hook, Andrew L.; Thissen, Helmut W.; Hayes, Jason P.; Voelcker, Nicolas H.

2005-02-01

The recent development of living microarrays as novel tools for the analysis of gene expression in an in-situ environment promises to unravel gene function within living organisms. In order to significantly enhance microarray performance, we are working towards electro-responsive DNA transfection chips. This study focuses on the control of DNA adsorption and desorption by appropriate surface modification of highly doped p++ silicon. Silicon was modified by plasma polymerisation of allylamine (ALAPP), a non-toxic surface that sustains cell growth. Subsequent high surface density grafting of poly(ethylene oxide) formed a layer resistant to biomolecule adsorption and cell attachment. Spatially controlled excimer laser ablation of the surface produced micron resolution patterns of re-exposed plasma polymer whilst the rest of the surface remained non-fouling. We observed electro-stimulated preferential adsorption of DNA to the ALAPP surface and subsequent desorption by the application of a negative bias. Cell culture experiments with HEK 293 cells demonstrated efficient and controlled transfection of cells using the expression of green fluorescent protein as a reporter. Thus, these chemically patterned surfaces are promising platforms for use as living microarrays.

PeakForce Tapping resolves individual microvilli on living cells.

PubMed

Schillers, Hermann; Medalsy, Izhar; Hu, Shuiqing; Slade, Andrea L; Shaw, James E

2016-02-01

Microvilli are a common structure found on epithelial cells that increase the apical surface thus enhancing the transmembrane transport capacity and also serve as one of the cell's mechanosensors. These structures are composed of microfilaments and cytoplasm, covered by plasma membrane. Epithelial cell function is usually coupled to the density of microvilli and its individual size illustrated by diseases, in which microvilli degradation causes malabsorption and diarrhea. Atomic force microscopy (AFM) has been widely used to study the topography and morphology of living cells. Visualizing soft and flexible structures such as microvilli on the apical surface of a live cell has been very challenging because the native microvilli structures are displaced and deformed by the interaction with the probe. PeakForce Tapping® is an AFM imaging mode, which allows reducing tip-sample interactions in time (microseconds) and controlling force in the low pico-Newton range. Data acquisition of this mode was optimized by using a newly developed PeakForce QNM-Live Cell probe, having a short cantilever with a 17-µm-long tip that minimizes hydrodynamic effects between the cantilever and the sample surface. In this paper, we have demonstrated for the first time the visualization of the microvilli on living kidney cells with AFM using PeakForce Tapping. The structures observed display a force dependence representing either the whole microvilli or just the tips of the microvilli layer. Together, PeakForce Tapping allows force control in the low pico-Newton range and enables the visualization of very soft and flexible structures on living cells under physiological conditions. © 2015 The Authors Journal of Molecular Recognition Published by John Wiley & Sons Ltd.

Polyelectrolytes with high charge density

NASA Technical Reports Server (NTRS)

Rembaum, A.; Yen, S. P. S.

1974-01-01

Polymers can be used as flocculants to clarify residential and industrial water supplies and as bactericidal and fungicidal agents. They can be used in preparation of electroconductive photocopy papers, to improve living cell adhesion to glass or plastic, and as anticancer agents.

An in situ probe for on-line monitoring of cell density and viability on the basis of dark field microscopy in conjunction with image processing and supervised machine learning.

PubMed

Wei, Ning; You, Jia; Friehs, Karl; Flaschel, Erwin; Nattkemper, Tim Wilhelm

2007-08-15

Fermentation industries would benefit from on-line monitoring of important parameters describing cell growth such as cell density and viability during fermentation processes. For this purpose, an in situ probe has been developed, which utilizes a dark field illumination unit to obtain high contrast images with an integrated CCD camera. To test the probe, brewer's yeast Saccharomyces cerevisiae is chosen as the target microorganism. Images of the yeast cells in the bioreactors are captured, processed, and analyzed automatically by means of mechatronics, image processing, and machine learning. Two support vector machine based classifiers are used for separating cells from background, and for distinguishing live from dead cells afterwards. The evaluation of the in situ experiments showed strong correlation between results obtained by the probe and those by widely accepted standard methods. Thus, the in situ probe has been proved to be a feasible device for on-line monitoring of both cell density and viability with high accuracy and stability. (c) 2007 Wiley Periodicals, Inc.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

PubMed Central

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-01-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min−1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics. PMID:27991512

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions

NASA Astrophysics Data System (ADS)

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-01

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min-1. The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions.

PubMed

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Szlag, Daniel; Feletti, Lely; Vandenberg, Wim; Dedecker, Peter; Hofkens, Johan; Leutenegger, Marcel; Lasser, Theo; Radenovic, Aleksandra

2016-12-19

Live-cell imaging of focal adhesions requires a sufficiently high temporal resolution, which remains a challenge for super-resolution microscopy. Here we address this important issue by combining photoactivated localization microscopy (PALM) with super-resolution optical fluctuation imaging (SOFI). Using simulations and fixed-cell focal adhesion images, we investigate the complementarity between PALM and SOFI in terms of spatial and temporal resolution. This PALM-SOFI framework is used to image focal adhesions in living cells, while obtaining a temporal resolution below 10 s. We visualize the dynamics of focal adhesions, and reveal local mean velocities around 190 nm min -1 . The complementarity of PALM and SOFI is assessed in detail with a methodology that integrates a resolution and signal-to-noise metric. This PALM and SOFI concept provides an enlarged quantitative imaging framework, allowing unprecedented functional exploration of focal adhesions through the estimation of molecular parameters such as fluorophore densities and photoactivation or photoswitching kinetics.

Direct observation of the effects of cellulose synthesis inhibitors using live cell imaging of Cellulose Synthase (CESA) in Physcomitrella patens.

PubMed

Tran, Mai L; McCarthy, Thomas W; Sun, Hao; Wu, Shu-Zon; Norris, Joanna H; Bezanilla, Magdalena; Vidali, Luis; Anderson, Charles T; Roberts, Alison W

2018-01-15

Results from live cell imaging of fluorescently tagged Cellulose Synthase (CESA) proteins in Cellulose Synthesis Complexes (CSCs) have enhanced our understanding of cellulose biosynthesis, including the mechanisms of action of cellulose synthesis inhibitors. However, this method has been applied only in Arabidopsis thaliana and Brachypodium distachyon thus far. Results from freeze fracture electron microscopy of protonemal filaments of the moss Funaria hygrometrica indicate that a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), fragments CSCs and clears them from the plasma membrane. This differs from Arabidopsis, in which DCB causes CSC accumulation in the plasma membrane and a different cellulose synthesis inhibitor, isoxaben, clears CSCs from the plasma membrane. In this study, live cell imaging of the moss Physcomitrella patens indicated that DCB and isoxaben have little effect on protonemal growth rates, and that only DCB causes tip rupture. Live cell imaging of mEGFP-PpCESA5 and mEGFP-PpCESA8 showed that DCB and isoxaben substantially reduced CSC movement, but had no measureable effect on CSC density in the plasma membrane. These results suggest that DCB and isoxaben have similar effects on CSC movement in P. patens and Arabidopsis, but have different effects on CSC intracellular trafficking, cell growth and cell integrity in these divergent plant lineages.

Collective interaction of microscale matters in natural analogy: human cancer cells vs. microspheres

NASA Astrophysics Data System (ADS)

Ahn, Sungsook; Lee, Sang Joon; Postech Team

2014-11-01

Collective behaviors have been considered both in living and lifeless things as a natural phenomenon. During the ordering process, a sudden and spontaneous transition is typically generated between an order and a disorder according to the population density of interacting elements. In a cellular level collective behavior, the cells are distributed in the characteristic patterns according to the population density and the mutual interaction of the individual cells undergo density-dependent diffusive motion. On the other hand, density-controlled surface-modified hollow microsphere suspension induces an overpopulation via buoyancy which provides a driving force to induce an assembly. The collective behaviors of the cells and microspheres in a designed liquid medium are explained in terms of the deviation from the interparticle distance distribution and the induced strength to organize the particle position in a specific distance range. as a result, microscale particulate matters exhibit high resemblance in their pair correlation and dynamical heterogeneity in the intermediate range between a single individual and an agglomerate. Therefore, it is suggested that biological systems are analogically explained to be dominated by physically interactive aspects.

Current density reversibly alters metabolic spatial structure of exoelectrogenic anode biofilms

NASA Astrophysics Data System (ADS)

Sun, Dan; Cheng, Shaoan; Zhang, Fang; Logan, Bruce E.

2017-07-01

Understanding how current densities affect electrogenic biofilm activity is important for wastewater treatment as current densities can substantially decrease at COD concentrations greater than those suitable for discharge to the environment. We examined the biofilm's response, in terms of viability and enzymatic activity, to different current densities using microbial electrolysis cells with a lower (0.7 V) or higher (0.9 V) added voltage to alter current production. Viability was assessed using florescent dyes, with dead cells identified on the basis of dye penetration due to a compromised cell outer-membrane (red), and live cells (intact membrane) fluorescing green. Biofilms operated with 0.7 V produced 2.4 ± 0.2 A m-2, and had an inactive layer near the electrode and a viable layer at the biofilm-solution interface. The lack of cell activity near the electrode surface was confirmed by using an additional dye that fluoresces only with enzymatic activity. Adding 0.9 V increased the current by 61%, and resulted in a single, more homogeneous and active biofilm layer. Switching biofilms between these two voltages produced outcomes associated with the new current rather than the previous biofilm conditions. These findings suggest that maintaining higher current densities will be needed to ensure long-term viability electrogenic biofilms.

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Direct fluorescent-dye labeling of α-tubulin in mammalian cells for live cell and superresolution imaging.

PubMed

Schvartz, Tomer; Aloush, Noa; Goliand, Inna; Segal, Inbar; Nachmias, Dikla; Arbely, Eyal; Elia, Natalie

2017-10-15

Genetic code expansion and bioorthogonal labeling provide for the first time a way for direct, site-specific labeling of proteins with fluorescent-dyes in live cells. Although the small size and superb photophysical parameters of fluorescent-dyes offer unique advantages for high-resolution microscopy, this approach has yet to be embraced as a tool in live cell imaging. Here we evaluated the feasibility of this approach by applying it for α-tubulin labeling. After a series of calibrations, we site-specifically labeled α-tubulin with silicon rhodamine (SiR) in live mammalian cells in an efficient and robust manner. SiR-labeled tubulin successfully incorporated into endogenous microtubules at high density, enabling video recording of microtubule dynamics in interphase and mitotic cells. Applying this labeling approach to structured illumination microscopy resulted in an increase in resolution, highlighting the advantages in using a smaller, brighter tag. Therefore, using our optimized assay, genetic code expansion provides an attractive tool for labeling proteins with a minimal, bright tag in quantitative high-resolution imaging. © 2017 Schvartz 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).

Horizontal transmission of Symbiodinium cells between adult and juvenile corals is aided by benthic sediment

NASA Astrophysics Data System (ADS)

Nitschke, Matthew R.; Davy, Simon K.; Ward, Selina

2016-03-01

Of all reef-building coral species, 80-85 % initially draw their intracellular symbionts (dinoflagellates of the genus Symbiodinium) from the environment. Although Symbiodinium cells are crucial for the growth of corals and the formation of coral reefs, little is known about how corals first encounter free-living Symbiodinium cells. We report how the supply of free-living Symbiodinium cells to the benthos by adult corals can increase the rate of horizontal symbiont acquisition for conspecific recruits. Three species of newly settled aposymbiotic (i.e., symbiont-free) corals were maintained in an open aquarium system containing: sterilized sediment and adult coral fragments combined; adult coral fragments alone; sterilized sediment alone; or seawater at Heron Island, Great Barrier Reef, Australia. In all instances, the combination of an adult coral and sediment resulted in the highest symbiont acquisition rates by juvenile corals (up to five-fold greater than seawater alone). Juvenile corals exposed to individual treatments of adult coral or sediment produced an intermediate acquisition response (<52 % of recruits), and symbiont acquisition from unfiltered seawater was comparatively low (<20 % of recruits). Additionally, benthic free-living Symbiodinium cells reached their highest densities in the adult coral + sediment treatment (up to 1.2 × 104 cells mL-1). Our results suggest that corals seed microhabitats with free-living Symbiodinium cells suitable for many coral species during the process of coral recruitment.

Poly(ethylene glycol) hydrogel microstructures encapsulating living cells

NASA Technical Reports Server (NTRS)

Koh, Won-Gun; Revzin, Alexander; Pishko, Michael V.

2002-01-01

We present an easy and effective method for the encapsulation of cells inside PEG-based hydrogel microstructures fabricated using photolithography. High-density arrays of three-dimensional microstructures were created on substrates using this method. Mammalian cells were encapsulated in cylindrical hydrogel microstructures of 600 and 50 micrometers in diameter or in cubic hydrogel structures in microfluidic channels. Reducing lateral dimension of the individual hydrogel microstructure to 50 micrometers allowed us to isolate 1-3 cells per microstructure. Viability assays demonstrated that cells remained viable inside these hydrogels after encapsulation for up to 7 days.

Trapping and assembling of particles and live cells on large-scale random gold nano-island substrates

PubMed Central

Kang, Zhiwen; Chen, Jiajie; Wu, Shu-Yuen; Chen, Kun; Kong, Siu-Kai; Yong, Ken-Tye; Ho, Ho-Pui

2015-01-01

We experimentally demonstrated the use of random plasmonic nano-islands for optical trapping and assembling of particles and live cells into highly organized pattern with low power density. The observed trapping effect is attributed to the net contribution due to near-field optical trapping force and long-range thermophoretic force, which overcomes the axial convective drag force, while the lateral convection pushes the target objects into the trapping zone. Our work provides a simple platform for on-chip optical manipulation of nano- and micro-sized objects, and may find applications in physical and life sciences. PMID:25928045

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

PubMed

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

2003-11-01

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

Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

PubMed

Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

2016-08-01

Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

PhotoGate microscopy: tracking single molecules in a cytoplasm (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yildiz, Ahmet

2016-02-01

Tracking single molecules inside cells reveals the dynamics of biological processes, including receptor trafficking, signaling and cargo transport. However, individual molecules often cannot be resolved inside cells due to their high density in the cellular environment. We developed a photobleaching gate assay, which controls the number of fluorescent particles in a region of interest by repeatedly photobleaching its boundary. Using this method, we tracked single particles at surface densities two orders of magnitude higher than the single-molecule detection limit. We observed ligand-induced dimerization of epidermal growth factor receptors (EGFR) on a live cell membrane. In addition, we tracked individual intraflagellar transport (IFT) trains along the length of a cilium and observed their remodeling at the ciliary tip.

Ratiometric sensing of fluoride and acetate anions based on a BODIPY-azaindole platform and its application to living cell imaging.

PubMed

Mahapatra, Ajit Kumar; Maji, Rajkishor; Maiti, Kalipada; Adhikari, Susanta Sekhar; Das Mukhopadhyay, Chitrangada; Mandal, Debasish

2014-01-07

A new BODIPY-azaindole based fluorescent sensor 1 was designed and synthesized as a new colorimetric and ratiometric fluorescent chemosensor for fluoride. The binding and sensing abilities of sensor 1 towards various anions were studied by absorption, emission and (1)H NMR titration spectroscopies. The spectral responses of 1 to fluoride in acetonitrile-water were studied: an approximately 69 nm red shift in absorption and ratiometric fluorescent response was observed. The striking light yellow to deep brown color change in ambient light and green to blue emission color change are thought to be due to the deprotonation of the indole moiety of the azaindole fluorophore. From the changes in the absorption, fluorescence, and (1)H NMR titration spectra, proton-transfer mechanisms were deduced. Density function theory and time-dependent density function theory calculations were conducted to rationalize the optical response of the sensor. Results were supported by confocal fluorescence imaging and MTT assay of live cells.

Statistical mechanics of tuned cell signalling: sensitive collective response by synthetic biological circuits

NASA Astrophysics Data System (ADS)

Voliotis, M.; Liverpool, T. B.

2017-03-01

Living cells sense and process environmental cues through noisy biochemical mechanisms. This apparatus limits the scope of engineering cells as viable sensors. Here, we highlight a mechanism that enables robust, population-wide responses to external stimulation based on cellular communication, known as quorum sensing. We propose a synthetic circuit consisting of two mutually repressing quorum sensing modules. At low cell densities the system behaves like a genetic toggle switch, while at higher cell densities the behaviour of nearby cells is coupled via diffusible quorum sensing molecules. We show by systematic coarse graining that at large length and timescales that the system can be described using the Ising model of a ferromagnet. Thus, in analogy with magnetic systems, the sensitivity of the population-wide response, or its ‘susceptibility’ to a change in the external signal, is highly enhanced for a narrow range of cell-cell coupling close to a critical value. We expect that our approach will be used to enhance the sensitivity of synthetic bio-sensing networks.

The effect of cell density, proximity, and time on the cytotoxicity of magnesium and galvanically coupled magnesium-titanium particles in vitro.

PubMed

Kim, Jua; Gilbert, Jeremy L

2018-05-01

Magnesium (Mg) and galvanically coupled magnesium-titanium (Mg-Ti) particles in vitro have been reported previously to kill cells in a dosage-dependent manner. Mg-Ti particles kill cells more effectively than Mg alone, due to the galvanic effect of Mg and Ti. This study further investigated the in vitro cytotoxicity of Mg and Mg-Ti in terms of particle concentration, cell density, time, and proximity. Cell density has an effect on cell viability only at low particle concentrations (below 250 µg/mL), where cell viability dropped only for lower cell densities (5000-10,000 cells/cm 2 ) and not for higher cell densities (20,000-30,000 cells/cm 2 ), showing that the particles cannot kill if there are more cells present. Cytotoxicity of Mg and Mg-Ti particles is quick and temporary, where the particles kill cells only during particle corrosion (first 24 h). Depending on the percentage of surviving cells, particle concentrations, and ongoing corrosion activity, the remaining live cells either proliferated and recovered, or just remained viable and quiescent. The particle killing is also proximity-dependent, where cell viability was significantly higher for cells far away from the particles (greater than ∼1 mm) compared to those close to the particles (less than ∼1 mm). Although the increase of pH does affect cell viability negatively, it is not the sole killing factor since cell viability is significantly dependent on particle type and proximity but not pH. Mg and Mg-Ti particles used in this study are large enough to prevent direct cell phagocytosis so that the cell killing effect may be attributed to solely electrochemical reactions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1428-1439, 2018. © 2018 Wiley Periodicals, Inc.

Mass Measurements of Focal Adhesions in Single Cells Using High Resolution Surface Plasmon Resonance Microscopy.

PubMed

Peterson, Alexander W; Halter, Michael; Tona, Alessandro; Plant, Anne L; Elliott, John T

2018-01-01

Surface plasmon resonance microscopy (SPRM) is a powerful label-free imaging technique with spatial resolution approaching the optical diffraction limit. The high sensitivity of SPRM to small changes in index of refraction at an interface allows imaging of dynamic protein structures within a cell. Visualization of subcellular features, such as focal adhesions (FAs), can be performed on live cells using a high numerical aperture objective lens with a digital light projector to precisely position the incident angle of the excitation light. Within the cell-substrate region of the SPRM image, punctate regions of high contrast are putatively identified as the cellular FAs. Optical parameter analysis is achieved by application of the Fresnel model to the SPRM data and resulting refractive index measurements are used to calculate protein density and mass. FAs are known to be regions of high protein density that reside at the cell-substratum interface. Comparing SPRM with fluorescence images of antibody stained for vinculin, a component in FAs, reveals similar measurements of FA size. In addition, a positive correlation between FA size and protein density is revealed by SPRM. Comparing SPRM images for two cell types reveals a distinct difference in the protein density and mass of their respective FAs. Application of SPRM to quantify mass can greatly aid monitoring basic processes that control FA mass and growth and contribute to accurate models that describe cell-extracellular interactions.

3D characterization of EMT cell density in developing cardiac cushions using optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Yu, Siyao; Gu, Shi; Zhao, Xiaowei; Liu, Yehe; Jenkins, Michael W.; Watanabe, Michiko; Rollins, Andrew M.

2017-02-01

Congenital heart defects (CHDs) are the most common birth defect, affecting between 4 and 75 per 1,000 live births depending on the inclusion criteria. Many of these defects can be traced to defects of cardiac cushions, critical structures during development that serve as precursors to many structures in the mature heart, including the atrial and ventricular septa, and all four sets of cardiac valves. Epithelial-mesenchymal transition (EMT) is the process through which cardiac cushions become populated with cells. Altered cushion size or altered cushion cell density has been linked to many forms of CHDs, however, quantitation of cell density in the complex 3D cushion structure poses a significant challenge to conventional histology. Optical coherence tomography (OCT) is a technique capable of 3D imaging of the developing heart, but typically lacks the resolution to differentiate individual cells. Our goal is to develop an algorithm to quantitatively characterize the density of cells in the developing cushion using 3D OCT imaging. First, in a heart volume, the atrioventricular (AV) cushions were manually segmented. Next, all voxel values in the region of interest were pooled together to generate a histogram. Finally, two populations of voxels were classified using either K-means classification, or a Gaussian mixture model (GMM). The voxel population with higher values represents cells in the cushion. To test the algorithm, we imaged and evaluated avian embryonic hearts at looping stages. As expected, our result suggested that the cell density increases with developmental stages. We validated the technique against scoring by expert readers.

Imaging optical sensor arrays.

PubMed

Walt, David R

2002-10-01

Imaging optical fibres have been etched to prepare microwell arrays. These microwells have been loaded with sensing materials such as bead-based sensors and living cells to create high-density sensor arrays. The extremely small sizes and volumes of the wells enable high sensitivity and high information content sensing capabilities.

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

NASA Astrophysics Data System (ADS)

Deschout, Hendrik; Lukes, Tomas; Sharipov, Azat; Feletti, Lely; Lasser, Theo; Radenovic, Aleksandra

2017-02-01

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM-SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

Live cell imaging at the Munich ion microbeam SNAKE - a status report.

PubMed

Drexler, Guido A; Siebenwirth, Christian; Drexler, Sophie E; Girst, Stefanie; Greubel, Christoph; Dollinger, Günther; Friedl, Anna A

2015-02-18

Ion microbeams are important tools in radiobiological research. Still, the worldwide number of ion microbeam facilities where biological experiments can be performed is limited. Even fewer facilities combine ion microirradiation with live-cell imaging to allow microscopic observation of cellular response reactions starting very fast after irradiation and continuing for many hours. At SNAKE, the ion microbeam facility at the Munich 14 MV tandem accelerator, a large variety of biological experiments are performed on a regular basis. Here, recent developments and ongoing research projects at the ion microbeam SNAKE are presented with specific emphasis on live-cell imaging experiments. An overview of the technical details of the setup is given, including examples of suitable biological samples. By ion beam focusing to submicrometer beam spot size and single ion detection it is possible to target subcellular structures with defined numbers of ions. Focusing of high numbers of ions to single spots allows studying the influence of high local damage density on recruitment of damage response proteins.

Simple and Inexpensive Paper-Based Astrocyte Co-culture to Improve Survival of Low-Density Neuronal Networks

PubMed Central

Aebersold, Mathias J.; Thompson-Steckel, Greta; Joutang, Adriane; Schneider, Moritz; Burchert, Conrad; Forró, Csaba; Weydert, Serge; Han, Hana; Vörös, János

2018-01-01

Bottom-up neuroscience aims to engineer well-defined networks of neurons to investigate the functions of the brain. By reducing the complexity of the brain to achievable target questions, such in vitro bioassays better control experimental variables and can serve as a versatile tool for fundamental and pharmacological research. Astrocytes are a cell type critical to neuronal function, and the addition of astrocytes to neuron cultures can improve the quality of in vitro assays. Here, we present cellulose as an astrocyte culture substrate. Astrocytes cultured on the cellulose fiber matrix thrived and formed a dense 3D network. We devised a novel co-culture platform by suspending the easy-to-handle astrocytic paper cultures above neuronal networks of low densities typically needed for bottom-up neuroscience. There was significant improvement in neuronal viability after 5 days in vitro at densities ranging from 50,000 cells/cm2 down to isolated cells at 1,000 cells/cm2. Cultures exhibited spontaneous spiking even at the very low densities, with a significantly greater spike frequency per cell compared to control mono-cultures. Applying the co-culture platform to an engineered network of neurons on a patterned substrate resulted in significantly improved viability and almost doubled the density of live cells. Lastly, the shape of the cellulose substrate can easily be customized to a wide range of culture vessels, making the platform versatile for different applications that will further enable research in bottom-up neuroscience and drug development. PMID:29535595

Phase separation like dynamics during Myxococcus xanthus fruiting body formation

NASA Astrophysics Data System (ADS)

Liu, Guannan; Thutupalli, Shashi; Wigbers, Manon; Shaevitz, Joshua

2015-03-01

Collective motion exists in many living organisms as an advantageous strategy to help the entire group with predation, forage, and survival. However, the principles of self-organization underlying such collective motions remain unclear. During various developmental stages of the soil-dwelling bacterium, Myxococcus xanthus, different types of collective motions are observed. In particular, when starved, M. xanthus cells eventually aggregate together to form 3-dimensional structures (fruiting bodies), inside which cells sporulate in response to the stress. We study the fruiting body formation process as an out of equilibrium phase separation process. As local cell density increases, the dynamics of the aggregation M. xanthus cells switch from a spatio-temporally random process, resembling nucleation and growth, to an emergent pattern formation process similar to a spinodal decomposition. By employing high-resolution microscopy and a video analysis system, we are able to track the motion of single cells within motile collective groups, while separately tuning local cell density, cell velocity and reversal frequency, probing the multi-dimensional phase space of M. xanthus development.

Very high cell density perfusion of CHO cells anchored in a non-woven matrix-based bioreactor.

PubMed

Zhang, Ye; Stobbe, Per; Silvander, Christian Orrego; Chotteau, Véronique

2015-11-10

Recombinant Chinese Hamster Ovary (CHO) cells producing IgG monoclonal antibody were cultivated in a novel perfusion culture system CellTank, integrating the bioreactor and the cell retention function. In this system, the cells were harbored in a non-woven polyester matrix perfused by the culture medium and immersed in a reservoir. Although adapted to suspension, the CHO cells stayed entrapped in the matrix. The cell-free medium was efficiently circulated from the reservoir into- and through the matrix by a centrifugal pump placed at the bottom of the bioreactor resulting in highly homogenous concentrations of the nutrients and metabolites in the whole system as confirmed by measurements from different sampling locations. A real-time biomass sensor using the dielectric properties of living cells was used to measure the cell density. The performances of the CellTank were studied in three perfusion runs. A very high cell density measured as 200 pF/cm (where 1 pF/cm is equivalent to 1 × 10(6)viable cells/mL) was achieved at a perfusion rate of 10 reactor volumes per day (RV/day) in the first run. In the second run, the effect of cell growth arrest by hypothermia at temperatures lowered gradually from 37 °C to 29 °C was studied during 13 days at cell densities above 100 pF/cm. Finally a production run was performed at high cell densities, where a temperature shift to 31 °C was applied at cell density 100 pF/cm during a production period of 14 days in minimized feeding conditions. The IgG concentrations were comparable in the matrix and in the harvest line in all the runs, indicating no retention of the product of interest. The cell specific productivity was comparable or higher than in Erlenmeyer flask batch culture. During the production run, the final harvested IgG production was 35 times higher in the CellTank compared to a repeated batch culture in the same vessel volume during the same time period. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells.

PubMed

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2013-01-01

Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50-60 nm on a time scale of 2.3 s. Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

PubMed Central

Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

2016-01-01

Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

Laser scanning in vivo confocal microscopy of the normal human corneoscleral limbus.

PubMed

Patel, Dipika V; Sherwin, Trevor; McGhee, Charles N J

2006-07-01

To elucidate the structure of the human corneoscleral limbus by in vivo laser scanning confocal microscopy and to correlate limbal epithelial dimensions and density with the central epithelium and in relation to age. Fifty adult subjects were recruited into one of two age groups: younger (age<45 years) and older (age>or=45 years). Fifty left eyes of these 50 healthy subjects were examined by laser scanning in vivo confocal microscopy, to assess the basal epithelium of the central cornea and inferior limbus. Mean epithelial cell diameter, area, and density were calculated for the central basal epithelium, limbus-corneal basal epithelium, and limbus-palisade epithelium. Data were analyzed in relation to the two age groups, group A, 30+/-6 years (n=25; mean+/-SD), and group B, 60+/-11 years (n=25; P<0.01). Mean epithelial density in the limbus-cornea and limbus-palisade regions decreased significantly with age: limbus-cornea group A=7253+/-1077 cells/mm2 group B=6614+/-987 cells/mm2, P=0.03; limbus palisade group A=5409+/-799 cells/mm2, group B=5055+/-722 cells/mm2, P=0.03). Central corneal epithelial density did not change with age: group A=6162+/-503 cells/mm2, group B=6362+/-614 cells/mm2, P=0.08. Mean epithelial density was greatest at the limbus-cornea (7010+/-1081 cells/mm2) and lowest at the limbus-palisades (5289+/-847 cells/mm2). The mean width of palisade ridges was 25.0+/-6.3 microm. This is the first study to image clearly the living human corneal limbus by laser scanning in vivo confocal microscopy and to demonstrate quantitative changes in the basal epithelium with age.

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

PubMed

Su, Yu-Wen; Wang, Wei

2018-04-12

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

Possible determination of the physical parameters of the first living cells based on the fundamental physical constants

NASA Astrophysics Data System (ADS)

Atanasov, Atanas Todorov

2016-12-01

Here is developed the hypothesis that the cell parameters of unicellular organisms (Prokaryotes and Eukaryotes) are determined by the gravitational constant (G, N.m2 /kg2), Planck constant (h, J.s) and growth rate of cells. By scaling analyses it was shown that the growth rate vgr(m/s) of unicellular bacteria and protozoa is relatively constant parameter, ranging in a narrow window of 10-12 - 10-10 m/s, in comparison to the diapason of cell mass, ranging 10 orders of magnitudes from 10-17 kg in bacteria to 10-7 kg in amoebas. By dimensional analyses it was shown that the combination between the growth rate of cells, gravitational constant and Planck constant gives equations with dimension of mass M(vgr)=(h.vgr/G)½ in kg, length L(v gr)=(hṡG/vgr3)1/2 in meter, time T(vgr)=(hṡG/vgr5)1/2 in seconds, and density ρ ((vgr)=vgr.3.5/hG2 in kg/m3 . For growth rate vgr in diapason of 1×10-11 m/s - 1×10-9.5 m/s the calculated numerical values for mass (3×10-18 -1×10-16 kg), length (5×10-8 -1×10-5 m), time (1×102 -1×106 s) and density (1×10-1 - 1×104 kg/m3) overlaps with diapason of experimentally measured values for cell mass (3×10-18 -1×10-15 kg), volume to surface ratio (1×10-7 -1×10-4 m), doubling time (1×103 -1×107 s), and density (1050 - 1300 kg/m3) in bacteria and protozoa. These equations show that appearance of the first living cells could be mutually connected to the physical constants.

Physiological considerations in applying laboratory-determined buoyant densities to predictions of bacterial and protozoan transport in groundwater: Results of in-situ and laboratory tests

USGS Publications Warehouse

Harvey, R.W.; Metge, D.W.; Kinner, N.; Mayberry, N.

1997-01-01

Buoyant densities were determined for groundwater bacteria and microflagellates (protozoa) from a sandy aquifer (Cape Cod, MA) using two methods: (1) density-gradient centrifugation (DGC) and (2) Stoke's law approximations using sedimentation rates observed during natural-gradient injection and recovery tests. The dwarf (average cell size, 0.3 ??m), unattached bacteria inhabiting a pristine zone just beneath the water table and a majority (~80%) of the morphologically diverse community of free- living bacteria inhabiting a 5-km-long plume of organically-contaminated groundwater had DGC-determined buoyant densities <1.019 g/cm3 before culturing. In the aquifer, sinking rates for the uncultured 2-??m size class of contaminant plume bacteria were comparable to that of the bromide tracer (1.9 x 10-3 M), also suggesting a low buoyant density. Culturing groundwater bacteria resulted in larger (0.8-1.3 ??m), less neutrally- buoyant (1.043-1.081 g/cm3) cells with potential sedimentation rates up to 64-fold higher than those predicted for the uncultured populations. Although sedimentation generally could be neglected in predicting subsurface transport for the community of free-living groundwater bacteria, it appeared to be important for the cultured isolates, at least until they readapt to aquifer conditions. Culturing-induced alterations in size of the contaminant-plume microflagellates (2-3 ??m) were ameliorated by using a lower nutrient, acidic (pH 5) porous growth medium. Buoyant densities of the cultured microflagellates were low, i.e., 1.024-1.034 g/cm3 (using the DGC assay) and 1.017-1.039 g/cm3 (estimated from in-situ sedimentation rates), suggesting good potential for subsurface transport under favorable conditions.

Modeling the chemical kinetics of atmospheric plasma for cell treatment in a liquid solution

NASA Astrophysics Data System (ADS)

Kim, H. Y.; Lee, H. W.; Kang, S. K.; Wk. Lee, H.; Kim, G. C.; Lee, J. K.

2012-07-01

Low temperature atmospheric pressure plasmas have been known to be effective for living cell inactivation in a liquid solution but it is not clear yet which species are key factors for the cell treatment. Using a global model, we elucidate the processes through which pH level in the solution is changed from neutral to acidic after plasma exposure and key components with pH and air variation. First, pH level in a liquid solution is changed by He+ and He(21S) radicals. Second, O3 density decreases as pH level in the solution decreases and air concentration decreases. It can be a method of removing O3 that causes chest pain and damages lung tissue when the density is very high. H2O2, HO2, and NO radicals are found to be key factors for cell inactivation in the solution with pH and air variation.

Diesel oil removal by immobilized Pseudoxanthomonas sp. RN402.

PubMed

Nopcharoenkul, Wannarak; Netsakulnee, Parichat; Pinyakong, Onruthai

2013-06-01

Pseudoxanthomonas sp. RN402 was capable of degrading diesel, crude oil, n-tetradecane and n-hexadecane. The RN402 cells were immobilized on the surface of high-density polyethylene plastic pellets at a maximum cell density of 10(8) most probable number (MPN) g(-1) of plastic pellets. The immobilized cells not only showed a higher efficacy of diesel oil removal than free cells but could also degrade higher concentrations of diesel oil. The rate of diesel oil removal by immobilized RN402 cells in liquid culture was 1,050 mg l(-1) day(-1). Moreover, the immobilized cells could maintain high efficacy and viability throughout 70 cycles of bioremedial treatment of diesel-contaminated water. The stability of diesel oil degradation in the immobilized cells resulted from the ability of living RN402 cells to attach to material surfaces by biofilm formation, as was shown by CLSM imaging. These characteristics of the immobilized RN402 cells, including high degradative efficacy, stability and flotation, make them suitable for the purpose of continuous wastewater bioremediation.

Common themes and cell type specific variations of higher order chromatin arrangements in the mouse

PubMed Central

Mayer, Robert; Brero, Alessandro; von Hase, Johann; Schroeder, Timm; Cremer, Thomas; Dietzel, Steffen

2005-01-01

Background Similarities as well as differences in higher order chromatin arrangements of human cell types were previously reported. For an evolutionary comparison, we now studied the arrangements of chromosome territories and centromere regions in six mouse cell types (lymphocytes, embryonic stem cells, macrophages, fibroblasts, myoblasts and myotubes) with fluorescence in situ hybridization and confocal laser scanning microscopy. Both species evolved pronounced differences in karyotypes after their last common ancestors lived about 87 million years ago and thus seem particularly suited to elucidate common and cell type specific themes of higher order chromatin arrangements in mammals. Results All mouse cell types showed non-random correlations of radial chromosome territory positions with gene density as well as with chromosome size. The distribution of chromosome territories and pericentromeric heterochromatin changed during differentiation, leading to distinct cell type specific distribution patterns. We exclude a strict dependence of these differences on nuclear shape. Positional differences in mouse cell nuclei were less pronounced compared to human cell nuclei in agreement with smaller differences in chromosome size and gene density. Notably, the position of chromosome territories relative to each other was very variable. Conclusion Chromosome territory arrangements according to chromosome size and gene density provide common, evolutionary conserved themes in both, human and mouse cell types. Our findings are incompatible with a previously reported model of parental genome separation. PMID:16336643

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

DOE PAGES

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.; ...

2016-11-16

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

One-Cell Doubling Evaluation by Living Arrays of Yeast, ODELAY!

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

Herricks, Thurston; Dilworth, David J.; Mast, Fred D.

Cell growth is a complex phenotype widely used in systems biology to gauge the impact of genetic and environmental perturbations. Due to the magnitude of genome-wide studies, resolution is often sacrificed in favor of throughput, creating a demand for scalable, time-resolved, quantitative methods of growth assessment. We present ODELAY (One-cell Doubling Evaluation by Living Arrays of Yeast), an automated and scalable growth analysis platform. High measurement density and single-cell resolution provide a powerful tool for large-scale multiparameter growth analysis based on the modeling of microcolony expansion on solid media. Pioneered in yeast but applicable to other colony forming organisms, ODELAYmore » extracts the three key growth parameters (lag time, doubling time, and carrying capacity) that define microcolony expansion from single cells, simultaneously permitting the assessment of population heterogeneity. The utility of ODELAY is illustrated using yeast mutants, revealing a spectrum of phenotypes arising from single and combinatorial growth parameter perturbations.« less

Development of a High-Throughput Ultrasound Technique for the Analysis of Tissue Engineering Constructs

PubMed Central

Stukel, Jessica; Goss, Monika; Zhou, Haoyan; Zhou, Wenda; Willits, Rebecca; Exner, Agata A.

2015-01-01

Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90± 2.58*10−5 a.u. compared to non-fiber forming intensity at 2.74± 0.36*10−5 a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5 ± 0.8*10−5 a.u. in 1 day live cells compared to 2.26 ± 0.39*10−5 a.u. in fixed cells at a concentration of 1*106 cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings. PMID:26577255

Development of a High-Throughput Ultrasound Technique for the Analysis of Tissue Engineering Constructs.

PubMed

Stukel, Jessica M; Goss, Monika; Zhou, Haoyan; Zhou, Wenda; Willits, Rebecca Kuntz; Exner, Agata A

2016-03-01

Development of hydrogel-based tissue engineering constructs is growing at a rapid rate, yet translation to patient use has been sluggish. Years of costly preclinical tests are required to predict clinical performance and safety of these devices. The tests are invasive, destructive to the samples and, in many cases, are not representative of the ultimate in vivo scenario. Biomedical imaging has the potential to facilitate biomaterial development by enabling longitudinal noninvasive device characterization directly in situ. Among the various available imaging modalities, ultrasound stands out as an excellent candidate due to low cost, wide availability, and a favorable safety profile. The overall goal of this work was to demonstrate the utility of clinical ultrasound in longitudinal characterization of 3D hydrogel matrices supporting cell growth. Specifically, we developed a quantitative technique using clinical B-mode ultrasound to differentiate collagen content and fibroblast density within poly(ethylene glycol) (PEG) hydrogels and validated it in an in vitro phantom environment. By manipulating the hydrogel gelation, differences in ultrasound signal intensity were found between gels with collagen fibers and those with non-fiber forming collagen, indicating that the technique was sensitive to the configuration of the protein. At a collagen density of 2.5 mg/mL collagen, fiber forming collagen had a significantly increased signal intensity of 14.90 ± 2.58 × 10(-5) a.u. compared to non-fiber forming intensity at 2.74 ± 0.36 × 10(-5) a.u. Additionally, differences in intensity were found between living and fixed fibroblasts, with an increased signal intensity detected in living cells (5.00 ± 0.80 × 10(-5) a.u. in 1 day live cells compared to 2.26 ± 0.39 × 10(-5) a.u.in fixed cells at a concentration of 1 × 10(6) cells/mL in gels containing collagen). Overall, there was a linear correlation >0.90 for ultrasound intensity with increasing cell density. Results demonstrate the feasibility of using clinical ultrasound for characterization of PEG-based hydrogels in a tissue-mimicking phantom. The approach is clinically-relevant and could, with further validation, be utilized to nondestructively monitor in vivo performance of implanted tissue engineering scaffolds over time in preclinical and clinical settings.

Poly-adenine-mediated fluorescent spherical nucleic acid probes for live-cell imaging of endogenous tumor-related mRNA.

PubMed

Zhu, Dan; Zhao, Dongxia; Huang, Jiaxuan; Zhu, Yu; Chao, Jie; Su, Shao; Li, Jiang; Wang, Lihua; Shi, Jiye; Zuo, Xiaolei; Weng, Lixing; Li, Qian; Wang, Lianhui

2018-05-16

Identification of tumor-related mRNA in living cells hold great promise for early cancer diagnosis and pathological research. Herein, we present poly-adenine (polyA)-mediated fluorescent spherical nucleic acid (FSNA) probes for intracellular mRNA detection with regulable sensitivities by programmably adjusting the loading density of DNA on gold nano-interface. Gold nanoparticles (AuNPs) functionalized with polyA-tailed recognition sequences were hybridized to fluorescent "reporter" strands to fabricate fluorescence-quenched FSNA probes. While exposed to target gene, the "reporter" strands were released from FSNA through strand displacement and fluorescence was recovered. With polyA20 tail as the attaching block, the detection limit of FSNA probes was calculated to be 0.31 nM, which is ~55 fold lower than that of thiolated probes without surface density regulation. Quantitative intracellular mRNA detection and imaging could be achieved with polyA-mediated FSNA probes within 2 hours, indicating their application potential in rapid and sensitive intracellular target imaging. Copyright © 2018. Published by Elsevier Inc.

A nu-space for image correlation spectroscopy: characterization and application to measure protein transport in live cells

NASA Astrophysics Data System (ADS)

Potvin-Trottier, Laurent; Chen, Lingfeng; Horwitz, Alan Rick; Wiseman, Paul W.

2013-08-01

We introduce a new generalized theoretical framework for image correlation spectroscopy (ICS). Using this framework, we extend the ICS method in time-frequency (ν, nu) space to map molecular flow of fluorescently tagged proteins in individual living cells. Even in the presence of a dominant immobile population of fluorescent molecules, nu-space ICS (nICS) provides an unbiased velocity measurement, as well as the diffusion coefficient of the flow, without requiring filtering. We also develop and characterize a tunable frequency-filter for spatio-temporal ICS (STICS) that allows quantification of the density, the diffusion coefficient and the velocity of biased diffusion. We show that the techniques are accurate over a wide range of parameter space in computer simulation. We then characterize the retrograde flow of adhesion proteins (α6- and αLβ2-GFP integrins and mCherry-paxillin) in CHO.B2 cells plated on laminin and intercellular adhesion molecule 1 (ICAM-1) ligands respectively. STICS with a tunable frequency filter, in conjunction with nICS, measures two new transport parameters, the density and transport bias coefficient (a measure of the diffusive character of a flow/biased diffusion), showing that molecular flow in this cell system has a significant diffusive component. Our results suggest that the integrin-ligand interaction, along with the internal myosin-motor generated force, varies for different integrin-ligand pairs, consistent with previous results.

Improved Separators For Rechargeable Lithium Cells

NASA Technical Reports Server (NTRS)

Shen, David; Surampudi, Subbarao; Huang, Chen-Kuo; Halpert, Gerald

1994-01-01

Improved pairs of separators proposed for use in rechargeable lithium cells operating at ambient temperature. Block growth of lithium dendrites and help prevent short circuits. Each cell contains one separator made of microporous polypropylene placed next to anode, and one separator made of microporous polytetrafluoroethylene (PTFE) next to cathode. Separators increase cycle lives of secondary lithium cells. Cells to which concept applicable those of Li/TiS(2), Li/NbSe(3), Li/CoO(2), Li/MoS(2), Li/VO(x), and Li/MnO(2) chemical systems. Advantageous in spacecraft, military, communications, automotive, and other applications in which high energy density and rechargeability needed.

Passive microrheology of normal and cancer cells after ML7 treatment by atomic force microscopy

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

Lyapunova, Elena, E-mail: lyapunova@icmm.ru; Ural Federal University, Kuibyishev Str. 48, Ekaterinburg, 620000; Nikituk, Alexander, E-mail: nas@icmm.ru

Mechanical properties of living cancer and normal thyroidal cells were investigated by atomic force microscopy (AFM). Cell mechanics was compared before and after treatment with ML7, which is known to reduce myosin activity and induce softening of cell structures. We recorded force curves with extended dwell time of 6 seconds in contact at maximum forces from 500 pN to 1 nN. Data were analyzed within different frameworks: Hertz fit was applied in order to evaluate differences in Young’s moduli among cell types and conditions, while the fluctuations of the cantilever in contact with cells were analyzed with both conventional algorithmsmore » (probability density function and power spectral density) and multifractal detrended fluctuation analysis (MF-DFA). We found that cancer cells were softer than normal cells and ML7 had a substantial softening effect on normal cells, but only a marginal one on cancer cells. Moreover, we observed that all recorded signals for normal and cancer cells were monofractal with small differences between their scaling parameters. Finally, the applicability of wavelet-based methods of data analysis for the discrimination of different cell types is discussed.« less

Interaction of fluorescently labeled pyrrole-imidazole polyamide probes with fixed and living murine and human cells.

PubMed

Nozeret, Karine; Loll, François; Cardoso, Gildas Mouta; Escudé, Christophe; Boutorine, Alexandre S

2018-06-01

Pericentromeric heterochromatin plays important roles in controlling gene expression and cellular differentiation. Fluorescent pyrrole-imidazole polyamides targeting murine pericentromeric DNA (major satellites) can be used for the visualization of pericentromeric heterochromatin foci in live mouse cells. New derivatives targeting human repeated DNA sequences (α-satellites) were synthesized and their interaction with target DNA was characterized. The possibility to use major satellite and α -satellite binding polyamides as tools for staining pericentromeric heterochromatin was further investigated in fixed and living mouse and human cells. The staining that was previously observed using the mouse model was further characterized and optimized, but remained limited regarding the fluorophores that can be used. The promising results regarding the staining in the mouse model could not be extended to the human model. Experiments performed in human cells showed chromosomal DNA staining without selectivity. Factors limiting the use of fluorescent polyamides, in particular probe aggregation in the cytoplasm, were investigated. Results are discussed with regards to structure and affinity of probes, density of target sites and chromatin accessibility in both models. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

In vitro cytotoxicity of galvanically coupled magnesium-titanium particles on human osteosarcoma SAOS2 cells: A potential cancer therapy.

PubMed

Kim, Jua; Gilbert, Jeremy L

2018-04-10

Osteosarcoma is a malignant bone cancer that occurs mostly in children and young adults. This study investigated the cytotoxicity of Mg and Mg-Ti microparticles to human osteosarcoma cells. Osteosarcoma cells were killed in a dosage-dependent manner when cells, with a cell seeding density of 30,000 cells/cm 2 , were cultured with 0 to 2500 µg/mL of Mg or Mg-Ti in cell culture media for 24-72 h. Mg-Ti killed cells more effectively, where 1250 µg/mL of Mg-Ti killed cells completely by 24 h, while 2500 µg/mL of Mg killed nearly all cells, but not all. Killing due to particle corrosion occurred mostly during the first 24 h, and so the percent cell viability between 24 and 72 h showed not much variability. However, the measurement of live and dead cell numbers, over the timeframe of 24-72 h, showed more insight, such as cell recovery. If particle concentrations were low, the number of live cells increased after 24 h, indicating cell proliferation. If particle concentrations were high, the number of live cells either remained steady or decreased, indicating cell quiescence or continued killing, respectively. Increase in the number of dead cells also indicated killing, while plateau meant discontinued killing. In addition, repeated killing of recovered cells exhibited the same dose-dependent killing profile as the initial experiment, implying little development of cell resistance to treatment. These results, together, show that osteosarcoma cells are susceptible to killing by way of exposure to corroding particles, showing highly effective killing using the galvanic couple of Mg-Ti. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc.

Increased illumination uniformity and reduced photodamage offered by the Lissajous scanning in fiber-optic two-photon endomicroscopy

NASA Astrophysics Data System (ADS)

Liang, Wenxuan; Murari, Kartikeya; Zhang, Yuying; Chen, Yongping; Li, Ming-Jun; Li, Xingde

2012-02-01

We compare the illumination uniformity and the associated effects of the spiral and Lissajous scanning patterns that are commonly used in an endomicroscope. Theoretical analyses and numerical simulations were first performed to quantitatively investigate the area illumination density in the spiral scanning pattern. The results revealed the potential problem of manifest photodamage due to the very high illumination density in the center of the spiral scan. Similar analyses of the Lissajous scanning pattern, which can be conveniently implemented on the same endomicroscope with no hardware modifications, showed a more uniform illumination density with about an 80-fold reduction in the peak illumination density. To underscore the benefit offered by the improved illumination uniformity, we conducted in vitro two-photon fluorescence imaging of cultured cells stained with a LIVE/DEAD viability assay using our home-built, fiber-optic, two-channel endomicroscopy system. Both the spiral and the Lissajous scans were implemented. Our experimental results showed that cells near the spiral scan center experienced obvious photodamage, whereas cells remained alive over the entire region under the Lissajous beam scanning, confirming the predicted advantage offered by the Lissajous scan over this spiral scan in an endomicroscopy setting.

Nickel-hydrogen bipolar battery system

NASA Technical Reports Server (NTRS)

Thaller, L. H.

1982-01-01

Rechargeable nickel-hydrogen systems are described that more closely resemble a fuel cell system than a traditional nickel-cadmium battery pack. This was stimulated by the currently emerging requirements related to large manned and unmanned low Earth orbit applications. The resultant nickel-hydrogen battery system should have a number of features that would lead to improved reliability, reduced costs as well as superior energy density and cycle lives as compared to battery systems constructed from the current state-of-the-art nickel-hydrogen individual pressure vessel cells.

First experimental results of a cryogenic stopping cell with short-lived, heavy uranium fragments produced at 1000 MeV/u

NASA Astrophysics Data System (ADS)

Purushothaman, S.; Reiter, M. P.; Haettner, E.; Dendooven, P.; Dickel, T.; Geissel, H.; Ebert, J.; Jesch, C.; Plass, W. R.; Ranjan, M.; Weick, H.; Amjad, F.; Ayet, S.; Diwisch, M.; Estrade, A.; Farinon, F.; Greiner, F.; Kalantar-Nayestanaki, N.; Knöbel, R.; Kurcewicz, J.; Lang, J.; Moore, I. D.; Mukha, I.; Nociforo, C.; Petrick, M.; Pfützner, M.; Pietri, S.; Prochazka, A.; Rink, A.-K.; Rinta-Antila, S.; Scheidenberger, C.; Takechi, M.; Tanaka, Y. K.; Winfield, J. S.; Yavor, M. I.

2013-11-01

A cryogenic stopping cell (CSC) has been commissioned with 238U projectile fragments produced at 1000 MeV/u. The spatial isotopic separation in flight was performed with the FRS applying a monoenergetic degrader. For the first time, a stopping cell was operated with exotic nuclei at cryogenic temperatures (70 to 100 K). A helium stopping gas density of up to 0.05\\ \\text{mg/cm}^3 was used, about two times higher than reached before for a stopping cell with RF ion repelling structures. An overall efficiency of up to 15%, a combined ion survival and extraction efficiency of about 50%, and extraction times of 24 ms were achieved for heavy α-decaying uranium fragments. Mass spectrometry with a multiple-reflection time-of-flight mass spectrometer has demonstrated the excellent cleanliness of the CSC. This setup has opened a new field for the spectroscopy of short-lived nuclei.

Nectar-living yeasts of a tropical host plant community: diversity and effects on community-wide floral nectar traits

PubMed Central

2017-01-01

We characterize the diversity of nectar-living yeasts of a tropical host plant community at different hierarchical sampling levels, measure the associations between yeasts and nectariferous plants, and measure the effect of yeasts on nectar traits. Using a series of hierarchically nested sampling units, we extracted nectar from an assemblage of host plants that were representative of the diversity of life forms, flower shapes, and pollinator types in the tropical area of Yucatan, Mexico. Yeasts were isolated from single nectar samples; their DNA was identified, the yeast cell density was estimated, and the sugar composition and concentration of nectar were quantified using HPLC. In contrast to previous studies from temperate regions, the diversity of nectar-living yeasts in the plant community was characterized by a relatively high number of equally common species with low dominance. Analyses predict highly diverse nectar yeast communities in a relatively narrow range of tropical vegetation, suggesting that the diversity of yeasts will increase as the number of sampling units increases at the level of the species, genera, and botanical families of the hosts. Significant associations between specific yeast species and host plants were also detected; the interaction between yeasts and host plants impacted the effect of yeast cell density on nectar sugars. This study provides an overall picture of the diversity of nectar-living yeasts in tropical host plants and suggests that the key factor that affects the community-wide patterns of nectar traits is not nectar chemistry, but rather the type of yeasts interacting with host plants. PMID:28717591

Chondrogenesis of Human Bone Marrow Mesenchymal Stem Cells in 3-Dimensional, Photocrosslinked Hydrogel Constructs: Effect of Cell Seeding Density and Material Stiffness

PubMed Central

Sun, Aaron X.; Lin, Hang; Fritch, Madalyn R.; Shen, He; Alexander, Pete G.; DeHart, Michael; Tuan, Rocky S.

2018-01-01

Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-L-lactic acid/polyethylene glycol/poly-L-lactic acid] (PLLA-PEG 1000) and [poly-D,L-lactic acid/polyethylene glycol/poly-D,L-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (~1,500 to 1,800 kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50 × 106/mL) and hydrogel stiffnesses (~150kPa to ~1,500 kPa Young’s moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20 × 106 cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects. PMID:28611002

The Effects of Microalgae as Live Food for Brachionus plicatilis (Rotifer) in Intensive Culture System

PubMed Central

Rahman, Abdull Razak Abd; Cob, Zaidi Che; Jamari, Zainoddin; Mohamed, Abdul Majid; Toda, Tatsuki; Ross, Othman Haji

2018-01-01

Brachionus plicatilis is used to feed fish and crustacean larvae in the aquaculture industry. It is well established that the type of microalgae may influence rotifer production. This experiment was conducted to determine the effect of five different locally available microalgae species at Fisheries Research Institute (FRI), Kampung Pulau Sayak, Kedah, Malaysia on the instantaneous growth rate (μ) of rotifer. Nannochloris sp., Tetraselmis sp., Isochrysis sp., Chlorella sp., and Nannochloropsis sp. were used as feed at different algae densities (0.1, 0.3, 0.7 and 1.5 × 106 cells/ml) and culture volumes (20, 70 and 210 ml). At algae densities ranging from 0.3 to 1.5 × 106 cells/ml, an average μ value of more than 0.90 per day were recorded for all algae species. However, at density of 0.1 × 106 cells/ml, only Tetraselmis sp. resulted in the significantly highest μ value compared with others (p < 0.05). In terms of volume, smaller culture volume of Tetraselmis sp. (20 ml) showed significantly higher μ compared with higher volume (70 and 210 ml cultures). PMID:29644020

Three Gorges Dam: Impact of Water Level Changes on the Density of Schistosome-Transmitting Snail Oncomelania hupensis in Dongting Lake Area, China

PubMed Central

Wu, Jin-Yi; Zhou, Yi-Biao; Chen, Yue; Liang, Song; Li, Lin-Han; Zheng, Sheng-Bang; Zhu, Shao-ping; Ren, Guang-Hui; Song, Xiu-Xia; Jiang, Qing-Wu

2015-01-01

Background Schistosomiasis remains an important public health issue in China and worldwide. Oncomelania hupensis is the unique intermediate host of schistosoma japonicum, and its change influences the distribution of S. japonica. The Three Gorges Dam (TGD) has substantially changed the ecology and environment in the Dongting Lake region. This study investigated the impact of water level and elevation on the survival and habitat of the snails. Methods Data were collected for 16 bottomlands around 4 hydrological stations, which included water, density of living snails (form the Anxiang Station for Schistosomiasis Control) and elevation (from Google Earth). Based on the elevation, sixteen bottomlands were divided into 3 groups. ARIMA models were built to predict the density of living snails in different elevation areas. Results Before closure of TGD, 7 out of 9 years had a water level beyond the warning level at least once at Anxiang hydrological station, compared with only 3 out of 10 years after closure of TGD. There were two severe droughts that happened in 2006 and 2011, with much fewer number of flooding per year compared with other study years. Overall, there was a correlation between water level changing and density of living snails variation in all the elevations areas. The density of living snails in all elevations areas was decreasing after the TGD was built. The relationship between number of flooding per year and the density of living snails was more pronounced in the medium and high elevation areas; the density of living snails kept decreasing from 2003 to 2014. In low elevation area however, the density of living snails decreased after 2003 first and turned to increase after 2011. Our ARIMA prediction models indicated that the snails would not disappear in the Dongting Lake region in the next 7 years. In the low elevation area, the density of living snails would increase slightly, and then stabilize after the year 2017. In the medium elevation region, the change of the density of living snails would be more obvious and would increase till the year 2020. In the high elevation area, the density of living snails would remain stable after the year 2015. Conclusion The TGD influenced water levels and reduced the risk of flooding and the density of living snails in the study region. Based on our prediction models, the density of living snails in all elevations tends to be stabilized. Control of S. japonica would continue to be an important task in the study area in the coming decade. PMID:26114956

Nickel-hydrogen bipolar battery systems

NASA Technical Reports Server (NTRS)

Thaller, L. H.

1982-01-01

Nickel-hydrogen cells are currently being manufactured on a semi-experimental basis. Rechargeable nickel-hydrogen systems are described that more closely resemble a fuel cell system than a traditional nickel-cadmium battery pack. This has been stimulated by the currently emerging requirements related to large manned and unmanned low earth orbit applications. The resultant nickel-hydrogen battery system should have a number of features that would lead to improved reliability, reduced costs as well as superior energy density and cycle lives as compared to battery systems constructed from the current state-of-the-art nickel-hydrogen individual pressure vessel cells.

Structure-function relationships in the stem cell's mechanical world B: emergent anisotropy of the cytoskeleton correlates to volume and shape changing stress exposure.

PubMed

Chang, Hana; Knothe Tate, Melissa L

2011-12-01

In the preceding study (Part A), we showed that prescribed seeding conditions as well as seeding density can be used to subject multipotent stem cells (MSCs) to volume changing stresses and that changes in volume of the cell are associated with changes in shape, but not volume, of the cell nucleus. In the current study, we aim to control the mechanical milieu of live cells using these prescribed seeding conditions concomitant to delivery of shape changing stresses via fluid flow, while observing adaptation of the cytoskeleton, a major cellular transducer that modulates cell shape, stiffness and remodeling. We hypothesize that the spatiotemporal organization of tubulin and actin elements of the cytoskeleton changes in response to volume and shape changing stresses emulating those during development, prior to the first beating of the heart or twitching of muscle. Our approach was to quantify the change over baseline in spatiotemporal distribution of actin and tubulin in live C3H/10T1/2 model stem cells subjected to volume changing stresses induced by seeding at density as well as low magnitude, short duration, shape changing (shear) stresses induced by fluid flow (0.5 or 1.0 dyne/cm2 for 30/60/90 minutes). Upon exposure to fluid flow, both tubulin thickness (height) and concentration (fluorescence intensity) change significantly over baseline, as a function of proximity to neighboring cells (density) and the substrate (apical-basal height). Given our recently published studies showing amplification of stress gradients (flow velocity) with increasing distance to nearest neighbors and the substrate, i.e. with decreasing density and toward the apical side of the cell, tubulin adaptation appears to depend significantly on the magnitude of the stress to which the cell is exposed locally. In contrast, adaptation of actin to the changing mechanical milieu is more global, exhibiting less significant differences attributable to nearest neighbors or boundaries than differences attributable to magnitude of the stress to which the cell is exposed globally (0.5 versus 1.0 dyne/cm2). Furthermore, changes in the actin cytoskeletal distribution correlate positively with one pre-mesenchymal condensation marker (Msx2) and negatively with early markers of chondrogenesis (ColIIaI alone, indicative of pre-hypertrophic chondrogenesis) and osteogenesis (Runx2). Changes in the tubulin cytoskeletal distribution correlate positively with a marker of pericondensation (Sox9 alone), negatively with chondrogenesis (ColIIaI) and positively with adipogenesis (Ppar-gamma 2). Taken as a whole, exposure of MSCs to volume and shape changing stresses results in emergent anisotropy of cytoskeletal architecture (structure), which relate to emergent cell fate (function).

Consumption, supply and transport: self-organization without direct communication

NASA Technical Reports Server (NTRS)

Kessler, J. O.

1996-01-01

Swimming bacteria of the species Bacillus subtilis require and consume oxygen. In static liquid cultures the cells' swimming behaviour leads them to accumulate up oxygen concentration gradients generated by consumption and supply. Since the density of bacterial cells exceeds that of the fluid in which they live, fluid regions where cells have accumulated are denser than depleted regions. These density variations cause convection. The fluid motion is dynamically maintained by the swimming of the cells toward regions of attraction: the air-fluid interface and the fluctuating advecting attractors, gradients of oxygen concentration that are embedded in the convecting fluid. Because of the fluid dynamical conservation laws, these complex physical and biological factors generate patterns ordered over distances > 10000 bacterial cell diameters. The convection enhances long-range transport and mixing of oxygen, cells and extracellular products by orders of magnitude. Thus, through the interplay of physical and biological factors, a population of undifferentiated selfish cells creates functional dynamic patterns. Populations of bacteria that have organised themselves into regularly patterned regions of vigorous convection and varying cell concentration interact with their environment as if they were one purposeful, coherent multicellular individual. The mathematical and experimental ingredients of these remarkable phenomena are presented here.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy

NASA Astrophysics Data System (ADS)

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-01

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Tracking molecular dynamics without tracking: image correlation of photo-activation microscopy.

PubMed

Pandžić, Elvis; Rossy, Jérémie; Gaus, Katharina

2015-03-09

Measuring protein dynamics in the plasma membrane can provide insights into the mechanisms of receptor signaling and other cellular functions. To quantify protein dynamics on the single molecule level over the entire cell surface, sophisticated approaches such as single particle tracking (SPT), photo-activation localization microscopy (PALM) and fluctuation-based analysis have been developed. However, analyzing molecular dynamics of fluorescent particles with intermittent excitation and low signal-to-noise ratio present at high densities has remained a challenge. We overcame this problem by applying spatio-temporal image correlation spectroscopy (STICS) analysis to photo-activated (PA) microscopy time series. In order to determine under which imaging conditions this approach is valid, we simulated PA images of diffusing particles in a homogeneous environment and varied photo-activation, reversible blinking and irreversible photo-bleaching rates. Further, we simulated data with high particle densities that populated mobile objects (such as adhesions and vesicles) that often interfere with STICS and fluctuation-based analysis. We demonstrated in experimental measurements that the diffusion coefficient of the epidermal growth factor receptor (EGFR) fused to PAGFP in live COS-7 cells can be determined in the plasma membrane and revealed differences in the time-dependent diffusion maps between wild-type and mutant Lck in activated T cells. In summary, we have developed a new analysis approach for live cell photo-activation microscopy data based on image correlation spectroscopy to quantify the spatio-temporal dynamics of single proteins.

Lon Protease of Azorhizobium caulinodans ORS571 Is Required for Suppression of reb Gene Expression

PubMed Central

Nakajima, Azusa; Tsukada, Shuhei; Siarot, Lowela; Ogawa, Tetsuhiro; Oyaizu, Hiroshi

2012-01-01

Bacterial Lon proteases play important roles in a variety of biological processes in addition to housekeeping functions. In this study, we focused on the Lon protease of Azorhizobium caulinodans, which can fix nitrogen both during free-living growth and in stem nodules of the legume Sesbania rostrata. The nitrogen fixation activity of an A. caulinodans lon mutant in the free-living state was not significantly different from that of the wild-type strain. However, the stem nodules formed by the lon mutant showed little or no nitrogen fixation activity. By microscopic analyses, two kinds of host cells were observed in the stem nodules formed by the lon mutant. One type has shrunken host cells containing a high density of bacteria, and the other type has oval or elongated host cells containing a low density or no bacteria. This phenotype is similar to a praR mutant highly expressing the reb genes. Quantitative reverse transcription-PCR analyses revealed that reb genes were also highly expressed in the lon mutant. Furthermore, a lon reb double mutant formed stem nodules showing higher nitrogen fixation activity than the lon mutant, and shrunken host cells were not observed in these stem nodules. These results suggest that Lon protease is required to suppress the expression of the reb genes and that high expression of reb genes in part causes aberrance in the A. caulinodans-S. rostrata symbiosis. In addition to the suppression of reb genes, it was found that Lon protease was involved in the regulation of exopolysaccharide production and autoagglutination of bacterial cells. PMID:22752172

Stem cell motility enables a density-dependent rate of fate commitment during scaled resizing of adult organs

NASA Astrophysics Data System (ADS)

Du, Xinxin; O'Brien, Lucy; Riedel-Kruse, Ingmar

Many adult organs grow or shrink to accommodate fluctuating levels of physiological demand. Specifically, the intestine of the fruit fly (the midgut) expands four-fold in the number of mature cells and, proportionally, the number of stem cells when the fly eats. However, the cellular behaviors that give rise to this stem scaling are not well-understood. Here we present a biophysical model of the adult fly midgut. A set of differential equations can recapitulate the physiological kinetics of cells during midgut growth and shrinkage as long as the rate of stem cell fate commitment depends on the stem cell number density in the tissue. To elucidate the source of this dependence, we model the tissue in a 2D simulation with soft spheres, where stem cells choose fate commitment through Delta-Notch pathway interactions with other stem cells, a known process in fly midguts. We find that as long as stem cells exhibit a large enough amplitude of random motion through the tissue (`stem cell motility'), and explore a large enough `territory' in their lifetime, stem cell scaling can occur. These model observations are confirmed through in vivo live-imaging, where we indeed see that stem cells are motile in the fly midgut.

Electrical characterization of single cells using polysilicon wire ion sensor in an isolation window.

PubMed

Wu, You-Lin; Hsu, Po-Yen; Hsu, Chung-Ping; Wang, Chih-Cheng; Lee, Li-Wen; Lin, Jing-Jenn

2011-10-01

A polysilicon wire (PSW) sensor can detect the H(+) ion density (pH value) of the medium coated on its surface, and different cells produce different extracellular acidification and hence different H(+) ion densities. Based on this, we used a PSW sensor in combination with a mold-cast polydimethylsiloxane (PDMS) isolation window to detect the adhesion, apoptosis and extracellular acidification of single normal cells and single cancer cells. Single living human normal cells WI38, MRC5, and BEAS-2B as well as non-small-cell lung cancer (NSCLC) cells A549, H1299, and CH27 were cultivated separately inside the isolation window. The current flowing through the PSW channel was measured. From the PSW channel current change as a function of time, we determined the cell adhesion time by observing the time required for the current change to saturate, since a stable extracellular ion density was established after the cells were completely adhered to the PSW surface. The apoptosis of cells can also be determined when the channel current change drops to zero. We found that all the NSCLC cells had a higher channel current change and hence a lower pH value than the normal cells anytime after they were seeded. The corresponding average pH values were 5.86 for A549, 6.00 for H1299, 6.20 for CH27, 6.90 for BEAS-2B, 6.96for MRC5, and 7.02 for WI38, respectively, after the cells were completely adhered to the PSW surface. Our results show that NSCLC cells have a stronger cell-substrate adhesion and a higher extracellular acidification rate than normal cells.

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

PubMed Central

Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

2016-01-01

Local surface charge density of lipid membranes influences membrane–protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values. PMID:27561322

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

NASA Astrophysics Data System (ADS)

Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

2016-08-01

Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

PubMed

Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

2016-08-26

Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values.

Dissecting Regional Variations in Stress Fiber Mechanics in Living Cells with Laser Nanosurgery

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

Tanner, Kandice; Boudreau, Aaron; Bissell, Mina J

The ability of a cell to distribute contractile stresses across the extracellular matrix in a spatially heterogeneous fashion underlies many cellular behaviors, including motility and tissue assembly. Here we investigate the biophysical basis of this phenomenon by using femtosecond laser nanosurgery to measure the viscoelastic recoil and cell-shape contributions of contractile stress fibers (SFs) located in specific compartments of living cells. Upon photodisruption and recoil, myosin light chain kinase-dependent SFs located along the cell periphery display much lower effective elasticities and higher plateau retraction distances than Rho-associated kinase-dependent SFs located in the cell center, with severing of peripheral fibers uniquelymore » triggering a dramatic contraction of the entire cell within minutes of fiber irradiation. Image correlation spectroscopy reveals that when one population of SFs is pharmacologically dissipated, actin density flows toward the other population. Furthermore, dissipation of peripheral fibers reduces the elasticity and increases the plateau retraction distance of central fibers, and severing central fibers under these conditions triggers cellular contraction. Together, these findings show that SFs regulated by different myosin activators exhibit different mechanical properties and cell shape contributions. They also suggest that some fibers can absorb components and assume mechanical roles of other fibers to stabilize cell shape.« less

Recombination in Perovskite Solar Cells: Significance of Grain Boundaries, Interface Traps, and Defect Ions.

PubMed

Sherkar, Tejas S; Momblona, Cristina; Gil-Escrig, Lidón; Ávila, Jorge; Sessolo, Michele; Bolink, Henk J; Koster, L Jan Anton

2017-05-12

Trap-assisted recombination, despite being lower as compared with traditional inorganic solar cells, is still the dominant recombination mechanism in perovskite solar cells (PSCs) and limits their efficiency. We investigate the attributes of the primary trap-assisted recombination channels (grain boundaries and interfaces) and their correlation to defect ions in PSCs. We achieve this by using a validated device model to fit the simulations to the experimental data of efficient vacuum-deposited p-i-n and n-i-p CH 3 NH 3 PbI 3 solar cells, including the light intensity dependence of the open-circuit voltage and fill factor. We find that, despite the presence of traps at interfaces and grain boundaries (GBs), their neutral (when filled with photogenerated charges) disposition along with the long-lived nature of holes leads to the high performance of PSCs. The sign of the traps (when filled) is of little importance in efficient solar cells with compact morphologies (fused GBs, low trap density). On the other hand, solar cells with noncompact morphologies (open GBs, high trap density) are sensitive to the sign of the traps and hence to the cell preparation methods. Even in the presence of traps at GBs, trap-assisted recombination at interfaces (between the transport layers and the perovskite) is the dominant loss mechanism. We find a direct correlation between the density of traps, the density of mobile ionic defects, and the degree of hysteresis observed in the current-voltage ( J - V ) characteristics. The presence of defect states or mobile ions not only limits the device performance but also plays a role in the J - V hysteresis.

Ionic imbalance, in addition to molecular crowding, abates cytoskeletal dynamics and vesicle motility during hypertonic stress

PubMed Central

Nunes, Paula; Roth, Isabelle; Meda, Paolo; Féraille, Eric; Brown, Dennis; Hasler, Udo

2015-01-01

Cell volume homeostasis is vital for the maintenance of optimal protein density and cellular function. Numerous mammalian cell types are routinely exposed to acute hypertonic challenge and shrink. Molecular crowding modifies biochemical reaction rates and decreases macromolecule diffusion. Cell volume is restored rapidly by ion influx but at the expense of elevated intracellular sodium and chloride levels that persist long after challenge. Although recent studies have highlighted the role of molecular crowding on the effects of hypertonicity, the effects of ionic imbalance on cellular trafficking dynamics in living cells are largely unexplored. By tracking distinct fluorescently labeled endosome/vesicle populations by live-cell imaging, we show that vesicle motility is reduced dramatically in a variety of cell types at the onset of hypertonic challenge. Live-cell imaging of actin and tubulin revealed similar arrested microfilament motility upon challenge. Vesicle motility recovered long after cell volume, a process that required functional regulatory volume increase and was accelerated by a return of extracellular osmolality to isosmotic levels. This delay suggests that, although volume-induced molecular crowding contributes to trafficking defects, it alone cannot explain the observed effects. Using fluorescent indicators and FRET-based probes, we found that intracellular ATP abundance and mitochondrial potential were reduced by hypertonicity and recovered after longer periods of time. Similar to the effects of osmotic challenge, isovolumetric elevation of intracellular chloride concentration by ionophores transiently decreased ATP production by mitochondria and abated microfilament and vesicle motility. These data illustrate how perturbed ionic balance, in addition to molecular crowding, affects membrane trafficking. PMID:26045497

Effect of probe diffusion on the SOFI imaging accuracy.

PubMed

Vandenberg, Wim; Dedecker, Peter

2017-03-23

Live-cell super-resolution fluorescence imaging is becoming commonplace for exploring biological systems, though sample dynamics can affect the imaging quality. In this work we evaluate the effect of probe diffusion on super-resolution optical fluctuation imaging (SOFI), using a theoretical model and numerical simulations based on the imaging of live cells labelled with photochromic fluorescent proteins. We find that, over a range of physiological conditions, fluorophore diffusion results in a change in the amplitude of the SOFI signal. The magnitude of this change is approximately proportional to the on-time ratio of the fluorophores. However, for photochromic fluorescent proteins this effect is unlikely to present a significant distortion in practical experiments in biological systems. Due to this lack of distortions, probe diffusion strongly enhances the SOFI imaging by avoiding spatial undersampling caused by the limited labeling density.

Rumen ciliate protozoa of domestic sheep (Ovis aries) and goat (Capra aegagrus hircus) in Kyrgyzstan.

PubMed

Gürelli, Gözde; Canbulat, Savaş; Aldayarov, Nurbek; Dehority, Burk A

2016-03-01

Species composition and concentration of rumen ciliate protozoa were investigated in the rumen contents of 14 domestic sheep and 1 goat living in Bishkek, Kyrgyzstan. This is the first report on rumen ciliates from ruminants living in Kyrgyzstan. In sheep 12 genera, 28 species and 12 morphotypes were detected, whereas in goat 8 genera, 12 species and 4 morphotypes were detected. The density of ciliates in sheep was (28.1 ± 20.0) × 10(4) cells mL(-1) and in goat was 37.0 × 10(4) cells mL(-1). Dasytricha ruminantium, Isotricha prostoma, Entodinium simulans and Ophryoscolex caudatus were major species (100%) in sheep, and for the first time, Diplodinium rangiferi was detected in a domestic goat. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Sensing dynamic cytoplasm refractive index changes of adherent cells with quantitative phase microscopy using incorporated microspheres as optical probes.

PubMed

Przibilla, Sabine; Dartmann, Sebastian; Vollmer, Angelika; Ketelhut, Steffi; Greve, Burkhard; von Bally, Gert; Kemper, Björn

2012-09-01

The intracellular refractive index is an important parameter that describes the optical density of the cytoplasm and the concentration of the intracellular solutes. The refractive index of adherently grown cells is difficult to access. We present a method in which silica microspheres in living cells are used to determine the cytoplasm refractive index with quantitative phase microscopy. The reliability of our approach for refractive index retrieval is shown by data from a comparative study on osmotically stimulated adherent and suspended human pancreatic tumor cells. Results from adherent human fibro sarcoma cells demonstrate the capability of the method for sensing of dynamic refractive index changes and its usage with microfluidics.

Titanium is not "the most biocompatible metal" under cathodic potential: The relationship between voltage and MC3T3 preosteoblast behavior on electrically polarized cpTi surfaces.

PubMed

Ehrensberger, Mark T; Sivan, Shiril; Gilbert, Jeremy L

2010-06-15

An electrochemically controlled system has been developed which allows for cell culture directly on electrically polarized metal surfaces with simultaneous control and assessment of the electrochemical current, potential, and impedance of the interface. This system was utilized in this study to assess the interactions between electrochemically polarized commercially pure titanium (cpTi) and MC3T3 preosteoblast cells. Cells were cultured on CpTi for 24 h at static potentials between -1000 mV and +1000 mV vs. Ag/AgCl and cell morphology (SEM and cell area) and viability (MTT and Live-Dead assay) were assessed along with the electrochemical current densities and surface oxide impedance properties. The results indicate that cathodic polarization in the range of -600 mV to -1000 mV markedly reduces the spreading and viability of cells cultured directly on cpTi within 24 h, while anodic polarization (-300 mV to +1000 mV) out to 72 h shows no difference in cell behavior as compared to the OCP condition. Analysis of the relationship between the cell outcomes and the electrochemical current densities and impedance indicated the presence of voltage-dependent electrochemical thresholds (cathodic current density, i(c) > 1.0 microA/cm(2), R(p) < 10(5) Omega cm(2)) which may control the biocompatibility of cpTi. In addition, these outcomes have direct clinical significance for modular orthopedic implants whose potential can shift, via fretting corrosion, down into the range of potentials exhibiting poor cell behavior. (c) 2009 Wiley Periodicals, Inc.

Oxidized-LDL induce morphological changes and increase stiffness of endothelial cells

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

Chouinard, Julie A.; Research Centre on Aging, Sherbrooke Geriatric University Institute, Sherbrooke, Quebec; Grenier, Guillaume

There is increasing evidence suggesting that oxidized low-density lipoproteins (ox-LDL) play a critical role in endothelial injury contributing to the age-related physio-pathological process of atherosclerosis. In this study, the effects of native LDL and ox-LDL on the mechanical properties of living human umbilical vein endothelial cells (HUVEC) were investigated by atomic force microscopy (AFM) force measurements. The contribution of filamentous actin (F-actin) and vimentin on cytoskeletal network organization were also examined by fluorescence microscopy. Our results revealed that ox-LDL had an impact on the HUVEC shape by interfering with F-actin and vimentin while native LDL showed no effect. AFM colloidalmore » force measurements on living individual HUVEC were successfully used to measure stiffness of cells exposed to native and ox-LDL. AFM results demonstrated that the cell body became significantly stiffer when cells were exposed for 24 h to ox-LDL while cells exposed for 24 h to native LDL displayed similar rigidity to that of the control cells. Young's moduli of LDL-exposed HUVEC were calculated using two models. This study thus provides quantitative evidence on biomechanical mechanisms related to endothelial cell dysfunction and may give new insight on strategies aiming to protect endothelial function in atherosclerosis.« less

Ionization balance in Titan's nightside ionosphere

NASA Astrophysics Data System (ADS)

Vigren, E.; Galand, M.; Yelle, R. V.; Wellbrock, A.; Coates, A. J.; Snowden, D.; Cui, J.; Lavvas, P.; Edberg, N. J. T.; Shebanits, O.; Wahlund, J.-E.; Vuitton, V.; Mandt, K.

2015-03-01

Based on a multi-instrumental Cassini dataset we make model versus observation comparisons of plasma number densities, nP = (nenI)1/2 (ne and nI being the electron number density and total positive ion number density, respectively) and short-lived ion number densities (N+, CH2+, CH3+, CH4+) in the southern hemisphere of Titan's nightside ionosphere over altitudes ranging from 1100 and 1200 km and from 1100 to 1350 km, respectively. The nP model assumes photochemical equilibrium, ion-electron pair production driven by magnetospheric electron precipitation and dissociative recombination as the principal plasma neutralization process. The model to derive short-lived-ion number densities assumes photochemical equilibrium for the short-lived ions, primary ion production by electron-impact ionization of N2 and CH4 and removal of the short-lived ions through reactions with CH4. It is shown that the models reasonably reproduce the observations, both with regards to nP and the number densities of the short-lived ions. This is contrasted by the difficulties in accurately reproducing ion and electron number densities in Titan's sunlit ionosphere.

Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

PubMed Central

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

2011-01-01

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507

Hydrodynamic size-dependent cellular uptake of aqueous QDs probed by fluorescence correlation spectroscopy.

PubMed

Dong, Chaoqing; Irudayaraj, Joseph

2012-10-11

Aqueous quantum dots (QDs) directly synthesized with various thiol ligands have been investigated as imaging probes in living cells. However, the effect of the surface chemistry of these ligands on QDs' cellular uptakes and their intracellular fate remains poorly understood. In this work, four CdTe QDs were directly synthesized under aqueous conditions using four different thiols as stabilizers and their interactions with cells were investigated. Fluorescence correlation spectroscopy (FCS), X-ray photoelectron spectroscopy (XPS), and zeta potential measurements on QDs primarily show that the surface structure of these QDs is highly dependent on the thiol ligands used in the preparation of QDs' precursors, including its layer thicknesses, densities, and surface charges. Subsequently, FCS integrated with the maximum-entropy-method-based FCS (MEMFCS) was used to investigate the concentration distribution and dynamics of these QDs in living A-427 cells. Our findings indicate that QDs' surface characteristics affect cell membrane adsorption and subsequent internalization. More critically, we show that the cellular uptake of aqueous QDs is dependent on their hydrodynamic diameter and might have the potential to escape trapped environments to accumulate in the cytoplasm.

Ratiometric Fluorescence Azide-Alkyne Cycloaddition for Live Mammalian Cell Imaging.

PubMed

Fu, Hongxia; Li, Yanru; Sun, Lingbo; He, Pan; Duan, Xinrui

2015-11-17

Click chemistry with metabolic labeling has been widely used for selectively imaging biomacromolecules in cells. The first example of azide-alkyne cycloaddition for ratiometric fluorescent imaging of live cells is reported. The precursor of the azido fluorophore (cresyl violet) has a fluorescence emission peak at 620 nm. The electron-rich nitrogen of the azido group blue-shifts the emission peak to 566 nm. When the click reaction occurs, an emission peak appears at 620 nm due to the lower electronic density of the newly formed triazole ring, which allows us to ratiometrically record fluorescence signals. This emission shift was applied to ratiometric imaging of propargylcholine- and dibenzocyclooctyne-labeled human breast cancer cells MCF-7 under laser confocal microscopy. Two typical triazole compounds were isolated for photophysical parameter measurements. The emission spectra presented a fluorescence emission peak around 620 nm for both click products. The results further confirmed the emission wavelength change was the result of azide-alkyne cycloaddition reaction. Since nearly all biomolecules can be metabolically labeled by reported alkyne-functionalized derivatives of native metabolites, our method can be readily applied to image these biomacromolecules.

The Effects of Microalgae as Live Food for Brachionus plicatilis (Rotifer) in Intensive Culture System.

PubMed

Rahman, Abdull Razak Abd; Cob, Zaidi Che; Jamari, Zainoddin; Mohamed, Abdul Majid; Toda, Tatsuki; Ross, Othman Haji

2018-03-01

Brachionus plicatilis is used to feed fish and crustacean larvae in the aquaculture industry. It is well established that the type of microalgae may influence rotifer production. This experiment was conducted to determine the effect of five different locally available microalgae species at Fisheries Research Institute (FRI), Kampung Pulau Sayak, Kedah, Malaysia on the instantaneous growth rate (μ) of rotifer. Nannochloris sp., Tetraselmis sp., Isochrysis sp., Chlorella sp., and Nannochloropsis sp. were used as feed at different algae densities (0.1, 0.3, 0.7 and 1.5 × 10 6 cells/ml) and culture volumes (20, 70 and 210 ml). At algae densities ranging from 0.3 to 1.5 × 10 6 cells/ml, an average μ value of more than 0.90 per day were recorded for all algae species. However, at density of 0.1 × 10 6 cells/ml, only Tetraselmis sp. resulted in the significantly highest μ value compared with others ( p < 0.05). In terms of volume, smaller culture volume of Tetraselmis sp. (20 ml) showed significantly higher μ compared with higher volume (70 and 210 ml cultures).

The relationship between high residential density in student dormitories and anxiety, binge eating and Internet addiction: a study of Chinese college students.

PubMed

Tao, Zhuoli; Wu, Gao; Wang, Zeyuan

2016-01-01

Although various studies have indicated that high residential density may affect health and psychological outcomes, to our knowledge, there have been no studies regarding the predictive nature of crowded living conditions on binge eating and the use of the Internet as coping strategies. A total of 1048 Chinese college students (540 males and 508 females) were randomly selected and asked to complete a battery of questionnaires that included the Zung's Self-Rating Anxiety Scale, the Internet Addiction Test, and Rosenbaum's Self-Control Scale. Binge eating behaviors and compensatory behaviors were also reported, and variables about residential density were measured. Among female participants, binge eating scores were significantly predicted by anxiety caused by high-density living conditions (P = 0.008), and similarly, the frequency of compensatory behaviors was significantly predicted by anxiety caused by high-density living conditions (P = 0.000) and self-control (P = 0.003). Furthermore, the Internet Addiction Test scores were significantly predicted by the anxiety caused by high -density living conditions (P = 0.000) and self-control (P = 0.000). Among male participants, not only were the binge eating scores significantly predicted by the anxiety caused by high-density living conditions (P = 0.000) and self-control (P = 0.000), but the frequency of compensatory behaviors was also significantly predicted by the anxiety caused by high-density living conditions (P = 0.000) and self-control (P = 0.01). Furthermore, Internet Addiction Test scores were significantly predicted by anxiety caused by high-density living conditions (P = 0.000) and self-control (P = 0.000). It was further found that for both genders, subjective factors such as self-control, and the anxiety caused by high-density living conditions had a stronger impact on Internet addiction than objective factors, such as the size of the student's dormitory room. Moreover, self-control was found to act as a moderator in the relationship between anxiety and Internet addiction among male participants. Binge eating and Internet use could be considered coping strategies for Chinese college students facing high residential density in their dormitories.

Electrochemical push-pull probe: from scanning electrochemical microscopy to multimodal altering of cell microenvironment.

PubMed

Bondarenko, Alexandra; Cortés-Salazar, Fernando; Gheorghiu, Mihaela; Gáspár, Szilveszter; Momotenko, Dmitry; Stanica, Luciana; Lesch, Andreas; Gheorghiu, Eugen; Girault, Hubert H

2015-04-21

To understand biological processes at the cellular level, a general approach is to alter the cells' environment and to study their chemical responses. Herein, we present the implementation of an electrochemical push-pull probe, which combines a microfluidic system with a microelectrode, as a tool for locally altering the microenvironment of few adherent living cells by working in two different perturbation modes, namely electrochemical (i.e., electrochemical generation of a chemical effector compound) and microfluidic (i.e., infusion of a chemical effector compound from the pushing microchannel, while simultaneously aspirating it through the pulling channel, thereby focusing the flow between the channels). The effect of several parameters such as flow rate, working distance, and probe inclination angle on the affected area of adherently growing cells was investigated both theoretically and experimentally. As a proof of concept, localized fluorescent labeling and pH changes were purposely introduced to validate the probe as a tool for studying adherent cancer cells through the control over the chemical composition of the extracellular space with high spatiotemporal resolution. A very good agreement between experimental and simulated results showed that the electrochemical perturbation mode enables to affect precisely only a few living cells localized in a high-density cell culture.

Effect of low level laser therapy and high intensity laser therapy on endothelial cell proliferation in vitro: preliminary communication

NASA Astrophysics Data System (ADS)

Lukowicz, Malgorzata; Szymanska, Justyna; Goralczyk, Krzysztof; Zajac, Andrzej; Rość, Danuta

2013-01-01

Background: The main purpose of this study was to analyze the influence of power intensity and wavelength of Low Level Laser Therapy (LLLT) and HILT (High Intensity Laser Therapy) on endothelial cell proliferation. Material and methods: The tests were done on human umbilical vein endothelial cells (HUVEC). Cultures were exposed to laser irradiation of 660 nm and 670 nm at different dosages, power output was 10 - 40 mW as well as 820 nm with power 100 mW and 808 nm with power 1500 mW. Energy density was from 0.28 to 11,43 J/cm2. Cell proliferation of a control and tested culture was evaluated with a colorimetric device to detect live cells. The tests were repeated 8 times. Results: We observed good effects of LLLT on live isolated ECs and no effects in experiments on previous deep-frozen cultures. Also HILT stimulated the proliferation of HUVEC. Conclusion: Endothelial cells play a key role in vascular homeostasis in humans. We observed the stimulatory effect of LLLT and HILT on proliferation of HUVEC. Many factors influence the proliferation of EC, so is it necessary to continue the experiment with different doses, intensity and cell concentration.

Planar Diamond-Based Multiarrays to Monitor Neurotransmitter Release and Action Potential Firing: New Perspectives in Cellular Neuroscience.

PubMed

Carabelli, Valentina; Marcantoni, Andrea; Picollo, Federico; Battiato, Alfio; Bernardi, Ettore; Pasquarelli, Alberto; Olivero, Paolo; Carbone, Emilio

2017-02-15

High biocompatibility, outstanding electrochemical responsiveness, inertness, and transparency make diamond-based multiarrays (DBMs) first-rate biosensors for in vitro detection of electrochemical and electrical signals from excitable cells together, with potential for in vivo applications as neural interfaces and prostheses. Here, we will review the electrochemical and physical properties of various DBMs and how these devices have been employed for recording released neurotransmitter molecules and all-or-none action potentials from living cells. Specifically, we will overview how DBMs can resolve localized exocytotic events from subcellular compartments using high-density microelectrode arrays (MEAs), or monitoring oxidizable neurotransmitter release from populations of cells in culture and tissue slices using low-density MEAs. Interfacing DBMs with excitable cells is currently leading to the promising opportunity of recording electrical signals as well as creating neuronal interfaces through the same device. Given the recent increasingly growing development of newly available DBMs of various geometries to monitor electrical activity and neurotransmitter release in a variety of excitable and neuronal tissues, the discussion will be limited to planar DBMs.

Automated, contour-based tracking and analysis of cell behaviour over long time scales in environments of varying complexity and cell density.

PubMed

Baker, Richard M; Brasch, Megan E; Manning, M Lisa; Henderson, James H

2014-08-06

Understanding single and collective cell motility in model environments is foundational to many current research efforts in biology and bioengineering. To elucidate subtle differences in cell behaviour despite cell-to-cell variability, we introduce an algorithm for tracking large numbers of cells for long time periods and present a set of physics-based metrics that quantify differences in cell trajectories. Our algorithm, termed automated contour-based tracking for in vitro environments (ACTIVE), was designed for adherent cell populations subject to nuclear staining or transfection. ACTIVE is distinct from existing tracking software because it accommodates both variability in image intensity and multi-cell interactions, such as divisions and occlusions. When applied to low-contrast images from live-cell experiments, ACTIVE reduced error in analysing cell occlusion events by as much as 43% compared with a benchmark-tracking program while simultaneously tracking cell divisions and resulting daughter-daughter cell relationships. The large dataset generated by ACTIVE allowed us to develop metrics that capture subtle differences between cell trajectories on different substrates. We present cell motility data for thousands of cells studied at varying densities on shape-memory-polymer-based nanotopographies and identify several quantitative differences, including an unanticipated difference between two 'control' substrates. We expect that ACTIVE will be immediately useful to researchers who require accurate, long-time-scale motility data for many cells. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

Coherent imaging with incoherent light in digital holographic microscopy

NASA Astrophysics Data System (ADS)

Chmelik, Radim

2012-01-01

Digital holographic microscope (DHM) allows for imaging with a quantitative phase contrast. In this way it becomes an important instrument, a completely non-invasive tool for a contrast intravital observation of living cells and a cell drymass density distribution measurement. A serious drawback of current DHMs is highly coherent illumination which makes the lateral resolution worse and impairs the image quality by a coherence noise and a parasitic interference. An uncompromising solution to this problem can be found in the Leith concept of incoherent holography. An off-axis hologram can be formed with arbitrary degree of light coherence in systems equipped with an achromatic interferometer and thus the resolution and the image quality typical for an incoherent-light wide-field microscopy can be achieved. In addition, advanced imaging modes based on limited coherence can be utilized. The typical example is a coherence-gating effect which provides a finite axial resolution and makes DHM image similar to that of a confocal microscope. These possibilities were described theoretically using the formalism of three-dimensional coherent transfer functions and proved experimentally by the coherence-controlled holographic microscope which is DHM based on the Leith achromatic interferometer. Quantitative-phase-contrast imaging is demonstrated with incoherent light by the living cancer cells observation and their motility evaluation. The coherence-gating effect was proved by imaging of model samples through a scattering layer and living cells inside an opalescent medium.

Sodium-metal chloride batteries

NASA Technical Reports Server (NTRS)

Ratnakumar, B. V.; Attia, A. I.; Halpert, G.

1992-01-01

It was concluded that rapid development in the technology of sodium metal chloride batteries has been achieved in the last decade mainly due to the: expertise available with sodium sulfur system; safety; and flexibility in design and fabrication. Long cycle lives of over 1000 and high energy densities of approx. 100 Wh/kg have been demonstrated in both Na/FeCl2 and Na/NiCl2 cells. Optimization of porous cathode and solid electrolyte geometries are essential for further enhancing the battery performance. Fundamental studies confirm the capabilities of these systems. Nickel dichloride emerges as the candidate cathode material for high power density applications such as electric vehicle and space.

Density of Diadema antillarum (Echinodermata: Echinoidea) on live coral patch reefs and dead Acropora cervicornis rubble patches near Loggerhead Key, Dry Tortugas National Park, Florida, USA

EPA Science Inventory

Density of adult Diadema antillarum was assessed on live coral patch reefs and dead Acropora cervicornis rubble patches next to Loggerhead Key, Dry Tortugas National Park, Florida, USA in June 2009. Mean density on live coral patch reefs (0.49 individuals m-2) was not statistical...

Can live tree size-density relationships provide a mechanism for predicting down and dead tree resources?

Treesearch

Christopher Woodall; James Westfall

2009-01-01

Live tree size-density relationships in forests have long provided a framework for understanding stand dynamics. There has been little examination of the relationship between the size-density attributes of live and standing/down dead trees (e.g., number and mean tree size per unit area, such information could help in large-scale efforts to estimate dead wood resources...

Game of Life on the Equal Degree Random Lattice

NASA Astrophysics Data System (ADS)

Shao, Zhi-Gang; Chen, Tao

2010-12-01

An effective matrix method is performed to build the equal degree random (EDR) lattice, and then a cellular automaton game of life on the EDR lattice is studied by Monte Carlo (MC) simulation. The standard mean field approximation (MFA) is applied, and then the density of live cells is given ρ=0.37017 by MFA, which is consistent with the result ρ=0.37±0.003 by MC simulation.

[Effects of Alexandrium tamarense and Prorocentrum donghaiense on rotifer Brachionus plicatilis population].

PubMed

Wang, Liping; Yan, Tian; Tan, Zhijun; Zhou, Mingjiang

2003-07-01

The effects of Prorocentrum donghaiense and Alexandrium sp., causative species of harmful algal bloom of East China Sea in May 2002, on rotifer Brachionus plicatilis population were studied in the laboratory. The results showed that Alexandrium tamarense (ATHK) had a lethal effect on B. plicatilis and the 48hLC50 was about 1300 cell.ml-1. The toxin comparison of different fractions showed that the algal culture and re-suspended algal cells had the adverse effects, and the alga at earlier growth phases showed a stronger impact, indicating that the inhibitory effect was related with the activity of the living algal cells. P. donghaiense at high densities (4 x 10(4), 5 x 10(4) and 10 x 10(4) cell.ml-1) had an adverse effect on B. plicatilis population, while at low densities (1 x 10(4), 2 x 10(4) and 3 x 10(4) cell.ml-1), the alga could be used as food for rotifer population. When the two algae were mixed, the lethal effect of A. tamarense could be decreased by P. donghaiense. The results indicated that the above HAB event could affect the micro-zooplankton population in the occurrence area of East China Sea.

Segmentation and classification of cell cycle phases in fluorescence imaging.

PubMed

Ersoy, Ilker; Bunyak, Filiz; Chagin, Vadim; Cardoso, M Christina; Palaniappan, Kannappan

2009-01-01

Current chemical biology methods for studying spatiotemporal correlation between biochemical networks and cell cycle phase progression in live-cells typically use fluorescence-based imaging of fusion proteins. Stable cell lines expressing fluorescently tagged protein GFP-PCNA produce rich, dynamically varying sub-cellular foci patterns characterizing the cell cycle phases, including the progress during the S-phase. Variable fluorescence patterns, drastic changes in SNR, shape and position changes and abundance of touching cells require sophisticated algorithms for reliable automatic segmentation and cell cycle classification. We extend the recently proposed graph partitioning active contours (GPAC) for fluorescence-based nucleus segmentation using regional density functions and dramatically improve its efficiency, making it scalable for high content microscopy imaging. We utilize surface shape properties of GFP-PCNA intensity field to obtain descriptors of foci patterns and perform automated cell cycle phase classification, and give quantitative performance by comparing our results to manually labeled data.

The different mechanisms of hypoxic acclimatization and adaptation in Lizard Phrynocephalus vlangalii living on Qinghai-Tibet Plateau.

PubMed

He, Jianzheng; Xiu, Minghui; Tang, Xiaolong; Yue, Feng; Wang, Ningbo; Yang, Shaobin; Chen, Qiang

2013-03-01

Phrynocephalus vlangalii is a species of lizard endemic in China, which lives on Qinghai-Tibet Plateau ranging from 2000 to 4600 m above sea level. In this study, P. vlangalii were collected from low altitude (2750 m) and high altitude (4564 m). The lizards from low altitude were acclimatized in simulated hypoxic chamber (equivalent to 4600 m) for 7, 15, and 30 days. The hematological parameters, heart weight, myocardial capillary density, and myocardial enzyme activities were examined. The results showed that acclimatization to hypoxia significantly increased hemoglobin concentration ([Hb]), hematocrit (Hct), heart weight (HW), heart weight to body mass (HW/BM), lactate dehydrogenase (LDH) activity, but markedly decreased mean corpuscular hemoglobin concentration (MCHC) and succinate dehydrogenase (SDH) activity. Red blood cell (RBC) count, body mass (BM), myocardial capillary density did not change markedly during hypoxic acclimatization. On the other hand, [Hb], Hct, MCHC, HW/BM, myocardium capillary density, and SDH activity of P. vlangalii from high altitude were remarkably higher than those from low-altitude; however, LDH activity of high-altitude P. vlangalii was lower than that of low-altitude lizards. There was no significant difference in HW or BM between populations of high-altitude and low-altitude. Based on the present data, we suggest that P. vlangalii has special anatomical, physiological, and biochemical accommodate mechanisms to live in hypoxic environment, and the regulative mechanisms are different between hypoxic acclimatization and adaptation. Copyright © 2013 Wiley Periodicals, Inc.

"Deep-media culture condition" promoted lumen formation of endothelial cells within engineered three-dimensional tissues in vitro.

PubMed

Sekiya, Sachiko; Shimizu, Tatsuya; Yamato, Masayuki; Okano, Teruo

2011-03-01

In the field of tissue engineering, the induction of microvessels into tissues is an important task because of the need to overcome diffusion limitations of oxygen and nutrients within tissues. Powerful methods to create vessels in engineered tissues are needed for creating real living tissues. In this study, we utilized three-dimensional (3D) highly cell dense tissues fabricated by cell sheet technology. The 3D tissue constructs are close to living-cell dense tissue in vivo. Additionally, creating an endothelial cell (EC) network within tissues promoted neovascularization promptly within the tissue after transplantation in vivo. Compared to the conditions in vivo, however, common in vitro cell culture conditions provide a poor environment for creating lumens within 3D tissue constructs. Therefore, for determining adequate conditions for vascularizing engineered tissue in vitro, our 3D tissue constructs were cultured under a "deep-media culture conditions." Compared to the control conditions, the morphology of ECs showed a visibly strained cytoskeleton, and the density of lumen formation within tissues increased under hydrostatic pressure conditions. Moreover, the increasing expression of vascular endothelial cadherin in the lumens suggested that the vessels were stabilized in the stimulated tissues compared with the control. These findings suggested that deep-media culture conditions improved lumen formation in engineered tissues in vitro.

Fluoro-luminometric real-time measurement of bacterial viability and killing.

PubMed

Lehtinen, Janne; Virta, Marko; Lilius, Esa Matti

2003-10-01

The viability and killing of Escherichia coli was measured on a real-time basis using a fluoro-luminometric device, which allows successive measurements of fluorescence and bioluminescence without user intervention. Bacteria were made fluorescent and bioluminescent by expression of gfp and insect luciferase (lucFF) genes. The green fluorescent protein (GFP) is a highly fluorescent, extremely stable protein, which accumulates in cells during growth, and therefore the measured fluorescence signal was proportional to the total number of cells. The luciferase reaction is dependent of ATP produced by living cells, so that the bioluminescence level was a direct measure of the viable cells. In contrast to the bacterial luciferase, the insect luciferase uses a water-soluble and nonvolatile substrate, which makes automated multi-well microplate assay possible. For the validation of the assay, the proportion of living and dead cell populations was experimentally modified by incubating E. coli cells in the presence of various ethanol concentrations. Bacterial viability and killing measured by a fluoro-luminometric assay correlated fairly well with the reference methods: conventional plate counting, optical density measurement and various flow cytometric analyses. The real-time assay described here allows following the changes in bacterial cultures and assessing the bactericidal and other effects of various chemical, immunological and physical agents simultaneously in large numbers of samples.

Pattern formation by curvature-inducing proteins on spherical membranes

NASA Astrophysics Data System (ADS)

Agudo-Canalejo, Jaime; Golestanian, Ramin

2017-12-01

Spatial organisation is a hallmark of all living cells, and recreating it in model systems is a necessary step in the creation of synthetic cells. It is therefore of both fundamental and practical interest to better understand the basic mechanisms underlying spatial organisation in cells. In this work, we use a continuum model of membrane and protein dynamics to study the behaviour of curvature-inducing proteins on membranes of spherical shape, such as living cells or lipid vesicles. We show that the interplay between curvature energy, entropic forces, and the geometric constraints on the membrane can result in the formation of patterns of highly-curved/protein-rich and weakly-curved/protein-poor domains on the membrane. The spontaneous formation of such patterns can be triggered either by an increase in the average density of curvature-inducing proteins, or by a relaxation of the geometric constraints on the membrane imposed by the membrane tension or by the tethering of the membrane to a rigid cell wall or cortex. These parameters can also be tuned to select the size and number of the protein-rich domains that arise upon pattern formation. The very general mechanism presented here could be related to protein self-organisation in many biological processes, ranging from (proto)cell division to the formation of membrane rafts.

A naphthalene exciplex based Al3+ selective on-type fluorescent probe for living cells at the physiological pH range: experimental and computational studies.

PubMed

Banerjee, Arnab; Sahana, Animesh; Das, Sudipta; Lohar, Sisir; Guha, Subarna; Sarkar, Bidisha; Mukhopadhyay, Subhra Kanti; Mukherjee, Asok K; Das, Debasis

2012-05-07

2-((Naphthalen-6-yl)methylthio)ethanol (HL) was prepared by one pot synthesis using 2-mercaptoethanol and 2-bromomethylnaphthalene. It was found to be a highly selective fluorescent sensor for Al(3+) in the physiological pH (pH 7.0-8.0). It could sense Al(3+) bound to cells through fluorescence microscopy. Metal ions like Mn(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Ag(+), Cd(2+), Hg(2+), Cr(3+) and Pb(2+) did not interfere. No interference was also observed with anions like Cl(-), Br(-), F(-), SO(4)(2-), NO(3)(-), CO(3)(2-), HPO(4)(2-) and SCN(-). Experimentally observed structural and spectroscopic features of HL and its Al(3+) complex have been substantiated by computational calculations using density functional theory (DFT) and time dependent density functional theory (TDDFT).

Light Irradiation And Response Of The Living Body - Effect Of Pain Relief And Promotion Of Wound Healing -

NASA Astrophysics Data System (ADS)

Taguchi, Yoshio; Kurokawa, Yoshimochi; Ohara, Itaru; Ueki, Hamaichi; Inaba, Humio

1989-09-01

The first report of laser irradiation for wound healing was done by Mester, E., et al. in 1968. From their reports, we can get many knowledges and suggestions as for laser irradiation. At that time he used ruby laser (694.3 nm wave length) for surgical wounds and burns on the back skin of mice. The condition of irradiation was studied with energy density between 0.5-10 J/cm2 twice a week. As a result, they noticed 1 J/cm2 irradiation was effective for those wounds. After a few experimental reports, they published their clinical studies in 1975. Clinically, they used He-Ne laser (632.8 nm wave length) irradiation. Human leg ulcers due to peripheral circulatory disturbance were treated with energy density of 4 J/cm2 twice a week. And they got good results, obtaining complete healing in two-thirds of the cases. We became strongly stimulated by those reports. We have been studying the effect of light on experimental and clinical wound healing as well as on various kinds of biological phenomena since 1980. Particularly, its effect according to the difference of light has been studied. In October 1982, the first clinical case was tried by Argon laser (514.5 nm wave length) irradiation for therapeutic purpose. A man had a chronic ulcer of the left first toe due to Buerger's disease for 5 months. Surprizingly, on the 14th day after 6 treatments of the light irradiation, his ulcer completely healed. During these treatments, the patient noticed that the pain completely disappeared after 2 treatments. Fifty Argon laser treatments were carried out on clinical cases after these experiences, we reached to a conclusion that light irradiation stimulated something in the injured tissues and lead to good clinical results. Several studies concerning mechanism for these effects i.e. peripheral circulation, histology of granulation, cell proliferation, chemistry and other studies were carried out. From these investigations, peripheral circulation was improved when in those who were suffering from vascular disorders but was not effective in normal subjects. From a study of cellular electrophoretic mobility, irradiated G0G1 cells increased their mobility, but irradiated G2M cells decreased. These results suggested light irradiation contributed to homeostasis of living cells, tissues, and body. Experiments concerning the light sources, i.e. wave length, energy density and polarization were done. As a result, linear polarization and right circular polarization improved wound healing, but incoherent light itself did not. ,According to our studies, no distinguished differences among various kinds of wave length were noticed. And light irradiation with energy density was very effective between 2 to 6 J/cm2. We strongly suggest the role of coherency is very important to do light irradiation on the living body. In conclusion, we like to propose our new viewpoint. That is, the light irradiation should be discussed with the structure of high molecular substances in the living body.

Possible metabolic impact of Ramadan fasting in healthy men.

PubMed

Vardarli, Mustafa Cumhur; Hammes, Hans-Peter; Vardarli, İrfan

2014-01-01

Insulin sensitivity and β-cell function during Ramadan fasting in healthy male subjects have not been investigated so far. We assessed the changes of these and other metabolic parameters to judge the potential metabolic benefits of Ramadan fasting. Twenty-four healthy males of Turkish origin living in Germany, with normal glucose tolerance, participated in this study during Ramadan of 2009; 19 who completed fasting were analyzed. Blood was drawn at sunset after a period of fasting lasting approximately 15 h on days 0, 16, and 30 of Ramadan, as well as 7 and 28 days later. Insulin sensitivity (Homeostasis Model Assessment, HOMA), β-cell function, and other parameters were assessed. Ramadan fasting was associated with a significant reduction (-) or increment (+) for the following variables: insulin sensitivity (-20%; P = 0.04), β-cell function (+10%; P = 0.049), high-density lipoprotein cholesterol (-23%; P = 0.0003), low-density lipoprotein cholesterol (+14%; P = 0.007), nonesterified fatty acids (-62%; P < 0.0001), resistin (-20%; P = 0.01), adiponectin (+16%; P = 0.003), and glucagon (-21%; P = 0.01). C-peptide, insulin, leptin, triglyceride, and very low-density lipoprotein cholesterol concentrations were not significantly changed. Ramadan fasting is associated with transiently impaired insulin sensitivity, compensated for by an increased β-cell function. However, the pattern of insulin resistance-mediating adipocytokines suggests a potentially beneficial metabolic effect of Ramadan fasting.

Automated Photoreceptor Cell Identification on Nonconfocal Adaptive Optics Images Using Multiscale Circular Voting.

PubMed

Liu, Jianfei; Jung, HaeWon; Dubra, Alfredo; Tam, Johnny

2017-09-01

Adaptive optics scanning light ophthalmoscopy (AOSLO) has enabled quantification of the photoreceptor mosaic in the living human eye using metrics such as cell density and average spacing. These rely on the identification of individual cells. Here, we demonstrate a novel approach for computer-aided identification of cone photoreceptors on nonconfocal split detection AOSLO images. Algorithms for identification of cone photoreceptors were developed, based on multiscale circular voting (MSCV) in combination with a priori knowledge that split detection images resemble Nomarski differential interference contrast images, in which dark and bright regions are present on the two sides of each cell. The proposed algorithm locates dark and bright region pairs, iteratively refining the identification across multiple scales. Identification accuracy was assessed in data from 10 subjects by comparing automated identifications with manual labeling, followed by computation of density and spacing metrics for comparison to histology and published data. There was good agreement between manual and automated cone identifications with overall recall, precision, and F1 score of 92.9%, 90.8%, and 91.8%, respectively. On average, computed density and spacing values using automated identification were within 10.7% and 11.2% of the expected histology values across eccentricities ranging from 0.5 to 6.2 mm. There was no statistically significant difference between MSCV-based and histology-based density measurements (P = 0.96, Kolmogorov-Smirnov 2-sample test). MSCV can accurately detect cone photoreceptors on split detection images across a range of eccentricities, enabling quick, objective estimation of photoreceptor mosaic metrics, which will be important for future clinical trials utilizing adaptive optics.

Phenotypic plasticity and longevity in plants and animals: cause and effect?

PubMed

Borges, Renee M

2009-10-01

Immobile plants and immobile modular animals outlive unitary animals. This paper discusses competing but not necessarily mutually exclusive theories to explain this extreme longevity, especially from the perspective of phenotypic plasticity. Stem cell immortality, vascular autonomy, and epicormic branching are some important features of the phenotypic plasticity of plants that contribute to their longevity. Monocarpy versus polycarpy can also influence the kind of senescent processes experienced by plants. How density-dependent phenomena affecting the establishment of juveniles in these immobile organisms can influence the evolution of senescence, and consequently longevity, is reviewed and discussed. Whether climate change scenarios will favour long-lived or short-lived organisms, with their attendant levels of plasticity, is also presented.

Study of the cell activity in three-dimensional cell culture by using Raman spectroscopy

NASA Astrophysics Data System (ADS)

Arunngam, Pakajiraporn; Mahardika, Anggara; Hiroko, Matsuyoshi; Andriana, Bibin Bintang; Tabata, Yasuhiko; Sato, Hidetoshi

2018-02-01

The purpose of this study is to develop a estimation technique of local cell activity in cultured 3D cell aggregate with gelatin hydrogel microspheres by using Raman spectroscopy. It is an invaluable technique allowing real-time, nondestructive, and invasive measurement. Cells in body generally exist in 3D structure, which physiological cell-cell interaction enhances cell survival and biological functions. Although a 3D cell aggregate is a good model of the cells in living tissues, it was difficult to estimate their physiological conditions because there is no effective technique to make observation of intact cells in the 3D structure. In this study, cell aggregates were formed by MC3T-E1 (pre-osteoblast) cells and gelatin hydrogel microspheres. In appropriate condition MC3T-E1 cells can differentiate into osteoblast. We assume that the activity of the cell would be different according to the location in the aggregate because the cells near the surface of the aggregate have more access to oxygen and nutrient. Raman imaging technique was applied to measure 3D image of the aggregate. The concentration of the hydroxyapatite (HA) is generated by osteoblast was estimated with a strong band at 950-970 cm-1 which assigned to PO43- in HA. It reflects an activity of the specific site in the cell aggregate. The cell density in this specific site was analyzed by multivariate analysis of the 3D Raman image. Hence, the ratio between intensity and cell density in the site represents the cell activity.

Flow cytometric cell cycle analysis of muscle precursor cells cultured within 3D scaffolds in a perfusion bioreactor.

PubMed

Flaibani, Marina; Luni, Camilla; Sbalchiero, Elisa; Elvassore, Nicola

2009-01-01

It has been widely demonstrated that perfusion bioreactors improve in vitro three-dimensional (3D) cultures in terms of high cell density and uniformity of cell distribution; however, the studies reported in literature were primarily based on qualitative analysis (histology, immunofluorescent staining) or on quantitative data averaged on the whole population (DNA assay, PCR). Studies on the behavior, in terms of cell cycle, of a cell population growing in 3D scaffolds in static or dynamic conditions are still absent. In this work, a perfusion bioreactor suitable to culture C(2)C(12) muscle precursor cells within 3D porous collagen scaffolds was designed and developed and a method based on flowcytometric analyses for analyzing the cell cycle in the cell population was established. Cells were extracted by enzymatic digestion of the collagen scaffolds after 4, 7, and 10 days of culture, and flow cytometric live/dead and cell cycle analyses were performed with Propidium Iodide. A live/dead assay was used for validating the method for cell extraction and staining. Moreover, to investigate spatial heterogeneity of the cell population under perfusion conditions, two stacked scaffolds in the 3D domain, of which only the upstream layer was seeded, were analyzed separately. All results were compared with those obtained from static 3D cultures. The live/dead assay revealed the presence of less than 20% of dead cells, which did not affect the cell cycle analysis. Cell cycle analyses highlighted the increment of cell fractions in proliferating phases (S/G(2)/M) owing to medium perfusion in long-term cultures. After 7-10 days, the percentage of proliferating cells was 8-12% for dynamic cultures and 3-5% for the static controls. A higher fraction of proliferating cells was detected in the downstream scaffold. From a general perspective, this method provided data with a small standard deviation and detected the differences between static and dynamic cultures and between upper and lower scaffolds. Our methodology can be extended to other cell types to investigate the influence of 3D culture conditions on the expression of other relevant cell markers.

Effect of Dedifferentiation on Time to Mutation Acquisition in Stem Cell-Driven Cancers

PubMed Central

Jilkine, Alexandra; Gutenkunst, Ryan N.

2014-01-01

Accumulating evidence suggests that many tumors have a hierarchical organization, with the bulk of the tumor composed of relatively differentiated short-lived progenitor cells that are maintained by a small population of undifferentiated long-lived cancer stem cells. It is unclear, however, whether cancer stem cells originate from normal stem cells or from dedifferentiated progenitor cells. To address this, we mathematically modeled the effect of dedifferentiation on carcinogenesis. We considered a hybrid stochastic-deterministic model of mutation accumulation in both stem cells and progenitors, including dedifferentiation of progenitor cells to a stem cell-like state. We performed exact computer simulations of the emergence of tumor subpopulations with two mutations, and we derived semi-analytical estimates for the waiting time distribution to fixation. Our results suggest that dedifferentiation may play an important role in carcinogenesis, depending on how stem cell homeostasis is maintained. If the stem cell population size is held strictly constant (due to all divisions being asymmetric), we found that dedifferentiation acts like a positive selective force in the stem cell population and thus speeds carcinogenesis. If the stem cell population size is allowed to vary stochastically with density-dependent reproduction rates (allowing both symmetric and asymmetric divisions), we found that dedifferentiation beyond a critical threshold leads to exponential growth of the stem cell population. Thus, dedifferentiation may play a crucial role, the common modeling assumption of constant stem cell population size may not be adequate, and further progress in understanding carcinogenesis demands a more detailed mechanistic understanding of stem cell homeostasis. PMID:24603301

Near-infrared luminescent cubic silicon carbide nanocrystals for in vivo biomarker applications: an ab initio study

NASA Astrophysics Data System (ADS)

Somogyi, Bálint; Zólyomi, Viktor; Gali, Adam

2012-11-01

Molecule-sized fluorescent emitters are much sought-after to probe biomolecules in living cells. We demonstrate here by time-dependent density functional calculations that the experimentally achievable 1-2 nm sized silicon carbide nanocrystals can emit light in the near-infrared region after introducing appropriate color centers in them. These near-infrared luminescent silicon carbide nanocrystals may act as ideal fluorophores for in vivo bioimaging.

High-Content Surface and Total Expression siRNA Kinase Library Screen with VX-809 Treatment Reveals Kinase Targets that Enhance F508del-CFTR Rescue.

PubMed

Perkins, Lydia A; Fisher, Gregory W; Naganbabu, Matharishwan; Schmidt, Brigitte F; Mun, Frederick; Bruchez, Marcel P

2018-03-05

The most promising F508del-CFTR corrector, VX-809, has been unsuccessful as an effective, stand-alone treatment for CF patients, but the rescue effect in combination with other drugs may confer an acceptable level of therapeutic benefit. Targeting cellular factors that modify trafficking may act to enhance the cell surface density of F508-CFTR with VX-809 correction. Our goal is to identify druggable kinases that enhance F508del-CFTR rescue and stabilization at the cell surface beyond that achievable with the VX-809 corrector alone. To achieve this goal, we implemented a new high-throughput screening paradigm that quickly and quantitatively measures surface density and total protein in the same cells. This allowed for rapid screening for increased surface targeting and proteostatic regulation. The assay utilizes fluorogen-activating-protein (FAP) technology with cell excluded and cell permeant fluorogenic dyes in a quick, wash-free fluorescent plate reader format on live cells to first measure F508del-CFTR expressed on the surface and then the total amount of F508del-CFTR protein present. To screen for kinase targets, we used Dharmacon's ON-TARGET plus SMARTpool siRNA Kinase library (715 target kinases) with and without 10 μM VX-809 treatment in triplicate at 37 °C. We identified several targets that had a significant interaction with VX-809 treatment in enhancing surface density with siRNA knockdown. Select small-molecule inhibitors of the kinase targets demonstrated augmented surface expression with VX-809 treatment.

Pyrene-Labeled Amphiphiles: Dynamic And Structural Probes Of Membranes And Lipoproteins

NASA Astrophysics Data System (ADS)

Pownall, Henry J.; Homan, Reynold; Massey, John B.

1987-01-01

Lipids and proteins are important functional and structural components of living organisms. Although proteins are frequently found as soluble components of plasma or the cell cytoplasm, many lipids are much less soluble and separate into complex assemblies that usually contain proteins. Cell membranes and plasma lipoproteins' are two important macro-molecular assemblies that contain both lipids and proteins. Cell membranes are composed of a variety of lipids and proteins that form an insoluble bilayer array that has relatively little curvature over distances of several nm. Plasma lipoproteins are different in that they are much smaller, water-soluble, and have highly curved surfaces. A model of a high density lipoprotein (HDL) is shown in Figure 1. This model (d - 10 nm) contains a surface of polar lipids and proteins that surrounds a small core of insoluble lipids, mostly triglycerides and cholesteryl esters. The low density (LDL) (d - 25 nm) and very low density (VLDL) (d 90 nm) lipoproteins have similar architectures, except the former has a cholesteryl ester core and the latter a core that is almost exclusively triglyceride (Figure 1). The surface proteins of HDL are amphiphilic and water soluble; the single protein of LDL is insoluble, whereas VLDL contains both soluble and insoluble proteins. The primary structures of all of these proteins are known.

Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro.

PubMed

Blair, Harry C; Larrouture, Quitterie C; Li, Yanan; Lin, Hang; Beer-Stoltz, Donna; Liu, Li; Tuan, Rocky S; Robinson, Lisa J; Schlesinger, Paul H; Nelson, Deborah J

2017-06-01

We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and density of osteoblasts and by endogenous and exogenous inhibitors.

Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro

PubMed Central

Larrouture, Quitterie C.; Li, Yanan; Lin, Hang; Beer-Stoltz, Donna; Liu, Li; Tuan, Rocky S.; Robinson, Lisa J.; Schlesinger, Paul H.; Nelson, Deborah J.

2017-01-01

We review the characteristics of osteoblast differentiation and bone matrix synthesis. Bone in air breathing vertebrates is a specialized tissue that developmentally replaces simpler solid tissues, usually cartilage. Bone is a living organ bounded by a layer of osteoblasts that, because of transport and compartmentalization requirements, produce bone matrix exclusively as an organized tight epithelium. With matrix growth, osteoblasts are reorganized and incorporated into the matrix as living cells, osteocytes, which communicate with each other and surface epithelium by cell processes within canaliculi in the matrix. The osteoblasts secrete the organic matrix, which are dense collagen layers that alternate parallel and orthogonal to the axis of stress loading. Into this matrix is deposited extremely dense hydroxyapatite-based mineral driven by both active and passive transport and pH control. As the matrix matures, hydroxyapatite microcrystals are organized into a sophisticated composite in the collagen layer by nucleation in the protein lattice. Recent studies on differentiating osteoblast precursors revealed a sophisticated proton export network driving mineralization, a gene expression program organized with the compartmentalization of the osteoblast epithelium that produces the mature bone matrix composite, despite varying serum calcium and phosphate. Key issues not well defined include how new osteoblasts are incorporated in the epithelial layer, replacing those incorporated in the accumulating matrix. Development of bone in vitro is the subject of numerous projects using various matrices and mesenchymal stem cell-derived preparations in bioreactors. These preparations reflect the structure of bone to variable extents, and include cells at many different stages of differentiation. Major challenges are production of bone matrix approaching the in vivo density and support for trabecular bone formation. In vitro differentiation is limited by the organization and density of osteoblasts and by endogenous and exogenous inhibitors. PMID:27846781

Live-cell imaging of ER-PM contact architecture by a novel TIRFM approach reveals extension of junctions in response to store-operated Ca2+-entry.

PubMed

Poteser, Michael; Leitinger, Gerd; Pritz, Elisabeth; Platzer, Dieter; Frischauf, Irene; Romanin, Christoph; Groschner, Klaus

2016-10-19

Nanometer-spaced appositions between endoplasmic reticulum and plasma membrane (ER-PM junctions) stabilized by membrane-joining protein complexes are critically involved in cellular Ca 2+ -handling and lipid trafficking. ER-PM junctional architecture and plasticity associated with inter-membrane communication are as yet barely understood. Here, we introduce a method to precisely characterize ER-PM junction morphology and dynamics with high temporal resolution and minimal disturbance of junctional intermembrane communication. We show that expression of soluble cytosolic fluorophores in combination with TIRFM enables to delineate ER and PM distance in the range of 10-150 nm. Live-cell imaging of sub-plasmalemmal structures in RBL-2H3 mast cells by this method, designated as fluorescence density mapping (FDM), revealed profound dynamics of ER-PM contact sites in response to store-depletion. We report the existence of a Ca 2+ -dependent process that expands the junctional ER to enlarge its contact surface with the PM, thereby promoting and stabilizing STIM1-Orai1 competent ER-PM junctions.

Estimation and uncertainty analysis of dose response in an inter-laboratory experiment

NASA Astrophysics Data System (ADS)

Toman, Blaza; Rösslein, Matthias; Elliott, John T.; Petersen, Elijah J.

2016-02-01

An inter-laboratory experiment for the evaluation of toxic effects of NH2-polystyrene nanoparticles on living human cancer cells was performed with five participating laboratories. Previously published results from nanocytoxicity assays are often contradictory, mostly due to challenges related to producing a reliable cytotoxicity assay protocol for use with nanomaterials. Specific challenges include reproducibility preparing nanoparticle dispersions, biological variability from testing living cell lines, and the potential for nano-related interference effects. In this experiment, such challenges were addressed by developing a detailed experimental protocol and using a specially designed 96-well plate layout which incorporated a range of control measurements to assess multiple factors such as nanomaterial interference, pipetting accuracy, cell seeding density, and instrument performance. Detailed data analysis of these control measurements showed that good control of the experiments was attained by all participants in most cases. The main measurement objective of the study was the estimation of a dose response relationship between concentration of the nanoparticles and metabolic activity of the living cells, under several experimental conditions. The dose curve estimation was achieved by imbedding a three parameter logistic curve in a three level Bayesian hierarchical model, accounting for uncertainty due to all known experimental conditions as well as between laboratory variability in a top-down manner. Computation was performed using Markov Chain Monte Carlo methods. The fit of the model was evaluated using Bayesian posterior predictive probabilities and found to be satisfactory.

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

PubMed Central

Teissié, J; Ramos, C

1998-01-01

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion. PMID:9545050

Correlation between electric field pulse induced long-lived permeabilization and fusogenicity in cell membranes.

PubMed

Teissié, J; Ramos, C

1998-04-01

Electric field pulses have been reported to induce long-lived permeabilization and fusogenicity on cell membranes. The two membrane property alterations are under the control of the field strength, the pulse duration, and the number of pulses. Experiments on mammalian cells pulsed by square wave form pulses and then brought into contact randomly through centrifugation revealed an even stronger analogy between the two processes. Permeabilization was known to affect well-defined regions of the cell surface. Fusion can be obtained only when permeabilized surfaces on the two partners were brought into contact. Permeabilization was under the control of the pulse duration and of the number of pulses. A similar relationship was observed as far as fusion is concerned. But a critical level of local permeabilization must be present for fusion to take place when contacts are created. The same conclusions are obtained from previous experiments on ghosts subjected to exponentially decaying field pulses and then brought into contact by dielectrophoresis. These observations are in agreement with a model of membrane fusion in which the merging of local random defects occurs when the two membranes are brought into contact. The local defects are considered part of the structural membrane reorganization induced by the external field. Their density is dependent on the pulse duration and number of pulses. They support the long-lived permeabilization. Their number must be very large to support the occurrence of membrane fusion.

Direct measurement of local oxygen concentration in the bone marrow of live animals

NASA Astrophysics Data System (ADS)

Spencer, Joel A.; Ferraro, Francesca; Roussakis, Emmanuel; Klein, Alyssa; Wu, Juwell; Runnels, Judith M.; Zaher, Walid; Mortensen, Luke J.; Alt, Clemens; Turcotte, Raphaël; Yusuf, Rushdia; Côté, Daniel; Vinogradov, Sergei A.; Scadden, David T.; Lin, Charles P.

2014-04-01

Characterization of how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for the therapeutic manipulation of stem cells. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis, expression of hypoxia inducible factor-1α (Hif-1α) and related genes, and staining with surrogate hypoxic markers (for example, pimonidazole). Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow of live mice. Using two-photon phosphorescence lifetime microscopy, we determined the absolute pO2 of the bone marrow to be quite low (<32 mm Hg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mm Hg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change markedly after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

Integrating cell phone imaging with magnetic levitation (i-LEV) for label-free blood analysis at the point-of-living

PubMed Central

Durmus, Naside Gozde; Davis, Ronald W.; Steinmetz, Lars M.; Demirci, Utkan

2016-01-01

There is an emerging need for portable, robust, inexpensive and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia and chronic fatigue syndrome. Here, we present a magnetic levitation-based diagnosis system in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, we introduce an easy-to-use, smartphone incorporated levitation system for cell analysis. Using our portable imaging magnetic levitation (i-LEV) system, we show that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. PMID:26523938

Integrating Cell Phone Imaging with Magnetic Levitation (i-LEV) for Label-Free Blood Analysis at the Point-of-Living.

PubMed

Baday, Murat; Calamak, Semih; Durmus, Naside Gozde; Davis, Ronald W; Steinmetz, Lars M; Demirci, Utkan

2016-03-02

There is an emerging need for portable, robust, inexpensive, and easy-to-use disease diagnosis and prognosis monitoring platforms to share health information at the point-of-living, including clinical and home settings. Recent advances in digital health technologies have improved early diagnosis, drug treatment, and personalized medicine. Smartphones with high-resolution cameras and high data processing power enable intriguing biomedical applications when integrated with diagnostic devices. Further, these devices have immense potential to contribute to public health in resource-limited settings where there is a particular need for portable, rapid, label-free, easy-to-use, and affordable biomedical devices to diagnose and continuously monitor patients for precision medicine, especially those suffering from rare diseases, such as sickle cell anemia, thalassemia, and chronic fatigue syndrome. Here, a magnetic levitation-based diagnosis system is presented in which different cell types (i.e., white and red blood cells) are levitated in a magnetic gradient and separated due to their unique densities. Moreover, an easy-to-use, smartphone incorporated levitation system for cell analysis is introduced. Using our portable imaging magnetic levitation (i-LEV) system, it is shown that white and red blood cells can be identified and cell numbers can be quantified without using any labels. In addition, cells levitated in i-LEV can be distinguished at single-cell resolution, potentially enabling diagnosis and monitoring, as well as clinical and research applications. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label free cell tracking in 3D tissue engineering constructs with high resolution imaging

NASA Astrophysics Data System (ADS)

Smith, W. A.; Lam, K.-P.; Dempsey, K. P.; Mazzocchi-Jones, D.; Richardson, J. B.; Yang, Y.

2014-02-01

Within the field of tissue engineering there is an emphasis on studying 3-D live tissue structures. Consequently, to investigate and identify cellular activities and phenotypes in a 3-D environment for all in vitro experiments, including shape, migration/proliferation and axon projection, it is necessary to adopt an optical imaging system that enables monitoring 3-D cellular activities and morphology through the thickness of the construct for an extended culture period without cell labeling. This paper describes a new 3-D tracking algorithm developed for Cell-IQ®, an automated cell imaging platform, which has been equipped with an environmental chamber optimized to enable capturing time-lapse sequences of live cell images over a long-term period without cell labeling. As an integral part of the algorithm, a novel auto-focusing procedure was developed for phase contrast microscopy equipped with 20x and 40x objectives, to provide a more accurate estimation of cell growth/trajectories by allowing 3-D voxels to be computed at high spatiotemporal resolution and cell density. A pilot study was carried out in a phantom system consisting of horizontally aligned nanofiber layers (with precise spacing between them), to mimic features well exemplified in cellular activities of neuronal growth in a 3-D environment. This was followed by detailed investigations concerning axonal projections and dendritic circuitry formation in a 3-D tissue engineering construct. Preliminary work on primary animal neuronal cells in response to chemoattractant and topographic cue within the scaffolds has produced encouraging results.

Membrane tension regulates clathrin-coated pit dynamics

NASA Astrophysics Data System (ADS)

Liu, Allen

2014-03-01

Intracellular organization depends on close communication between the extracellular environment and a network of cytoskeleton filaments. The interactions between cytoskeletal filaments and the plasma membrane lead to changes in membrane tension that in turns help regulate biological processes. Endocytosis is thought to be stimulated by low membrane tension and the removal of membrane increases membrane tension. While it is appreciated that the opposing effects of exocytosis and endocytosis have on keeping plasma membrane tension to a set point, it is not clear how membrane tension affects the dynamics of clathrin-coated pits (CCPs), the individual functional units of clathrin-mediated endocytosis. Furthermore, although it was recently shown that actin dynamics counteracts membrane tension during CCP formation, it is not clear what roles plasma membrane tension plays during CCP initiation. Based on the notion that plasma membrane tension is increased when the membrane area increases during cell spreading, we designed micro-patterned surfaces of different sizes to control the cell spreading sizes. Total internal reflection fluorescence microscopy of living cells and high content image analysis were used to quantify the dynamics of CCPs. We found that there is an increased proportion of CCPs with short (<20s) lifetime for cells on larger patterns. Interestingly, cells on larger patterns have higher CCP initiation density, an effect unexpected based on the conventional view of decreasing endocytosis with increasing membrane tension. Furthermore, by analyzing the intensity profiles of CCPs that were longer-lived, we found CCP intensity decreases with increasing cell size, indicating that the CCPs are smaller with increasing membrane tension. Finally, disruption of actin dynamics significantly increased the number of short-lived CCPs, but also decreased CCP initiation rate. Together, our study reveals new mechanistic insights into how plasma membrane tension regulates the dynamics of CCPs.

On the distribution of Na+ pump sites in the frog skin

PubMed Central

Mills, JW; DiBona, DR

1977-01-01

Exposure of the outside of the isolated frog skin to a Ringer's solution, made hypertonic by the addition of mannitol, causes a rapid and sustained increase in transepithelial permeability through a structural distortion-a focal blistering-of the "tight" junctions of the outermost living cell layer. [(3)H]ouabain, used as an autoradiographic marker for the Na+-pump (Na+-K+-adenosine triphosphatase), is usually unable to penetrate the frog skin from the outside solution, but when added to a hypertonic mannitol- Ringer's solution in the outside bath it readily penetrates the epithelium, presumably through the opened shunt pathway. Radioautographic analysis of [(3)H]ouabain binding sites revealed that most of ouabain enters from the outside solution binds to the sites on the cell membranes of the stratum spinosum, as was the case when it was applied from the inside bath in an earlier study. The outer living cell layer, the first to be exposed to ouabain, does not appear to be the major site for the Na+-pump, and therefore, is not likely to be responsible for most of the active pumping of Na+. This result demonstrates that previous failure to show a high density of Na+-pump sites on the cells of the outermost layer, when [(3)H]ouabain was applied from the inside solution, was not due to the inability of the marker to reach these cells at a sufficient concentration to reveal all pump sites. These results provide further support for a model of Na+-transport across the frog skin which distributes the active pump step on the inward facing membranes of all living cells. PMID:144738

An Experimental Determination of Static Magnetic Fields Induced Noise in Living Systems

NASA Astrophysics Data System (ADS)

Brady, Megan; Laramee, Craig

2013-03-01

Living systems are constantly exposed to static magnetic fields (SMFs) from both natural and man-made sources. Exposures vary in dose and duration ranging from geomagnetic (~50 μT) to residential and industrial (~10s of mT) fields. Efforts to characterize responses to SMFs have yielded conflicting results, showing a dependence on experimental variables used. Here we argue that low to moderate SMF exposure is a sub-threshold perturbation operating below thermal noise, and assays that evaluate statistical characteristics of a single cell may identify responses not consistently found by population averaging approaches. Recent studies of gene expression show that it is a stochastic process capable of producing bursting dynamics. Moreover, theoretical and experimental methods have also been developed to allow quantitative estimates of the associated biophysical parameters. These developments provide a new way to assess responses of living systems to SMFs. In this work, we report on our efforts to use single molecule fluorescence in situ hybridization to assess responses of NIH-3T3 cells to SMF exposure at flux densities ranging from 1 to 440 mT for 48 hours. Results will contribute to determining mechanisms by which SMF exposure influences gene expression.

Sequential glycan profiling at single cell level with the microfluidic lab-in-a-trench platform: a new era in experimental cell biology.

PubMed

O'Connell, Tríona M; King, Damien; Dixit, Chandra K; O'Connor, Brendan; Walls, Dermot; Ducrée, Jens

2014-09-21

It is now widely recognised that the earliest changes that occur on a cell when it is stressed or becoming diseased are alterations in its surface glycosylation. Current state-of-the-art technologies in glycoanalysis include mass spectrometry, protein microarray formats, techniques in cytometry and more recently, glyco-quantitative polymerase chain reaction (Glyco-qPCR). Techniques for the glycoprofiling of the surfaces of single cells are either limited to the analysis of large cell populations or are unable to handle multiple and/or sequential probing. Here, we report a novel approach of single live cell glycoprofiling enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform for performing capture and retention of cells, along with shear-free reagent loading and washing. The significant technical improvement on state-of-the-art is the demonstration of consecutive, spatio-temporally profiling of glycans on a single cell by sequential elution of the previous lectin probe using their corresponding free sugar. We have qualitatively analysed glycan density on the surface of individual cells. This has allowed us to qualitatively co-localise the observed glycans. This approach enables exhaustive glycoprofiling and glycan mapping on the surface of individual live cells with multiple lectins. The possibility of sequentially profiling glycans on cells will be a powerful new tool to add to current glycoanalytical techniques. The LiaT platform will enable cell biologists to perform many high sensitivity assays and also will also make a significant impact on biomarker research.

A new visible-light-excitable ICT-CHEF-mediated fluorescence 'turn-on' probe for the selective detection of Cd(2+) in a mixed aqueous system with live-cell imaging.

PubMed

Goswami, Shyamaprosad; Aich, Krishnendu; Das, Sangita; Das Mukhopadhyay, Chitrangada; Sarkar, Deblina; Mondal, Tapan Kumar

2015-03-28

A new quinoline based sensor was developed and applied for the selective detection of Cd(2+) both in vitro and in vivo. The designed probe displays a straightforward approach for the selective detection of Cd(2+) with a prominent fluorescence enhancement along with a large red shift (∼38 nm), which may be because of the CHEF (chelation-enhanced fluorescence) and ICT (internal charge transfer) processes after interaction with Cd(2+). The interference from other biologically important competing metal ions, particularly Zn(2+), has not been observed. The visible-light excitability of the probe merits in the viewpoint of its biological application. The probe enables the detection of intracellular Cd(2+) with non-cytotoxic effects, which was demonstrated with the live RAW cells. The experimentally observed change in the structure and electronic properties of the sensor after the addition of Cd(2+) were modelled by the density functional theory (DFT) and time-dependent density functional theory (TDDFT) computational calculations, respectively. Moreover, the test strip experiment with this sensor exhibits both absorption and fluorescence color changes when exposed to Cd(2+) in a mixed aqueous solution, which also makes the probe more useful. The minimum limit of detection of Cd(2+) by the probe was in the range of 9.9 × 10(-8) M level.

Low bone mineral density among HIV-infected patients in Brazil

PubMed Central

Chaba, Daniela Cardeal da Silva; Soares, Lisméia R.; Pereira, Rosa M. R.; Rutherford, George W.; Assone, Tatiane; Takayama, Liliam; Fonseca, Luiz A. M.; Duarte, Alberto J. S.; Casseb, Jorge

2017-01-01

ABSTRACT Decrease in bone mineral density (BMD) has been a complication among people living with HIV/AIDS. To investigate the prevalence of osteopenia/osteoporosis among HIV-infected people living in São Paulo city, we studied 108 HIV-infected patients (79 men and 29 women). We extracted data from patients’ medical records and BMD was measured by dual-energy X-ray absorptiometry (DXA). Median age of participants was 42 years (interquartile range [IQR] 36-48 years), and the median time since HIV diagnosis was 4.01 years (IQR 2-11 years). Patients had acquired HIV primarily by the sexual route (men who have sex with men 44%, heterosexual 49%). Median age, duration of HIV infection, duration of ART and CD4 nadir were similar for men and women. Plasma viral load was undetectable for 53 patients (49%). Median CD4 T cell count was 399 cells/µL (IQR 247 - 568). Twenty five patients (23%) had LBMD, and there was no statistically significant difference between men and women (<-1). The associated risk factors for LBMD were older age (≥ 50 years old) and smoking with a RR of 3.87 and 2.80, respectively. Thus, despite the lack of statistically significant relationship between the use of ART and LBMD or between duration of ART and LBMD, these factors should be addressed in larger studies. PMID:29267597

Fast live cell imaging at nanometer scale using annihilating filter-based low-rank Hankel matrix approach

NASA Astrophysics Data System (ADS)

Min, Junhong; Carlini, Lina; Unser, Michael; Manley, Suliana; Ye, Jong Chul

2015-09-01

Localization microscopy such as STORM/PALM can achieve a nanometer scale spatial resolution by iteratively localizing fluorescence molecules. It was shown that imaging of densely activated molecules can accelerate temporal resolution which was considered as major limitation of localization microscopy. However, this higher density imaging needs to incorporate advanced localization algorithms to deal with overlapping point spread functions (PSFs). In order to address this technical challenges, previously we developed a localization algorithm called FALCON1, 2 using a quasi-continuous localization model with sparsity prior on image space. It was demonstrated in both 2D/3D live cell imaging. However, it has several disadvantages to be further improved. Here, we proposed a new localization algorithm using annihilating filter-based low rank Hankel structured matrix approach (ALOHA). According to ALOHA principle, sparsity in image domain implies the existence of rank-deficient Hankel structured matrix in Fourier space. Thanks to this fundamental duality, our new algorithm can perform data-adaptive PSF estimation and deconvolution of Fourier spectrum, followed by truly grid-free localization using spectral estimation technique. Furthermore, all these optimizations are conducted on Fourier space only. We validated the performance of the new method with numerical experiments and live cell imaging experiment. The results confirmed that it has the higher localization performances in both experiments in terms of accuracy and detection rate.

Flocking Transition in Confluent Tissues

NASA Astrophysics Data System (ADS)

Paoluzzi, Matteo; Giavazzi, Fabio; Macchi, Marta; Scita, Giorgio; Cerbino, Roberto; Manning, Lisa; Marchetti, Cristina

The emerging of collective migration in biological tissues plays a pivotal role in embryonic morphogenesis, wound healing and cancer invasion. While many aspects of single cell movements are well established, the mechanisms leading to coherent displacements of cohesive cell groups are still poorly understood. Some of us recently proposed a Self-Propelled Voronoi (SPV) model of dense tissues that combines self-propelled particle models and vertex models of confluent cell layers and exhibits a liquid-solid transition as a function of cell shape and cell motility. We now examine the role of cell polarization on collective cell dynamics by introducing an orientation mechanism that aligns cell polarization with local cell motility. The model predicts a density-independent flocking transition tuned by the strength of the aligning interaction, with both solid and liquid flocking states existing in different regions of parameter space. MP and MCM were supported by the Simons Foundation Targeted Grant in the Mathematical Modeling of Living Systems Number: 342354 and by the Syracuse Soft Matter Program.

Hydroxyapatite electrodeposition on anodized titanium nanotubes for orthopedic applications

NASA Astrophysics Data System (ADS)

Parcharoen, Yardnapar; Kajitvichyanukul, Puangrat; Sirivisoot, Sirinrath; Termsuksawad, Preecha

2014-08-01

Nanotubes modification for orthopedic implants has shown interesting biological performances (such as improving cell adhesion, cell differentiation, and enhancing osseointegration). The purpose of this study is to investigate effect of titanium dioxide (TiO2) nanotube feature on performance of hydroxyapatite-coated titanium (Ti) bone implants. TiO2 nanotubes were prepared by anodization using ammonium fluoride electrolyte (NH4F) with and without modifiers (PEG400 and Glycerol) at various potential forms, and times. After anodization, the nanotubes were subsequently annealed. TiO2 nanotubes were characterized by scanning electron microscope and X-ray diffractometer. The amorphous to anatase transformation due to annealing was observed. Smooth and highly organized TiO2 nanotubes were found when high viscous electrolyte, NH4F in glycerol, was used. Negative voltage (-4 V) during anodization was confirmed to increase nanotube thickness. Length of the TiO2 nanotubes was significantly increased by times. The TiO2 nanotube was electrodeposited with hydroxyapatite (HA) and its adhesion was estimated by adhesive tape test. The result showed that nanotubes with the tube length of 560 nm showed excellent adhesion. The coated HA were tested for biological test by live/dead cell straining. HA coated on TiO2 nanotubes showed higher cells density, higher live cells, and more spreading of MC3T3-E1 cells than that growing on titanium plate surface.

Macro to Nano: A Simple Method for Transporting Cultured Cells from Milliliter Scale to Nanoliter Scale

PubMed Central

Seale, Kevin T.; Faley, Shannon L.; Chamberlain, Jeff; Wikswo, John P.

2013-01-01

Microfluidic devices are well suited for the study of metabolism and paracrine and autocrine signaling because they allow steady or intermittent perfusion of biological cells at cell densities that approach those in living tissue. They also enable the study of small populations of rare cells. However, it can be difficult to introduce the cells into a microfluidic device to achieve and control such densities without damaging or clumping the cells. We describe simple procedures that address the problem of efficient introduction of cells and cell culture media into microfluidic devices using small bore polyetheretherketone (PEEK) tubing and Hamilton gastight syringes. Suspension or adherent cells grown in cell culture flasks are centrifuged and extracted directly from the centrifuge pellet into the end of the PEEK tubing by aspiration. The tube end is then coupled to pre-punched channels in the polydimethylsiloxane (PDMS) microfluidic device by friction fitting. Controlled depression of the syringe plunger expels the cells into the microfluidic device only seconds following aspiration. The gastight syringes and PEEK tubing with PEEK fittings provide a noncompliant source of pressure and suction with a rapid response time that is well suited for short-term intra-microfluidic cellular studies. The benefits of this method are its simplicity, modest expense, the short preparation time required for loading appropriate numbers of cells, and the applicability of the technique to small quantities of rare or expensive cells. This should in turn lead to new applications of microfludic devices to biology and medicine. PMID:20511682

Automated Photoreceptor Cell Identification on Nonconfocal Adaptive Optics Images Using Multiscale Circular Voting

PubMed Central

Liu, Jianfei; Jung, HaeWon; Dubra, Alfredo; Tam, Johnny

2017-01-01

Purpose Adaptive optics scanning light ophthalmoscopy (AOSLO) has enabled quantification of the photoreceptor mosaic in the living human eye using metrics such as cell density and average spacing. These rely on the identification of individual cells. Here, we demonstrate a novel approach for computer-aided identification of cone photoreceptors on nonconfocal split detection AOSLO images. Methods Algorithms for identification of cone photoreceptors were developed, based on multiscale circular voting (MSCV) in combination with a priori knowledge that split detection images resemble Nomarski differential interference contrast images, in which dark and bright regions are present on the two sides of each cell. The proposed algorithm locates dark and bright region pairs, iteratively refining the identification across multiple scales. Identification accuracy was assessed in data from 10 subjects by comparing automated identifications with manual labeling, followed by computation of density and spacing metrics for comparison to histology and published data. Results There was good agreement between manual and automated cone identifications with overall recall, precision, and F1 score of 92.9%, 90.8%, and 91.8%, respectively. On average, computed density and spacing values using automated identification were within 10.7% and 11.2% of the expected histology values across eccentricities ranging from 0.5 to 6.2 mm. There was no statistically significant difference between MSCV-based and histology-based density measurements (P = 0.96, Kolmogorov-Smirnov 2-sample test). Conclusions MSCV can accurately detect cone photoreceptors on split detection images across a range of eccentricities, enabling quick, objective estimation of photoreceptor mosaic metrics, which will be important for future clinical trials utilizing adaptive optics. PMID:28873173

Tracking quasi-stationary flow of weak fluorescent signals by adaptive multi-frame correlation.

PubMed

Ji, L; Danuser, G

2005-12-01

We have developed a novel cross-correlation technique to probe quasi-stationary flow of fluorescent signals in live cells at a spatial resolution that is close to single particle tracking. By correlating image blocks between pairs of consecutive frames and integrating their correlation scores over multiple frame pairs, uncertainty in identifying a globally significant maximum in the correlation score function has been greatly reduced as compared with conventional correlation-based tracking using the signal of only two consecutive frames. This approach proves robust and very effective in analysing images with a weak, noise-perturbed signal contrast where texture characteristics cannot be matched between only a pair of frames. It can also be applied to images that lack prominent features that could be utilized for particle tracking or feature-based template matching. Furthermore, owing to the integration of correlation scores over multiple frames, the method can handle signals with substantial frame-to-frame intensity variation where conventional correlation-based tracking fails. We tested the performance of the method by tracking polymer flow in actin and microtubule cytoskeleton structures labelled at various fluorophore densities providing imagery with a broad range of signal modulation and noise. In applications to fluorescent speckle microscopy (FSM), where the fluorophore density is sufficiently low to reveal patterns of discrete fluorescent marks referred to as speckles, we combined the multi-frame correlation approach proposed above with particle tracking. This hybrid approach allowed us to follow single speckles robustly in areas of high speckle density and fast flow, where previously published FSM analysis methods were unsuccessful. Thus, we can now probe cytoskeleton polymer dynamics in living cells at an entirely new level of complexity and with unprecedented detail.

Laparoscopic ovarian biopsy pick-up method for goats.

PubMed

Brandão, Fabiana A S; Alves, Benner G; Alves, Kele A; Souza, Samara S; Silva, Yago P; Freitas, Vicente J F; Teixeira, Dárcio I A; Gastal, Eduardo L

2018-02-01

Biopsy pick-up (BPU) has been considered a safe method to harvest ovarian fragments from live animals. However, no studies have been reported on the use of BPU to collect in vivo ovarian tissue in goats. The goals of this study were: (i) to test different biopsy needle sizes to collect ovarian tissue in situ using the BPU method (Experiment 1), and (ii) to study ovarian tissue features such as preantral follicle density, morphology, class distribution, and stromal cell density in ovarian fragments obtained in vivo through a laparoscopic BPU method (Experiment 2). In Experiment 1, goat ovaries (n = 20) were collected in a slaughterhouse and subjected to in situ BPU. Three needles (16, 18, and 20G) were tested. In Experiment 2, the most efficient biopsy needle from Experiment 1 was used to perform laparoscopic BPU in goats (n = 8). In Experiment 1, the recovery rate was greater (P < 0.05; range 50-62%) with 16G and 18G needles than the 20G (17%) needle. The mean weight of ovarian fragments collected by the 16G needle was greater (P < 0.05) than the 18G and the 20G needle. In Experiment 2, 62 biopsy attempts were performed and 52 ovarian fragments were collected (90% success rate). Overall, 2054 preantral follicles were recorded in 5882 histological sections analyzed. Mean preantral follicular density was 28.4 ± 1.3 follicles per cm 2 . The follicular density differed (P < 0.05) among animals and ovarian fragments within the same animal. The mean stromal cell density in the ovarian fragments was 37.1 ± 0.5 cells per 2500 μm 2 , and differed (P < 0.05) among animals. Moreover, preantral follicle density and stromal cell density were associated (P < 0.001). The percentage of morphologically normal follicles was 70.1 ± 1.2, and differed (P < 0.05) among animals. The majority (79%) of the morphologically normal follicles was classified as primordial follicles, and differed (P < 0.05) among animals and between ovaries. In summary, a laparoscopic BPU method has been developed to harvest ovarian tissue in vivo with a satisfactory success rate in goats. Furthermore, this study described for the first time that goat ovarian biopsy fragments have a high heterogeneity in follicular density, morphology, class distribution, and stromal cell density. Copyright © 2017 Elsevier Inc. All rights reserved.

Arrays of microscopic organic LEDs for high-resolution optogenetics

PubMed Central

Steude, Anja; Witts, Emily C.; Miles, Gareth B.; Gather, Malte C.

2016-01-01

Optogenetics is a paradigm-changing new method to study and manipulate the behavior of cells with light. Following major advances of the used genetic constructs over the last decade, the light sources required for optogenetic control are now receiving increased attention. We report a novel optogenetic illumination platform based on high-density arrays of microscopic organic light-emitting diodes (OLEDs). Because of the small dimensions of each array element (6 × 9 μm2) and the use of ultrathin device encapsulation, these arrays enable illumination of cells with unprecedented spatiotemporal resolution. We show that adherent eukaryotic cells readily proliferate on these arrays, and we demonstrate specific light-induced control of the ionic current across the membrane of individual live cells expressing different optogenetic constructs. Our work paves the way for the use of OLEDs for cell-specific optogenetic control in cultured neuronal networks and for acute brain slices, or as implants in vivo. PMID:27386540

Cell phone radiations affect early growth of Vigna radiata (mung bean) through biochemical alterations.

PubMed

Sharma, Ved Parkash; Singh, Harminder Pal; Batish, Daizy Rani; Kohli, Ravinder Kumar

2010-01-01

The indiscriminate use of wireless technologies, particularly of cell phones, has increased the health risks among living organisms including plants. We investigated the impact of cell phone electromagentic field (EMF) radiations (power density, 8.55 microW cm(-2)) on germination, early growth, proteins and carbohydrate contents, and activities of some enzymes in Vigna radiata. Cell phone EMF radiations significantly reduced the seedling length and dry weight of V radiata after exposure for 0.5, 1, 2, and 4 h. Furthermore, the contents of proteins and carbohydrates were reduced in EMF-exposed plants. However, the activities of proteases, alpha-amylases, beta-amylases, polyphenol oxidases, and peroxidases were enhanced in EMF-exposed radicles indicating their role in providing protection against EMF-induced stress. The study concludes that cell phone EMFs impair early growth of V radiata seedlings by inducing biochemical changes.

Population density and youth antisocial behavior.

PubMed

Harden, K Paige; D'Onofrio, Brian M; Van Hulle, Carol; Turkheimer, Eric; Rodgers, Joseph L; Waldman, Irwin D; Lahey, Benjamin B

2009-08-01

Theoretical models concerning how neighborhood contexts adversely influence juvenile antisocial behavior frequently focus on urban neighborhoods; however, previous studies comparing urban and rural areas on the prevalence of youth antisocial behavior have yielded mixed results. The current study uses longitudinal data on the offspring of a nationally representative sample of mothers (N = 4,886) in the US. There was no relation between density and mother-reported child conduct problems across ages 4-13 years, but youth living in areas of greater population density exhibited more youth self-reported delinquency across 10-17 years. Families often moved to counties with greater or lesser population density, but longitudinal analyses treating population density as a time-varying covariate did not support the hypothesis that living in densely populated counties influenced youth delinquency. Rather, the association between population density and delinquency appears to be due to unmeasured selection variables that differ between families who live in more or less densely populated counties.

Effect of TheraCyte-encapsulated parathyroid cells on lumbar fusion in a rat model.

PubMed

Chen, Sung-Hsiung; Huang, Shun-Chen; Lui, Chun-Chung; Lin, Tzu-Ping; Chou, Fong-Fu; Ko, Jih-Yang

2012-09-01

Implantation of TheraCyte 4 × 10(6) live parathyroid cells can increase the bone marrow density of the spine of ovariectomized rats. There has been no published study examining the effect of such implantation on spinal fusion outcomes. The purpose of this study was to examine the effect of TheraCyte-encapsulated parathyroid cells on posterolateral lumbar fusions in a rat model. Forty Sprague-Dawley rats underwent single-level, intertransverse process spinal fusions using iliac crest autograft. The rats were randomly assigned to two groups: Group 1 rats received sham operations on their necks (control; N = 20); Group 2 rats were implanted with TheraCyte-encapsulated 4 × 10(6) live parathyroid cells into the subcutis of their necks (TheraCyte; N = 20). Six weeks after surgery the rats were killed. Fusion was assessed by inspection, manual palpation, radiography, and histology. Blood was drawn to measure the serum levels of calcium, phosphorus, and intact parathyroid hormone (iPTH). Based on manual palpation, the control group had a fusion rate of 33 % (6/18) and the TheraCyte group had a fusion rate of 72 % (13/18) (P = 0.044). Histology confirmed the manual palpation results. Serum iPTH levels were significantly higher in the TheraCyte group compared with the control group (P < 0.05); neither serum calcium nor phosphorus levels were significantly different between the two groups. This pilot animal study revealed that there were more fusions in rats that received TheraCyte-encapsulated 4 × 10(6) live parathyroid cells than in control rats without significant change in serum calcium or phosphorus concentrations. As with any animal study, the results may not extrapolate to a higher species. Further studies are needed to determine if these effects are clinically significant.

Origin of Polar Order in Dense Suspensions of Phototactic Micro-Swimmers

PubMed Central

Furlan, Silvano; Comparini, Diego; Ciszak, Marzena; Beccai, Lucia; Mancuso, Stefano; Mazzolai, Barbara

2012-01-01

A main question for the study of collective motion in living organisms is the origin of orientational polar order, i.e., how organisms align and what are the benefits of such collective behaviour. In the case of micro-organisms swimming at a low Reynolds number, steric repulsion and long-range hydrodynamic interactions are not sufficient to explain a homogeneous polar order state in which the direction of motion is aligned. An external symmetry-breaking guiding field such as a mechanism of taxis appears necessary to understand this phonemonon. We have investigated the onset of polar order in the velocity field induced by phototaxis in a suspension of a motile micro-organism, the algae Chlamydomonas reinhardtii, for density values above the limit provided by the hydrodynamic approximation of a force dipole model. We show that polar order originates from a combination of both the external guiding field intensity and the population density. In particular, we show evidence for a linear dependence of a phototactic guiding field on cell density to determine the polar order for dense suspensions and demonstrate the existence of a density threshold for the origin of polar order. This threshold represents the density value below which cells undergoing phototaxis are not able to maintain a homogeneous polar order state and marks the transition to ordered collective motion. Such a transition is driven by a noise dominated phototactic reorientation where the noise is modelled as a normal distribution with a variance that is inversely proportional to the guiding field strength. Finally, we discuss the role of density in dense suspensions of phototactic micro-swimmers. PMID:22723904

Hybrid Tissue Engineering Scaffolds by Combination of Three-Dimensional Printing and Cell Photoencapsulation.

PubMed

Markovic, Marica; Van Hoorick, Jasper; Hölzl, Katja; Tromayer, Maximilian; Gruber, Peter; Nürnberger, Sylvia; Dubruel, Peter; Van Vlierberghe, Sandra; Liska, Robert; Ovsianikov, Aleksandr

2015-05-01

Three-dimensional (3D) printing offers versatile possibilities for adapting the structural parameters of tissue engineering scaffolds. However, it is also essential to develop procedures allowing efficient cell seeding independent of scaffold geometry and pore size. The aim of this study was to establish a method for seeding the scaffolds using photopolymerizable cell-laden hydrogels. The latter facilitates convenient preparation, and handling of cell suspension, while distributing the hydrogel precursor throughout the pores, before it is cross-linked with light. In addition, encapsulation of living cells within hydrogels can produce constructs with high initial cell loading and intimate cell-matrix contact, similar to that of the natural extra-cellular matrix (ECM). Three dimensional scaffolds were produced from poly(lactic) acid (PLA) by means of fused deposition modeling. A solution of methacrylamide-modified gelatin (Gel-MOD) in cell culture medium containing photoinitiator Li-TPO-L was used as a hydrogel precursor. Being an enzymatically degradable derivative of natural collagen, gelatin-based matrices are biomimetic and potentially support the process of cell-induced remodeling. Preosteoblast cells MC3T3-E1 at a density of 10 × 10 6 cells per 1 mL were used for testing the seeding procedure and cell proliferation studies. Obtained results indicate that produced constructs support cell survival and proliferation over extended duration of our experiment. The established two-step approach for scaffold seeding with the cells is simple, rapid, and is shown to be highly reproducible. Furthermore, it enables precise control of the initial cell density, while yielding their uniform distribution throughout the scaffold. Such hybrid tissue engineering constructs merge the advantages of rigid 3D printed constructs with the soft hydrogel matrix, potentially mimicking the process of ECM remodeling.

Cells Respond to Mechanical Stress by Rapid Disassembly of Caveolae

PubMed Central

Sinha, Bidisha; Köster, Darius; Ruez, Richard; Gonnord, Pauline; Bastiani, Michele; Abankwa, Daniel; Stan, Radu. V.; Butler-Browne, Gillian; Vedie, Benoit; Johannes, Ludger; Morone, Nobuhiro; Parton, Robert G.; Raposo, Graça; Sens, Pierre; Lamaze, Christophe; Nassoy, Pierre

2011-01-01

SUMMARY The precise role of caveolae, the characteristic plasma membrane invaginations present in many cells, still remains debated. The high density of caveolae in cells experiencing mechanical stress led us to investigate their role in membrane-mediated mechanical response. Acute mechanical stress induced by cell osmotic swelling or by uniaxial stretching results in the immediate disappearance of caveolae, which is associated with a reduced caveolin/Cavin1 interaction, and an increase of free caveolins at the plasma membrane. Tether pulling force measurements in live cells and in plasma membrane spheres demonstrate that caveola flattening and disassembly is the primary actin and ATP-independent cell response which buffers membrane tension surges during mechanical stress. Conversely, stress release leads to complete caveola reassembly in an actin and ATP-dependent process. The absence of a functional caveola reservoir in myotubes from muscular dystrophic patients enhanced membrane fragility under mechanical stress. Our findings support a new role for caveolae as a physiological membrane reservoir that allows cells to quickly accommodate sudden and acute mechanical stresses. PMID:21295700

Out-of-equilibrium dynamics in the cytoskeleton of the living cell

NASA Astrophysics Data System (ADS)

Lenormand, Guillaume; Bursac, Predrag; Butler, James P.; Fredberg, Jeffrey J.

2007-10-01

We report here measurements of rheological properties of the human airway smooth muscle cell using forced nanoscale motions of Arg-Gly-Asp RGD-coated microbeads tightly bound to the cytoskeleton. With changes of forcing amplitude, the storage modulus showed small but systematic nonlinearities, especially after treatment with a contractile agonist. In a dose-dependent manner, a large oscillatory shear applied from a few seconds up to 400s caused the cytoskeleton matrix to soften, a behavior comparable to physical rejuvenation observed in certain inert soft materials; the stiffness remained constant for as long as the large oscillatory shear was maintained, but suddenly fell with shear cessation. Stiffness then followed a slow scale-free recovery, a phenomenon comparable to physical aging. However, acetylated low-density lipoprotein acLDL-coated microbeads, which connect mainly to scavenger receptors, did not show similar out-of-equilibrium behaviors. Taken together, these data demonstrate in the cytoskeleton of the living cell behaviors with all the same signatures as that of soft inert condensed systems. This unexpected intersection of condensed matter physics and cytoskeletal biology suggests that trapping, intermittency, and approach to kinetic arrest represent central mesoscale features linking underlying molecular events to integrative cellular functions.

Enrichment of free-living amoebae in biofilms developed at upper water levels in drinking water storage towers: An inter- and intra-seasonal study.

PubMed

Taravaud, Alexandre; Ali, Myriam; Lafosse, Bernard; Nicolas, Valérie; Féliers, Cédric; Thibert, Sylvie; Lévi, Yves; Loiseau, Philippe M; Pomel, Sébastien

2018-08-15

Free-living amoebae (FLA) are ubiquitous organisms present in various natural and artificial environments, such as drinking water storage towers (DWST). Some FLA, such as Acanthamoeba sp., Naegleria fowleri, and Balamuthia mandrillaris, can cause severe infections at ocular or cerebral level in addition to being potential reservoirs of other pathogens. In this work, the abundance and diversity of FLA was evaluated in two sampling campaigns: one performed over five seasons in three DWST at three different levels (surface, middle and bottom) in water and biofilm using microscopy and PCR, and one based on the kinetics analysis in phase contrast and confocal microscopy of biofilm samples collected every two weeks during a 3-month period at the surface and at the bottom of a DWST. In the seasonal study, the FLA were detected in each DWST water in densities of ~20 to 25amoebaeL -1 . A seasonal variation of amoeba distribution was observed in water samples, with maximal densities in summer at ~30amoebaeL -1 and minimal densities in winter at ~16amoebaeL -1 . The FLA belonging to the genus Acanthamoeba were detected in two spring sampling campaigns, suggesting a possible seasonal appearance of this potentially pathogenic amoeba. Interestingly, a 1 log increase of amoebae density was observed in biofilm samples collected at the surface of all DWST compared to the middle and the bottom where FLA were at 0.1-0.2amoebae/cm 2 . In the kinetics study, an increase of amoebae density, total cell density, and biofilm thickness was observed as a function of time at the surface of the DWST, but not at the bottom. To our knowledge, this study describes for the first time a marked higher FLA density in biofilms collected at upper water levels in DWST, constituting a potential source of pathogenic micro-organisms. Copyright © 2018 Elsevier B.V. All rights reserved.

Relationships between the attributes of standing live and dead down trees in forests of the Lake States

Treesearch

Christopher W. Woodall; Linda Nagel

2007-01-01

Refining the understanding of the relationship between a stand's standing live and down dead trees in terms of size, density, and biomass attributes may aid efforts to predict fuel loadings based on standing tree attributes. Therefore, the objective of this study was to compare down dead and standing live tree attributes (size, density, and biomass) for inventory...

ROCK Inhibitor Enhances Adhesion and Wound Healing of Human Corneal Endothelial Cells

PubMed Central

Pipparelli, Aurélien; Arsenijevic, Yvan; Thuret, Gilles; Gain, Philippe

2013-01-01

Maintenance of corneal transparency is crucial for vision and depends mainly on the endothelium, a non-proliferative monolayer of cells covering the inner part of the cornea. When endothelial cell density falls below a critical threshold, the barrier and “pump” functions of the endothelium are compromised which results in corneal oedema and loss of visual acuity. The conventional treatment for such severe disorder is corneal graft. Unfortunately, there is a worldwide shortage of donor corneas, necessitating amelioration of tissue survival and storage after harvesting. Recently it was reported that the ROCK inhibitor Y-27632 promotes adhesion, inhibits apoptosis, increases the number of proliferating monkey corneal endothelial cells in vitro and enhance corneal endothelial wound healing both in vitro and in vivo in animal models. Using organ culture human cornea (N = 34), the effect of ROCK inhibitor was evaluated in vitro and ex vivo. Toxicity, corneal endothelial cell density, cell proliferation, apoptosis, cell morphometry, adhesion and wound healing process were evaluated by live/dead assay standard cell counting method, EdU labelling, Ki67, Caspase3, Zo-1 and Actin immunostaining. We demonstrated for the first time in human corneal endothelial cells ex vivo and in vitro, that ROCK inhibitor did not induce any toxicity effect and did not alter cell viability. ROCK inhibitor treatment did not induce human corneal endothelial cells proliferation. However, ROCK inhibitor significantly enhanced adhesion and wound healing. The present study shows that the selective ROCK inhibitor Y-27632 has no effect on human corneal endothelial cells proliferative capacities, but alters cellular behaviours. It induces changes in cell shape, increases cell adhesion and enhances wound healing ex vivo and in vitro. Its absence of toxicity, as demonstrated herein, is relevant for its use in human therapy. PMID:23626771

Extreme Mechanics of Growing Matter

NASA Astrophysics Data System (ADS)

Kuhl, Ellen

2013-03-01

Growth is a distinguishing feature of all living things. Unlike standard materials, living matter can autonomously respond to alterations in its environment. As a result of a continuous ultrastructural turnover and renewal of cells and extracellular matrix, living matter can undergo extreme changes in composition, size, and shape within the order of months, weeks, or days. While hard matter typically adapts by increasing its density to grow strong, soft matter adapts by increasing its volume to grow large. Here we provide a state-of-the-art review of growing matter, and compare existing mathematical models for growth and remodeling of living systems. Applications are plentiful ranging from plant growth to tumor growth, from asthma in the lungs to restenosis in the vasculature, from plastic to reconstructive surgery, and from skeletal muscle adaptation to heart failure. Using these examples, we discuss current challenges and potential future directions. We hope to initiate critical discussions around the biophysical modeling of growing matter as a powerful tool to better understand biological systems in health and disease. This research has been supported by the NSF CAREER award CMMI 0952021.

Growth dynamic of Naegleria fowleri in a microbial freshwater biofilm.

PubMed

Goudot, Sébastien; Herbelin, Pascaline; Mathieu, Laurence; Soreau, Sylvie; Banas, Sandrine; Jorand, Frédéric

2012-09-01

The presence of pathogenic free-living amoebae (FLA) such as Naegleria fowleri in freshwater environments is a potential public health risk. Although its occurrence in various water sources has been well reported, its presence and associated factors in biofilm remain unknown. In this study, the density of N. fowleri in biofilms spontaneously growing on glass slides fed by raw freshwater were followed at 32 °C and 42 °C for 45 days. The biofilms were collected with their substrata and characterized for their structure, numbered for their bacterial density, thermophilic free-living amoebae, and pathogenic N. fowleri. The cell density of N. fowleri within the biofilms was significantly affected both by the temperature and the nutrient level (bacteria/amoeba ratio). At 32 °C, the density remained constantly low (1-10 N. fowleri/cm(2)) indicating that the amoebae were in a survival state, whereas at 42 °C the density reached 30-900 N. fowleri/cm(2) indicating an active growth phase. The nutrient level, as well, strongly affected the apparent specific growth rate (μ) of N. fowleri in the range of 0.03-0.23 h(-1). At 42 °C a hyperbolic relationship was found between μ and the bacteria/amoeba ratio. A ratio of 10(6) to 10(7) bacteria/amoeba was needed to approach the apparent μ(max) value (0.23 h(-1)). Data analysis also showed that a threshold for the nutrient level of close to 10(4) bacteria/amoeba is needed to detect the growth of N. fowleri in freshwater biofilm. This study emphasizes the important role of the temperature and bacteria as prey to promote not only the growth of N. fowleri, but also its survival. Copyright © 2012 Elsevier Ltd. All rights reserved.

Live to cheat another day: bacterial dormancy facilitates the social exploitation of β-lactamases

PubMed Central

Medaney, Frances; Dimitriu, Tatiana; Ellis, Richard J; Raymond, Ben

2016-01-01

The breakdown of antibiotics by β-lactamases may be cooperative, since resistant cells can detoxify their environment and facilitate the growth of susceptible neighbours. However, previous studies of this phenomenon have used artificial bacterial vectors or engineered bacteria to increase the secretion of β-lactamases from cells. Here, we investigated whether a broad-spectrum β-lactamase gene carried by a naturally occurring plasmid (pCT) is cooperative under a range of conditions. In ordinary batch culture on solid media, there was little or no evidence that resistant bacteria could protect susceptible cells from ampicillin, although resistant colonies could locally detoxify this growth medium. However, when susceptible cells were inoculated at high densities, late-appearing phenotypically susceptible bacteria grew in the vicinity of resistant colonies. We infer that persisters, cells that have survived antibiotics by undergoing a period of dormancy, founded these satellite colonies. The number of persister colonies was positively correlated with the density of resistant colonies and increased as antibiotic concentrations decreased. We argue that detoxification can be cooperative under a limited range of conditions: if the toxins are bacteriostatic rather than bacteridical; or if susceptible cells invade communities after resistant bacteria; or if dormancy allows susceptible cells to avoid bactericides. Resistance and tolerance were previously thought to be independent solutions for surviving antibiotics. Here, we show that these are interacting strategies: the presence of bacteria adopting one solution can have substantial effects on the fitness of their neighbours. PMID:26505830

Sorption and precipitation of Mn2+ by viable and autoclaved Shewanella putrefaciens: Effect of contact time

NASA Astrophysics Data System (ADS)

Chubar, Natalia; Visser, Tom; Avramut, Cristina; de Waard, Helen

2013-01-01

The sorption of Mn(II) by viable and inactivated cells of Shewanella putrefaciens, a non-pathogenic, facultative anaerobic, gram-negative bacterium characterised as a Mn(IV) and Fe(III) reducer, was studied under aerobic conditions, as a function of pH, bacterial density and metal loading. During a short contact time (3-24 h), the adsorptive behaviour of live and dead bacteria toward Mn(II) was sufficiently similar, an observation that was reflected in the studies on adsorption kinetics at various metal loadings, effects of pH, bacteria density, isotherms and drifting of pH during adsorption. Continuing the experiment for an additional 2-30 days demonstrated that the Mn(II) sorption by suspensions of viable and autoclaved cells differed significantly from one another. The sorption to dead cells was characterised by a rapid equilibration and was described by an isotherm. In contrast, the sorption (uptake) to live bacteria exhibited a complex time-dependent uptake. This uptake began as adsorption and ion exchange processes followed by bioprecipitation, and it was accompanied by the formation of polymeric sugars (EPS) and the release of dissolved organic substances. FTIR, EXAFS/XANES and XPS demonstrated that manganese(II) phosphate was the main precipitate formed in 125 ml batches, which is the first evidence of the ability of microbes to synthesise manganese phosphates. XPS and XANES spectra did not detect Mn(II) oxidation. Although the release of protein-like compounds by the viable bacteria increased in the presence of Mn2+ (and, by contrast, the release of carbohydrates did not change), electrochemical analyses did not indicate any aqueous complexation of Mn(II) by the organic ligands.

Unliganded fibroblast growth factor receptor 1 forms density-independent dimers.

PubMed

Comps-Agrar, Laëtitia; Dunshee, Diana Ronai; Eaton, Dan L; Sonoda, Junichiro

2015-10-02

Fibroblast growth factors receptors (FGFRs) are thought to initiate intracellular signaling cascades upon ligand-induced dimerization of the extracellular domain. Although the existence of unliganded FGFR1 dimers on the surface of living cells has been proposed, this notion remains rather controversial. Here, we employed time-resolved Förster resonance energy transfer combined with SNAP- and ACP-tag labeling in COS7 cells to monitor dimerization of full-length FGFR1 at the cell-surface with or without the coreceptor βKlotho. Using this approach we observed homodimerization of unliganded FGFR1 that is independent of its surface density. The homo-interaction signal observed for FGFR1 was indeed as robust as that obtained for epidermal growth factor receptor (EGFR) and was further increased by the addition of activating ligands or pathogenic mutations. Mutational analysis indicated that the kinase and the transmembrane domains, rather than the extracellular domain, mediate the ligand-independent FGFR1 dimerization. In addition, we observed a formation of a higher order ligand-independent complex by the c-spliced isoform of FGFR1 and βKlotho. Collectively, our approach provides novel insights into the assembly and dynamics of the full-length FGFRs on the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Interplay between Antibiotic Efficacy and Drug-Induced Lysis Underlies Enhanced Biofilm Formation at Subinhibitory Drug Concentrations

PubMed Central

Yu, Wen; Hallinen, Kelsey M.

2017-01-01

ABSTRACT Subinhibitory concentrations of antibiotics have been shown to enhance biofilm formation in multiple bacterial species. While antibiotic exposure has been associated with modulated expression of many biofilm-related genes, the mechanisms of drug-induced biofilm formation remain a focus of ongoing research efforts and may vary significantly across species. In this work, we investigate antibiotic-induced biofilm formation in Enterococcus faecalis, a leading cause of nosocomial infections. We show that biofilm formation is enhanced by subinhibitory concentrations of cell wall synthesis inhibitors but not by inhibitors of protein, DNA, folic acid, or RNA synthesis. Furthermore, enhanced biofilm is associated with increased cell lysis, increases in extracellular DNA (eDNA) levels, and increases in the density of living cells in the biofilm. In addition, we observe similar enhancement of biofilm formation when cells are treated with nonantibiotic surfactants that induce cell lysis. These findings suggest that antibiotic-induced biofilm formation is governed by a trade-off between drug toxicity and the beneficial effects of cell lysis. To understand this trade-off, we developed a simple mathematical model that predicts changes in antibiotic-induced biofilm formation due to external perturbations, and we verified these predictions experimentally. Specifically, we demonstrate that perturbations that reduce eDNA (DNase treatment) or decrease the number of living cells in the planktonic phase (a second antibiotic) decrease biofilm induction, while chemical inhibitors of cell lysis increase relative biofilm induction and shift the peak to higher antibiotic concentrations. Overall, our results offer experimental evidence linking cell wall synthesis inhibitors, cell lysis, increased eDNA levels, and biofilm formation in E. faecalis while also providing a predictive quantitative model that sheds light on the interplay between cell lysis and antibiotic efficacy in developing biofilms. PMID:29061740

Ethnic density as a buffer for psychotic experiences: findings from a national survey (EMPIRIC).

PubMed

Das-Munshi, Jayati; Bécares, Laia; Boydell, Jane E; Dewey, Michael E; Morgan, Craig; Stansfeld, Stephen A; Prince, Martin J

2012-10-01

Aetiological mechanisms underlying ethnic density associations with psychosis remain unclear. To assess potential mechanisms underlying the observation that minority ethnic groups experience an increased risk of psychosis when living in neighbourhoods of lower own-group density. Multilevel analysis of nationally representative community-level data (from the Ethnic Minorities Psychiatric Illness Rates in the Community survey), which included the main minority ethnic groups living in England, and a White British group. Structured instruments assessed discrimination, chronic strains and social support. The Psychosis Screening Questionnaire ascertained psychotic experiences. For every ten percentage point reduction in own-group density, the relative odds of reporting psychotic experiences increased 1.07 times (95% CI 1.01-1.14, P = 0.03 (trend)) for the total minority ethnic sample. In general, people living in areas of lower own-group density experienced greater social adversity that was in turn associated with reporting psychotic experiences. People resident in neighbourhoods of higher own-group density experience 'buffering' effects from the social risk factors for psychosis.

GABA Signaling Promotes Synapse Elimination and Axon Pruning in Developing Cortical Inhibitory Interneurons

PubMed Central

Wu, Xiaoyun; Fu, Yu; Knott, Graham; Lu, Jiangteng; Di Cristo, Graziella

2012-01-01

Accumulating evidence indicates that GABA acts beyond inhibitory synaptic transmission and regulates the development of inhibitory synapses in the vertebrate brain, but the underlying cellular mechanism is not well understood. We have combined live imaging of cortical GABAergic axons across time scales from minutes to days with single-cell genetic manipulation of GABA release to examine its role in distinct steps of inhibitory synapse formation in the mouse neocortex. We have shown previously, by genetic knockdown of GABA synthesis in developing interneurons, that GABA signaling promotes the maturation of inhibitory synapses and axons. Here we found that a complete blockade of GABA release in basket interneurons resulted in an opposite effect, a cell-autonomous increase in axon and bouton density with apparently normal synapse structures. These results not only demonstrate that GABA is unnecessary for synapse formation per se but also uncover a novel facet of GABA in regulating synapse elimination and axon pruning. Live imaging revealed that developing GABAergic axons form a large number of transient boutons, but only a subset was stabilized. Release blockade led to significantly increased bouton stability and filopodia density, increased axon branch extension, and decreased branch retraction. Our results suggest that a major component of GABA function in synapse development is transmission-mediated elimination of subsets of nascent contacts. Therefore, GABA may regulate activity-dependent inhibitory synapse formation by coordinately eliminating certain nascent contacts while promoting the maturation of other nascent synapses. PMID:22219294

Spatiotemporal mapping of diffusion dynamics and organization in plasma membranes

NASA Astrophysics Data System (ADS)

Bag, Nirmalya; Ng, Xue Wen; Sankaran, Jagadish; Wohland, Thorsten

2016-09-01

Imaging fluorescence correlation spectroscopy (FCS) and the related FCS diffusion law have been applied in recent years to investigate the diffusion modes of lipids and proteins in membranes. These efforts have provided new insights into the membrane structure below the optical diffraction limit, new information on the existence of lipid domains, and on the influence of the cytoskeleton on membrane dynamics. However, there has been no systematic study to evaluate how domain size, domain density, and the probe partition coefficient affect the resulting imaging FCS diffusion law parameters. Here, we characterize the effects of these factors on the FCS diffusion law through simulations and experiments on lipid bilayers and live cells. By segmenting images into smaller 7  ×  7 pixel areas, we can evaluate the FCS diffusion law on areas smaller than 2 µm and thus provide detailed maps of information on the membrane structure and heterogeneity at this length scale. We support and extend this analysis by deriving a mathematical expression to calculate the mean squared displacement (MSDACF) from the autocorrelation function of imaging FCS, and demonstrate that the MSDACF plots depend on the existence of nanoscopic domains. Based on the results, we derive limits for the detection of domains depending on their size, density, and relative viscosity in comparison to the surroundings. Finally, we apply these measurements to bilayers and live cells using imaging total internal reflection FCS and single plane illumination microscopy FCS.

Interferometric and nonlinear-optical spectral-imaging techniques for outer space and live cells

NASA Astrophysics Data System (ADS)

Itoh, Kazuyoshi

2015-12-01

Multidimensional signals such as the spectral images allow us to have deeper insights into the natures of objects. In this paper the spectral imaging techniques that are based on optical interferometry and nonlinear optics are presented. The interferometric imaging technique is based on the unified theory of Van Cittert-Zernike and Wiener-Khintchine theorems and allows us to retrieve a spectral image of an object in the far zone from the 3D spatial coherence function. The retrieval principle is explained using a very simple object. The promising applications to space interferometers for astronomy that are currently in progress will also be briefly touched on. An interesting extension of interferometric spectral imaging is a 3D and spectral imaging technique that records 4D information of objects where the 3D and spectral information is retrieved from the cross-spectral density function of optical field. The 3D imaging is realized via the numerical inverse propagation of the cross-spectral density. A few techniques suggested recently are introduced. The nonlinear optical technique that utilizes stimulated Raman scattering (SRS) for spectral imaging of biomedical targets is presented lastly. The strong signals of SRS permit us to get vibrational information of molecules in the live cell or tissue in real time. The vibrational information of unstained or unlabeled molecules is crucial especially for medical applications. The 3D information due to the optical nonlinearity is also the attractive feature of SRS spectral microscopy.

Ultraporous, Compressible, Wettable Polylactide/Polycaprolactone Sponges for Tissue Engineering.

PubMed

Mader, Michael; Jérôme, Valérie; Freitag, Ruth; Agarwal, Seema; Greiner, Andreas

2018-05-14

Ultraporous, degradable sponges made of either polylactide or of blends of polylactide/poly(ε-caprolactone) are prepared by freeze-drying of dispersions of short electrospun fibers and subsequent thermal annealing. The sponges feature ultrahigh porosity (99.6%), a hierarchical cellular structure, and high reversible compressibility with fast recovery from deformation in the dry as well as in the wet state. The sponge properties depend on the fiber dispersion concentration and the annealing temperature. Sponge characteristics like fiber density (2.5-20 mg/cm 3 ), size, shape, crystallinity, mechanical strength, wetability, and structural integrity are user adjustable. Cell culture experiments were successfully performed with Jurkat cells with Confocal Laser Scanning Microscopy and MTT staining showing rapid cell proliferation. Live/Dead staining demonstrated high viability of the seeded cells. The sponge characteristics and modifications investigated and presented here reveal that these sponges are highly promising for tissue engineering applications.

Joint level-set and spatio-temporal motion detection for cell segmentation.

PubMed

Boukari, Fatima; Makrogiannis, Sokratis

2016-08-10

Cell segmentation is a critical step for quantification and monitoring of cell cycle progression, cell migration, and growth control to investigate cellular immune response, embryonic development, tumorigenesis, and drug effects on live cells in time-lapse microscopy images. In this study, we propose a joint spatio-temporal diffusion and region-based level-set optimization approach for moving cell segmentation. Moving regions are initially detected in each set of three consecutive sequence images by numerically solving a system of coupled spatio-temporal partial differential equations. In order to standardize intensities of each frame, we apply a histogram transformation approach to match the pixel intensities of each processed frame with an intensity distribution model learned from all frames of the sequence during the training stage. After the spatio-temporal diffusion stage is completed, we compute the edge map by nonparametric density estimation using Parzen kernels. This process is followed by watershed-based segmentation and moving cell detection. We use this result as an initial level-set function to evolve the cell boundaries, refine the delineation, and optimize the final segmentation result. We applied this method to several datasets of fluorescence microscopy images with varying levels of difficulty with respect to cell density, resolution, contrast, and signal-to-noise ratio. We compared the results with those produced by Chan and Vese segmentation, a temporally linked level-set technique, and nonlinear diffusion-based segmentation. We validated all segmentation techniques against reference masks provided by the international Cell Tracking Challenge consortium. The proposed approach delineated cells with an average Dice similarity coefficient of 89 % over a variety of simulated and real fluorescent image sequences. It yielded average improvements of 11 % in segmentation accuracy compared to both strictly spatial and temporally linked Chan-Vese techniques, and 4 % compared to the nonlinear spatio-temporal diffusion method. Despite the wide variation in cell shape, density, mitotic events, and image quality among the datasets, our proposed method produced promising segmentation results. These results indicate the efficiency and robustness of this method especially for mitotic events and low SNR imaging, enabling the application of subsequent quantification tasks.

Development of biologically modified anodes for energy harvesting using microbial fuel cells

NASA Astrophysics Data System (ADS)

Sumner, James J.; Ganguli, Rahul; Chmelka, Brad

2012-06-01

Biological fuel cells hold promise as an alternative energy source to batteries for unattended ground sensor applications due to the fact that they can be extremely long lived. This lifetime can be extended over batteries by scavenging fuel from the deployed environment. Microbial fuel cells (MFC) are one class of such sources that produce usable energy from small organic compounds (i.e. sugars, alcohols, organic acids, and biopolymers) which can be easily containerized or scavenged from the environment. The use of microorganisms as the anodic catalysts is what makes these systems unique from other biofuel cell designs. One of the main drawbacks of engineering a sensor system powered by an MFC is that power densities and current flux are extremely low in currently reported systems. The power density is limited by the mass transfer of the fuel source to the catalyst, the metabolism of the microbial catalysts and the electron transfer from the organism to the anode. This presentation will focus on the development of a new style of microbially-modified anodes which will increase power density to a level where a practical power source can be engineered. This is being achieved by developing a three dimensional matrix as an artificial, conductive biofilm. These artificial biofilms will allow the capture of a consortium of microbes designed for efficient metabolism of the available fuel source. Also it will keep the microbes close to the electrode allowing ready access by fuel and providing a low resistance passage of the liberated electrons from fuel oxidation.

Associations of Alcohol Availability and Neighborhood Socioeconomic Characteristics With Drinking: Cross-Sectional Results From the Multi-Ethnic Study of Atherosclerosis (MESA).

PubMed

Brenner, Allison B; Diez Roux, Ana V; Barrientos-Gutierrez, Tonatiuh; Borrell, Luisa N

2015-01-01

Living in neighborhoods with a high density of alcohol outlets and socioeconomic disadvantage may increase residents' alcohol use. Few researchers have studied these exposures in relation to multiple types of alcohol use, including beverage-specific consumption, and how individual demographic factors influence these relationships. To examine the relationships of alcohol outlet density and neighborhood disadvantage with alcohol consumption, and to investigate differences in these associations by race/ethnicity and income. Using cross-sectional data (N = 5,873) from the Multi-ethnic Study of Atherosclerosis in 2002, we examine associations of residential alcohol outlet density and neighborhood socioeconomic disadvantage with current, total weekly and heaviest daily alcohol use in gender-specific regression models, as well as moderation by race/ethnicity and income. Drinking men living near high densities of alcohol outlets had 23%-29% more weekly alcohol use than men in low density areas. Among women who drank, those living near a moderate density of alcohol outlets consumed approximately 40% less liquor each week than those in low density areas, but higher outlet densities were associated with more wine consumption (35%-49%). Living in highly or moderately disadvantaged neighborhoods was associated with a lower probability of being a current drinker, but with higher rates of weekly beer consumption. Income moderated the relationship between neighborhood context and weekly alcohol use. Neighborhood disadvantage and alcohol outlet density may influence alcohol use with effects varying by gender and income. Results from this research may help target interventions and policy to groups most at risk for greater weekly consumption.

Glass-like dynamics in confined and congested ant traffic.

PubMed

Gravish, Nick; Gold, Gregory; Zangwill, Andrew; Goodisman, Michael A D; Goldman, Daniel I

2015-09-07

The collective movement of animal groups often occurs in confined spaces. As animal groups are challenged to move at high density, their mobility dynamics may resemble the flow of densely packed non-living soft materials such as colloids, grains, or polymers. However, unlike inert soft-materials, self-propelled collective living systems often display social interactions whose influence on collective mobility are only now being explored. In this paper, we study the mobility of bi-directional traffic flow in a social insect (the fire ant Solenopsis invicta) as we vary the diameter of confining foraging tunnels. In all tunnel diameters, we observe the emergence of spatially heterogeneous regions of fast and slow traffic that are induced through two phenomena: physical obstruction, arising from the inability of individual ants to interpenetrate, and time-delay resulting from social interaction in which ants stop to briefly antennate. Density correlation functions reveal that the relaxation dynamics of high density traffic fluctuations scale linearly with fluctuation size and are sensitive to tunnel diameter. We separate the roles of physical obstruction and social interactions in traffic flow using cellular automata based simulation. Social interaction between ants is modeled as a dwell time (Tint) over which interacting ants remain stationary in the tunnel. Investigation over a range of densities and Tint reveals that the slowing dynamics of collective motion in social living systems are consistent with dynamics near a fragile glass transition in inert soft-matter systems. In particular, flow is relatively insensitive to density until a critical density is reached. As social interaction affinity is increased (increasing Tint) traffic dynamics change and resemble a strong glass transition. Thus, social interactions play an important role in the mobility of collective living systems at high density. Our experiments and model demonstrate that the concepts of soft-matter physics aid understanding of the mobility of collective living systems, and motivate further inquiry into the dynamics of densely confined social living systems.

People like us: ethnic group density effects on health.

PubMed

Pickett, Kate E; Wilkinson, Richard G

2008-09-01

Studies examining the effects of neighbourhood characteristics have reported what has been called a 'group density' effect on health, such that members of low status minority communities living in an area with a higher proportion of their own racial or ethnic group tend to have better health than those who live in areas with a lower proportion. In this paper we survey published research on ethnic group density and health with the aim of stimulating further research. We situate the research question in the context of contemporary social epidemiology and provide a narrative review of studies of ethnic density and health. We go on to discuss processes which may underlie ethnic density effects, and highlight gaps in the literature and opportunities for further research. Although first recognised in studies of mental illness, 'group density' effects on physical health have been shown more recently. Generally, given individual material circumstances, living in a poorer area is associated with worse health. Members of ethnic minorities who live in areas where there are few like themselves are likely to be materially better off, and living in better neighbourhoods, than those who live in areas with a higher concentration. However, through the eyes of the majority community, they may be made more aware of belonging to a low status minority group, and the psychosocial effects of stigma may offset any advantage. If the psychological effects of stigma are sometimes powerful enough to override material advantage, this may have implications for our understanding of how low social status affects health more generally. As well as highlighting the importance of low social status, cultural isolation and social support to health and quality of life, this paper shows how an understanding of group density effects also has something to offer to our understanding of issues of prejudice, segregation, assimilation and integration in diverse societies.

Production of heterotrophic bacteria inhabiting macroscopic organic aggregates (marine snow) from surface waters

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

Alldredge, A.L.; Cole, J.J.; Caron, D.A.

1986-01-01

Macroscopic detrital aggregates, known as marine snow, are a ubiquitous and abundant component of the marine pelagic zone. Descriptions of microbial communities occurring at densities 2-5 orders of magnitude higher on these particles than in the surrounding seawater have led to the suggestion that marine snow may be a site of intense heterotrophic activity. The authors tested this hypothesis using incorporation of (/sup 3/H)thymidine into macromolecules as a measure of bacterial growth occurring on marine snow from oceanic waters in the North Atlantic and from neritic waters off southern California. Abundances of marine snow ranged from 0.1 to 4.3 aggregatesmore » per liter. However, only 0.1-4% ration per cell on aggregates was generally equal to or lower than that of bacteria found free-living in the surrounding seawater, indicating that attached bacteria were not growing more rapidly than free-living bacteria. Bacteria inhabiting aggregates were up to 25 times larger than free-living forms.« less

Self-sustainable, high-power-density bio-solar cells for lab-on-a-chip applications.

PubMed

Liu, Lin; Choi, Seokheun

2017-11-07

A microfluidic lab-on-a-chip system that generates its own power is essential for stand-alone, independent, self-sustainable point-of-care diagnostic devices to work in limited-resource and remote regions. Miniaturized biological solar cells (or micro-BSCs) can be the most suitable power source for those lab-on-a-chip applications because the technique resembles the earth's natural ecosystem - living organisms work in conjunction with non-living components of their environment to create a self-assembling and self-maintaining system. Micro-BSCs can continuously generate electricity from microbial photosynthetic and respiratory activities over day-night cycles, offering a clean and renewable power source with self-sustaining potential. However, the promise of this technology has not been translated into practical applications because of its relatively low power (∼nW cm -2 ) and current short lifetimes (∼a couple of hours). In this work, we enabled high-performance, self-sustaining, long-life micro-BSCs by using fundamental breakthroughs of device architectures and electrode materials. A 3-D biocompatible, conductive, and porous anode demonstrated great microbial biofilm formation and a high rate of bacterial extracellular electron transfer, which led to greater power generation. Furthermore, our micro-BSCs promoted gas exchange to the bacteria through a gas-permeable PDMS membrane in a well-controlled, tightly enclosed micro-chamber, substantially enhancing sustainability. Through photosynthetic reactions of the cyanobacteria Synechocystis sp. PCC 6803 without additional organic fuel, the 90 μL single-chambered bio-solar cell generated a maximum power density of 43.8 μW cm -2 and sustained consistent power production of ∼18.6 μW cm -2 during the day and ∼11.4 μW cm -2 at night for 20 days, which is the highest and longest reported success of any existing micro-scale bio-solar cells.

Cardiac Muscle-cell Based Actuator and Self-stabilizing Biorobot - PART 1.

PubMed

Holley, Merrel T; Nagarajan, Neerajha; Danielson, Christian; Zorlutuna, Pinar; Park, Kidong

2017-07-11

Biological machines often referred to as biorobots, are living cell- or tissue-based devices that are powered solely by the contractile activity of living components. Due to their inherent advantages, biorobots are gaining interest as alternatives to traditional fully artificial robots. Various studies have focused on harnessing the power of biological actuators, but only recently studies have quantitatively characterized the performance of biorobots and studied their geometry to enhance functionality and efficiency. Here, we demonstrate the development of a self-stabilizing swimming biorobot that can maintain its pitch, depth, and roll without external intervention. The design and fabrication of the PDMS scaffold for the biological actuator and biorobot followed by the functionalization with fibronectin is described in this first part. In the second part of this two-part article, we detail the incorporation of cardiomyocytes and characterize the biological actuator and biorobot function. Both incorporate a base and tail (cantilever) which produce fin-based propulsion. The tail is constructed with soft lithography techniques using PDMS and laser engraving. After incorporating the tail with the device base, it is functionalized with a cell adhesive protein and seeded confluently with cardiomyocytes. The base of the biological actuator consists of a solid PDMS block with a central glass bead (acts as a weight). The base of the biorobot consists of two composite PDMS materials, Ni-PDMS and microballoon-PDMS (MB-PDMS). The nickel powder (in Ni-PDMS) allows magnetic control of the biorobot during cells seeding and stability during locomotion. Microballoons (in MB-PDMS) decrease the density of MB-PDMS, and enable the biorobot to float and swim steadily. The use of these two materials with different mass densities, enabled precise control over the weight distribution to ensure a positive restoration force at any angle of the biorobot. This technique produces a magnetically controlled self-stabilizing swimming biorobot.

Adaptive optical imaging through complex living plant cells

NASA Astrophysics Data System (ADS)

Tamada, Yosuke; Hayano, Yutaka; Murata, Takashi; Oya, Shin; Honma, Yusuke; Kanazawa, Minoru; Miura, Noriaki; Hasebe, Mitsuyasu; Kamei, Yasuhiro; Hattori, Masayuki

2017-04-01

Live-cell imaging using fluorescent molecules is now essential for biological researches. However, images of living cells are accompanied with blur, which becomes stronger according to the depth inside the cells and tissues. This image blur is caused by the disturbance on light that goes through optically inhomogeneous living cells and tissues. Here, we show adaptive optics (AO) imaging of living plant cells. AO has been developed in astronomy to correct the disturbance on light caused by atmospheric turbulence. We developed AO microscope effective for the observation of living plant cells with strong disturbance by chloroplasts, and successfully obtained clear images inside plant cells.

Microneedle Array Design Determines the Induction of Protective Memory CD8+ T Cell Responses Induced by a Recombinant Live Malaria Vaccine in Mice

PubMed Central

Carey, John B.; Pearson, Frances E.; Vrdoljak, Anto; McGrath, Marie G.; Crean, Abina M.; Walsh, Patrick T.; Doody, Timothy; O'Mahony, Conor; Hill, Adrian V. S.; Moore, Anne C.

2011-01-01

Background Vaccine delivery into the skin has received renewed interest due to ease of access to the immune system and microvasculature, however the stratum corneum (SC), must be breached for successful vaccination. This has been achieved by removing the SC by abrasion or scarification or by delivering the vaccine intradermally (ID) with traditional needle-and-syringes or with long microneedle devices. Microneedle patch-based transdermal vaccine studies have predominantly focused on antibody induction by inactivated or subunit vaccines. Here, our principal aim is to determine if the design of a microneedle patch affects the CD8+ T cell responses to a malaria antigen induced by a live vaccine. Methodology and Findings Recombinant modified vaccinia virus Ankara (MVA) expressing a malaria antigen was percutaneously administered to mice using a range of silicon microneedle patches, termed ImmuPatch, that differed in microneedle height, density, patch area and total pore volume. We demonstrate that microneedle arrays that have small total pore volumes induce a significantly greater proportion of central memory T cells that vigorously expand to secondary immunization. Microneedle-mediated vaccine priming induced significantly greater T cell immunity post-boost and equivalent protection against malaria challenge compared to ID vaccination. Notably, unlike ID administration, ImmuPatch-mediated vaccination did not induce inflammatory responses at the site of immunization or in draining lymph nodes. Conclusions/Significance This study demonstrates that the design of microneedle patches significantly influences the magnitude and memory of vaccine-induced CD8+ T cell responses and can be optimised for the induction of desired immune responses. Furthermore, ImmuPatch-mediated delivery may be of benefit to reducing unwanted vaccine reactogenicity. In addition to the advantages of low cost and lack of pain, the development of optimised microneedle array designs for the induction of T cell responses by live vaccines aids the development of solutions to current obstacles of immunization programmes. PMID:21799855

How the climate limits the wood density of angiosperms

NASA Astrophysics Data System (ADS)

Choi, Jin Woo; Kim, Ho-Young

2017-11-01

Flowering trees have various types of wood structure to perform multiple functions under their environmental conditions. In addition to transporting water from the roots to the canopy and providing mechanical support, the structure should provide resistance to embolism to maintain soil-plant-atmosphere continuum. By investigating existing data of the resistivity to embolism and wood density of 165 angiosperm species, here we show that the climate can limit the intrinsic properties of trees. Trees living in the dry environments require a high wood density to slow down the pressure decrease as it loses water relatively fast by evaporation. However, building too much tissues will result in the decrease of hydraulic conductivity and moisture concentration around mesophyll cells. To rationalize the biologically observed lower bound of the wood density, we construct a mechanical model to predict the wood density as a function of the vulnerability to embolism and the time for the recovery. Also, we build an artificial system using hydrogel microchannels that can test the probability of embolism as a function of conduit distributions. Our theoretical prediction is shown to be consistent with the results obtained from the artificial system and the biological data.

Ultrastructural characterization of the hemocytes of Lasiodora sp. (Koch, 1850) (Araneae: Theraphosidae).

PubMed

Soares, Tatiana; Cavalcanti, Marília Gabriela Dos Santos; Ferreira, Felipe Roberto Borba; Cavalcanti, Maria do Socorro de Mendonça; Alves, Luiz Carlos; Brayner, Fábio André; Paiva, Patrícia Maria Guedes

2013-05-01

This paper is the first descriptive review of hemolymph cell types in the circulation of the tarantula spider Lasiodora sp. These animals are more long-lived than other arthropods, and may live for approximately twenty years. Such remarkable longevity may result from a highly successful immune system, which in turn is directly correlated with hemocyte function. Since the literature on the genus Lasiodora sp. is limited, the main goal of the present study was to identify the different cell types by optical and transmission microscope. Six hemocyte types were characterized and called prohemocyte, granulocyte type I, granulocyte type II, spherulocyte, oenocytoid and plasmatocyte. Prohemocytes presented a large nucleus, elongated granulocytes type I showed the nucleus with the same cell format, elliptical granulocytes type II showed the central nucleus of identical shape, spherulocytes exhibited the nucleus filling almost the whole cell, oval oenocytoids showed eccentric nucleus and less dense cytoplasm, and irregular plasmatocytes showed a nucleus and no granules in cytoplasm. These polymorphic granulocytes presented a round, elongated, elliptical, oval or irregular profile with large and varied numbers of granules, except for plasmatocytes, that were agranular. Different densities and different concentrations of these granules were found at the periphery of the cell. The possible reasons and implications of differences and similarities between arthropods hemocytes are discussed. It can be concluded that there are six cell types in Lasiodora sp. This study is of the first step in the elucidation of the role these cells play in the circulatory and immune system in spiders. Copyright © 2013 Elsevier Ltd. All rights reserved.

Impact of physical confinement on nuclei geometry and cell division dynamics in 3D spheroids.

PubMed

Desmaison, Annaïck; Guillaume, Ludivine; Triclin, Sarah; Weiss, Pierre; Ducommun, Bernard; Lobjois, Valérie

2018-06-08

Multicellular tumour spheroids are used as a culture model to reproduce the 3D architecture, proliferation gradient and cell interactions of a tumour micro-domain. However, their 3D characterization at the cell scale remains challenging due to size and cell density issues. In this study, we developed a methodology based on 3D light sheet fluorescence microscopy (LSFM) image analysis and convex hull calculation that allows characterizing the 3D shape and orientation of cell nuclei relative to the spheroid surface. By using this technique and optically cleared spheroids, we found that in freely growing spheroids, nuclei display an elongated shape and are preferentially oriented parallel to the spheroid surface. This geometry is lost when spheroids are grown in conditions of physical confinement. Live 3D LSFM analysis of cell division revealed that confined growth also altered the preferential cell division axis orientation parallel to the spheroid surface and induced prometaphase delay. These results provide key information and parameters that help understanding the impact of physical confinement on cell proliferation within tumour micro-domains.

Effect of tributyltin (TBT) in the metabolic activity of TBT-resistant and sensitive estuarine bacteria.

PubMed

Cruz, Andreia; Oliveira, Vanessa; Baptista, Inês; Almeida, Adelaide; Cunha, Angela; Suzuki, Satoru; Mendo, Sónia

2012-01-01

The effect of tributyltin (TBT) on growth and metabolic activity of three estuarine bacteria with different TBT resistance profiles was investigated in an organic-rich culture medium (TSB) and in phosphate buffered saline (PBS) buffer. Exposure to TBT was assessed by determining its effect on growth (OD(600 nm) measurement), bacterial productivity (leucine incorporation), viability (CFU counts), aggregation and cell size (from Live/Dead analysis), ATP and NADH concentrations. TBT exposure resulted in decrease of bacterial density, cell size, and metabolic activity. In addition, cell aggregates were observed in the TBT-treated cultures. TBT strongly affected bacterial cell metabolism and seemed to exert an effect on its equilibrium, interfering with cell activity. Also, TBT toxicity was lower when cells were grown in TSB than in PBS, suggesting that a nutrient-rich growth medium can protect cells from TBT toxicity. This study contributes to our understanding of the TBT-resistant cell behavior reflected in its physiology and metabolic activity. This information is of utmost importance for further studies of TBT bioremediation. Copyright © 2010 Wiley Periodicals, Inc.

Childhood exposure to constricted living space: a possible environmental threat for myopia development.

PubMed

Choi, Kai Yip; Yu, Wing Yan; Lam, Christie Hang I; Li, Zhe Chuang; Chin, Man Pan; Lakshmanan, Yamunadevi; Wong, Francisca Siu Yin; Do, Chi Wai; Lee, Paul Hong; Chan, Henry Ho Lung

2017-09-01

People in Hong Kong generally live in a densely populated area and their homes are smaller compared with most other cities worldwide. Interestingly, East Asian cities with high population densities seem to have higher myopia prevalence, but the association between them has not been established. This study investigated whether the crowded habitat in Hong Kong is associated with refractive error among children. In total, 1075 subjects [Mean age (S.D.): 9.95 years (0.97), 586 boys] were recruited. Information such as demographics, living environment, parental education and ocular status were collected using parental questionnaires. The ocular axial length and refractive status of all subjects were measured by qualified personnel. Ocular axial length was found to be significantly longer among those living in districts with a higher population density (F 2,1072  = 6.15, p = 0.002) and those living in a smaller home (F 2,1072  = 3.16, p = 0.04). Axial lengths were the same among different types of housing (F 3,1071  = 1.24, p = 0.29). Non-cycloplegic autorefraction suggested a more negative refractive error in those living in districts with a higher population density (F 2,1072  = 7.88, p < 0.001) and those living in a smaller home (F 2,1072  = 4.25, p = 0.02). After adjustment for other confounding covariates, the population density and home size also significantly predicted axial length and non-cycloplegic refractive error in the multiple linear regression model, while axial length and refractive error had no relationship with types of housing. Axial length in children and childhood refractive error were associated with high population density and small home size. A constricted living space may be an environmental threat for myopia development in children. © 2017 The Authors Ophthalmic & Physiological Optics © 2017 The College of Optometrists.

Pyrrole-coupled salicylimine-based fluorescence "turn on" probe for highly selective recognition of Zn²⁺ ions in mixed aqueous media: Application in living cell imaging.

PubMed

Bhosale, Jitendra; Fegade, Umesh; Bondhopadhyay, Banashree; Kaur, Simanpreet; Singh, Narinder; Basu, Anupam; Dabur, Rajesh; Bendre, Ratnamala; Kuwar, Anil

2015-06-01

Cation sensing behaviour of a pyrrole-based derivative (2-hydroxyl 3 methyl 6 isopropyl benzaldehyde}-3,4-dimethyl-1H-pyrrole-2-carbohydrazide (receptor 3) has been explored and is found to be selective towards Zn(2+) over a variety of tested cations. The receptor 3 has shown high selectivity and sensitivity towards Zn(2+) over the other alkali, alkaline earth and transition metal ions. In the presence of Zn(2+), absorption band of receptor 3 has shown the red shift. The sensing behaviour has been suggested to continue via enhancement process which has further been supported by UV-vis absorption and theoretical density functional theory (DFT) calculations indicating the formation of a 1:1 complex between the pyrrole based receptor 3 and Zn(2+). The present work is presenting a highly selective dual channel colorimetric sensor for zinc with great sensitivity. The developed sensor was successfully applied to image intracellular Zn(2+) in living cells. Copyright © 2015 John Wiley & Sons, Ltd.

Controlled power delivery for super-resolution imaging of biological samples using digital micromirror device

NASA Astrophysics Data System (ADS)

Valiya Peedikakkal, Liyana; Cadby, Ashley

2017-02-01

Localization based super resolution images of a biological sample is generally achieved by using high power laser illumination with long exposure time which unfortunately increases photo-toxicity of a sample, making super resolution microscopy, in general, incompatible with live cell imaging. Furthermore, the limitation of photobleaching reduces the ability to acquire time lapse images of live biological cells using fluorescence microscopy. Digital Light Processing (DLP) technology can deliver light at grey scale levels by flickering digital micromirrors at around 290 Hz enabling highly controlled power delivery to samples. In this work, Digital Micromirror Device (DMD) is implemented in an inverse Schiefspiegler telescope setup to control the power and pattern of illumination for super resolution microscopy. We can achieve spatial and temporal patterning of illumination by controlling the DMD pixel by pixel. The DMD allows us to control the power and spatial extent of the laser illumination. We have used this to show that we can reduce the power delivered to the sample to allow for longer time imaging in one area while achieving sub-diffraction STORM imaging in another using higher power densities.

A Postmortem Study of Frontal and Temporal Gyri Thickness and Cell Number in Human Obesity.

PubMed

Gómez-Apo, Erick; García-Sierra, Adrián; Silva-Pereyra, Juan; Soto-Abraham, Virgilia; Mondragón-Maya, Alejandra; Velasco-Vales, Verónica; Pescatello, Linda S

2018-01-01

This study aimed to compare cortex thickness and neuronal cell density in postmortem brain tissue from people with overweight or obesity and normal weight. The cortex thickness and neuron density of eight donors with overweight or obesity (mean = 31.6 kg/m 2 ; SD = 4.35; n = 8; 6 male) and eight donors with normal weight (mean = 21.8 kg/m 2 ; SD = 1.5; n = 8; 5 male) were compared. All participants were Mexican and lived in Mexico City. Randomly selected thickness measures of different cortex areas from the frontal and temporal lobes were analyzed based on high-resolution real-size photographs. A histological analysis of systematic-random fields was used to quantify the number of neurons in postmortem left and right of the first, second, and third gyri of frontal and temporal lobe brain samples. No statistical difference was found in cortical thickness between donors with overweight or obesity and individuals with normal weight. A smaller number of neurons was found among the donors with overweight or obesity than the donors with normal weight at different frontal and temporal areas. A lower density of neurons is associated with overweight or obesity. The morphological basis for structural brain changes in obesity requires further investigation. © 2017 The Obesity Society.

Regional distribution of T-tubule density in left and right atria in dogs.

PubMed

Arora, Rishi; Aistrup, Gary L; Supple, Stephen; Frank, Caleb; Singh, Jasleen; Tai, Shannon; Zhao, Anne; Chicos, Laura; Marszalec, William; Guo, Ang; Song, Long-Sheng; Wasserstrom, J Andrew

2017-02-01

The peculiarities of transverse tubule (T-tubule) morphology and distribution in the atrium-and how they contribute to excitation-contraction coupling-are just beginning to be understood. The objectives of this study were to determine T-tubule density in the intact, live right and left atria in a large animal and to determine intraregional differences in T-tubule organization within each atrium. Using confocal microscopy, T-tubules were imaged in both atria in intact, Langendorf-perfused normal dog hearts loaded with di-4-ANEPPS. T-tubules were imaged in large populations of myocytes from the endocardial surface of each atrium. Computerized data analysis was performed using a new MatLab (Mathworks, Natick, MA) routine, AutoTT. There was a large percentage of myocytes that had no T-tubules in both atria with a higher percentage in the right atrium (25.1%) than in the left atrium (12.5%) (P < .02). The density of transverse and longitudinal T-tubule elements was low in cells that did contain T-tubules, but there were no significant differences in density between the left atrial appendage, the pulmonary vein-posterior left atrium, the right atrial appendage, and the right atrial free wall. In contrast, there were significant differences in sarcomere spacing and cell width between different regions of the atria. There is a sparse T-tubule network in atrial myocytes throughout both dog atria, with significant numbers of myocytes in both atria-the right atrium more so than the left atrium-having no T-tubules at all. These regional differences in T-tubule distribution, along with differences in cell width and sarcomere spacing, may have implications for the emergence of substrate for atrial fibrillation. Copyright © 2016 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.

Effects of trap type, placement and ash distribution on emerald ash borer captures in a low density site.

PubMed

McCullough, Deborah G; Siegert, Nathan W; Poland, Therese M; Pierce, Steven J; Ahn, Su Zie

2011-10-01

Effective methods for early detection of newly established, low density emerald ash borer (Agrilus planipennis Fairmaire) infestations are critically needed in North America. We assessed adult A. planipennis captures on four types of traps in a 16-ha site in central Michigan. The site was divided into 16 blocks, each comprised of four 50- by 50-m cells. Green ash trees (Fraxinus pennsylvanica Marshall) were inventoried by diameter class and ash phloem area was estimated for each cell. One trap type was randomly assigned to each cell in each block. Because initial sampling showed that A. planipennis density was extremely low, infested ash logs were introduced into the center of the site. In total, 87 beetles were captured during the summer. Purple double-decker traps baited with a blend of ash leaf volatiles, Manuka oil, and ethanol captured 65% of all A. planipennis beetles. Similarly baited, green double-decker traps captured 18% of the beetles, whereas sticky bands on girdled trees captured 11% of the beetles. Purple traps baited with Manuka oil and suspended in the canopies of live ash trees captured only 5% of the beetles. At least one beetle was captured on 81% of the purple double-decker traps, 56% of the green double-decker traps, 42% of sticky bands, and 25% of the canopy traps. Abundance of ash phloem near traps had no effect on captures and trap location and sun exposure had only weak effects on captures. Twelve girdled and 29 nongirdled trees were felled and sampled in winter. Current-year larvae were present in 100% of the girdled trees and 72% of the nongirdled trees, but larval density was five times higher on girdled than nongirdled trees.

Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass

NASA Technical Reports Server (NTRS)

Benardini, James N.; LaDuc, Myron T.; Diamond, Rochelle

2012-01-01

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Testing spatial measures of alcohol outlet density with self-rated health in the Australian context: Implications for policy and practice.

PubMed

Badland, Hannah; Mavoa, Suzanne; Livingston, Michael; David, Stephanie; Giles-Corti, Billie

2016-05-01

Reducing access to alcohol is an important and cost-effective strategy for decreasing alcohol consumption and associated harm. Yet this is a less common approach to alcohol control in Australia. The aim of this research was to ascertain which alcohol outlet density spatial measures were related to long-term health outcomes, and the extent to which this differs for those living in more or less disadvantaged neighbourhoods. Existing Australian state-level spatial alcohol policies were reviewed. No appropriate spatial policies were identified; therefore, the literature was used to identify potential alcohol-related spatial measures. Spatial measures of alcohol outlet density were generated in a geographical information system and linked with health survey data drawn from 3141 adults living in metropolitan Melbourne. Logistic regression analysis was used to examine associations between alcohol outlet density measures, self-rated health and area-level disadvantage. Twelve spatial measures of alcohol outlet density were generated. Alcohol outlet density and self-rated health associations varied by area-level disadvantage. For those living in more disadvantaged areas, not having off-licenses available within 800 m, or on-licenses available within 400 m were protective of self-rated health. Local alcohol outlet density may have a more detrimental effect on self-rated health for those living in more disadvantaged neighbourhoods, compared with those living in more advantaged areas. There is a need for spatial alcohol policies to help reduce alcohol-related harm. This research proposes a set of spatial measures to generate a more consistent understanding of alcohol availability in Australia. [Badland H, Mavoa S, Livingston M, David S, Giles-Corti B. Testing spatial measures of alcohol outlet density with self-rated health in the Australian context: Implications for policy and practice. Drug Alcohol Rev 2016;35:298-306]. © 2015 Australasian Professional Society on Alcohol and other Drugs.

Functionalized Buckyballs for Visualizing Microbial Species in Different States and Environments

DOE PAGES

Cheng, Qingsu; Aravind, Ashwin; Buckley, Matthew; ...

2015-09-08

To date, in situ visualization of microbial density has remained an open problem. Here, functionalized buckyballs (e.g., C60-pyrrolidine tris acid) are shown to be a versatile platform that allows internalization within a microorganism without either adhering to the cell wall and cell membrane or binding to a matrix substrate such as soil. These molecular probes are validated via multi-scale imaging, to show association with microorganisms via fluorescence microscopy, positive cellular uptake via electron microscopy, and non-specific binding to the substrates through a combination of fluorescence and autoradiography imaging. In conclusion, we also demonstrate that cysteine-functionalized C60- pyrrolidine tris acid canmore » differentiate live and dead microorganisms.« less

Ethnic density as a buffer for psychotic experiences: findings from a national survey (EMPIRIC)†

PubMed Central

Das-Munshi, Jayati; Bécares, Laia; Boydell, Jane E.; Dewey, Michael E.; Morgan, Craig; Stansfeld, Stephen A.; Prince, Martin J.

2012-01-01

Background Aetiological mechanisms underlying ethnic density associations with psychosis remain unclear. Aims To assess potential mechanisms underlying the observation that minority ethnic groups experience an increased risk of psychosis when living in neighbourhoods of lower own-group density. Method Multilevel analysis of nationally representative community-level data (from the Ethnic Minorities Psychiatric Illness Rates in the Community survey), which included the main minority ethnic groups living in England, and a White British group. Structured instruments assessed discrimination, chronic strains and social support. The Psychosis Screening Questionnaire ascertained psychotic experiences. Results For every ten percentage point reduction in own-group density, the relative odds of reporting psychotic experiences increased 1.07 times (95% CI 1.01–1.14, P = 0.03 (trend)) for the total minority ethnic sample. In general, people living in areas of lower own-group density experienced greater social adversity that was in turn associated with reporting psychotic experiences. Conclusions People resident in neighbourhoods of higher own-group density experience ‘buffering’ effects from the social risk factors for psychosis. PMID:22844021

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment

PubMed Central

Schipler, Agnes; Mladenova, Veronika; Soni, Aashish; Nikolov, Vladimir; Saha, Janapriya; Mladenov, Emil; Iliakis, George

2016-01-01

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect. PMID:27257076

Possible roles of mechanical cell elimination intrinsic to growing tissues from the perspective of tissue growth efficiency and homeostasis.

PubMed

Lee, Sang-Woo; Morishita, Yoshihiro

2017-07-01

Cell competition is a phenomenon originally described as the competition between cell populations with different genetic backgrounds; losing cells with lower fitness are eliminated. With the progress in identification of related molecules, some reports described the relevance of cell mechanics during elimination. Furthermore, recent live imaging studies have shown that even in tissues composed of genetically identical cells, a non-negligible number of cells are eliminated during growth. Thus, mechanical cell elimination (MCE) as a consequence of mechanical cellular interactions is an unavoidable event in growing tissues and a commonly observed phenomenon. Here, we studied MCE in a genetically-homogeneous tissue from the perspective of tissue growth efficiency and homeostasis. First, we propose two quantitative measures, cell and tissue fitness, to evaluate cellular competitiveness and tissue growth efficiency, respectively. By mechanical tissue simulation in a pure population where all cells have the same mechanical traits, we clarified the dependence of cell elimination rate or cell fitness on different mechanical/growth parameters. In particular, we found that geometrical (specifically, cell size) and mechanical (stress magnitude) heterogeneities are common determinants of the elimination rate. Based on these results, we propose possible mechanical feedback mechanisms that could improve tissue growth efficiency and density/stress homeostasis. Moreover, when cells with different mechanical traits are mixed (e.g., in the presence of phenotypic variation), we show that MCE could drive a drastic shift in cell trait distribution, thereby improving tissue growth efficiency through the selection of cellular traits, i.e. intra-tissue "evolution". Along with the improvement of growth efficiency, cell density, stress state, and phenotype (mechanical traits) were also shown to be homogenized through growth. More theoretically, we propose a mathematical model that approximates cell competition dynamics, by which the time evolution of tissue fitness and cellular trait distribution can be predicted without directly simulating a cell-based mechanical model.

Possible roles of mechanical cell elimination intrinsic to growing tissues from the perspective of tissue growth efficiency and homeostasis

PubMed Central

2017-01-01

Cell competition is a phenomenon originally described as the competition between cell populations with different genetic backgrounds; losing cells with lower fitness are eliminated. With the progress in identification of related molecules, some reports described the relevance of cell mechanics during elimination. Furthermore, recent live imaging studies have shown that even in tissues composed of genetically identical cells, a non-negligible number of cells are eliminated during growth. Thus, mechanical cell elimination (MCE) as a consequence of mechanical cellular interactions is an unavoidable event in growing tissues and a commonly observed phenomenon. Here, we studied MCE in a genetically-homogeneous tissue from the perspective of tissue growth efficiency and homeostasis. First, we propose two quantitative measures, cell and tissue fitness, to evaluate cellular competitiveness and tissue growth efficiency, respectively. By mechanical tissue simulation in a pure population where all cells have the same mechanical traits, we clarified the dependence of cell elimination rate or cell fitness on different mechanical/growth parameters. In particular, we found that geometrical (specifically, cell size) and mechanical (stress magnitude) heterogeneities are common determinants of the elimination rate. Based on these results, we propose possible mechanical feedback mechanisms that could improve tissue growth efficiency and density/stress homeostasis. Moreover, when cells with different mechanical traits are mixed (e.g., in the presence of phenotypic variation), we show that MCE could drive a drastic shift in cell trait distribution, thereby improving tissue growth efficiency through the selection of cellular traits, i.e. intra-tissue “evolution”. Along with the improvement of growth efficiency, cell density, stress state, and phenotype (mechanical traits) were also shown to be homogenized through growth. More theoretically, we propose a mathematical model that approximates cell competition dynamics, by which the time evolution of tissue fitness and cellular trait distribution can be predicted without directly simulating a cell-based mechanical model. PMID:28704373

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

PubMed

Lee, Yong Chan; Chuang, Chun-Yu; Lee, Pak-Kei; Lee, Jin-Soo; Harper, Richart W; Buckpitt, Alan B; Wu, Reen; Oslund, Karen

2008-05-01

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

In vitro and in vivo evaluation of a new nanocomposite, containing high density polyethylene, tricalcium phosphate, hydroxyapatite, and magnesium oxide nanoparticles.

PubMed

Pourdanesh, Fereydoun; Jebali, Ali; Hekmatimoghaddam, Seyedhossein; Allaveisie, Azra

2014-07-01

In this study, a new nanocomposite, which contained high density polyethylene (HDPE), tricalcium phosphate (Ca3(PO4)2) nanoparticles (TCP NPs), hydroxyapatite nanoparticles (HA NPs), and magnesium oxide nanoparticles (MgO NPs) was prepared. As in vitro experiment, human osteoblasts (HOB) cells were exposed to pristine HDPE and its nanocomposite for a period of 1, 4, and 7 days at 37 °C, and then different assays were carried out, including osteoblast cell proliferation, Trypan blue staining, cell viability, alkaline phosphatase (ALP), and cell adhesion. Antibacterial property of pristine HDPE and its nanocomposite was evaluated, and also their mechanical properties were measured after 2 and 4 months. As in vivo experiment, pristine HDPE and its nanocomposite were separately implanted on calvarium bone of rabbits, and tissue inflammation and osteogenesis were investigated after 2, 4, and 6 months. In case of HOB cells treated with HDPE or nanocomposite, as incubation time was increased, cell proliferation, live/dead ratio, and cell viability were decreased. But, the ALP activity and cell adhesion of HOB cells which treated with nanocomposite were raised after increase of incubation time. This study demonstrated that although the mechanical properties of nanocomposite were similar to HDPE sheet, but their antibacterial property was not similar. The in vivo experiment showed that both pristine HDPE and its nanocomposite had same inflammation responses. Interestingly, osteogenesis was observed after 2 months at bone/nanocomposite interface, and was highly increased after 4 and 6 months. It must be noted that such pattern was not seen at bone/HDPE interface. Copyright © 2014 Elsevier B.V. All rights reserved.

Premixed calcium phosphate cements: synthesis, physical properties, and cell cytotoxicity.

PubMed

Xu, Hockin H K; Carey, Lisa E; Simon, Carl G; Takagi, Shozo; Chow, Laurence C

2007-04-01

Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder-liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. PCPCs were formulated from CPC powder+non-aqueous liquid+gelling agent+hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. Setting time (mean+/-S.D.; n=4) for PCPC-Tartaric was 8.2+/-0.8 min, significantly less than the 61.7+/-1.5 min for the Premixed Control developed previously (p<0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5+/-0.8 MPa, higher than 4.5+/-0.8 MPa of Premixed Control (p=0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p=0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p>0.1) from that of the non-premixed CPC control. PCPCs will eliminate the powder-liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs.

Imaging human retinal pigment epithelium cells using adaptive optics optical coherence tomography

NASA Astrophysics Data System (ADS)

Liu, Zhuolin; Kocaoglu, Omer P.; Turner, Timothy L.; Miller, Donald T.

2016-03-01

Retinal pigment epithelium (RPE) cells are vital to health of the outer retina, but are often compromised in ageing and major ocular diseases that lead to blindness. Early manifestation of RPE disruption occurs at the cellular level, and while biomarkers at this scale hold considerable promise, RPE cells have proven extremely challenging to image in the living human eye. We present a novel method based on optical coherence tomography (OCT) equipped with adaptive optics (AO) that overcomes the associated technical obstacles. The method takes advantage of the 3D resolution of AO-OCT, but more critically sub-cellular segmentation and registration that permit organelle motility to be used as a novel contrast mechanism. With this method, we successfully visualized RPE cells and characterized their 3D reflectance profile in every subject and retinal location (3° and 7° temporal to the fovea) imaged to date. We have quantified RPE packing geometry in terms of cell density, cone-to-RPE ratio, and number of nearest neighbors using Voronoi and power spectra analyses. RPE cell density (cells/mm2) showed no significant difference between 3° (4,892+/-691) and 7° (4,780+/-354). In contrast, cone-to- RPE ratio was significantly higher at 3° (3.88+/-0.52:1) than 7° (2.31+/- 0.23:1). Voronoi analysis also showed most RPE cells have six nearest neighbors, which was significantly larger than the next two most prevalent associations: five and seven. Averaged across the five subjects, prevalence of cells with six neighbors was 51.4+/-3.58% at 3°, and 54.58+/-3.01% at 7°. These results are consistent with histology and in vivo studies using other imaging modalities.

In Situ Spatiotemporal Mapping of Flow Fields around Seeded Stem Cells at the Subcellular Length Scale

PubMed Central

Song, Min Jae; Dean, David; Knothe Tate, Melissa L.

2010-01-01

A major hurdle to understanding and exploiting interactions between the stem cell and its environment is the lack of a tool for precise delivery of mechanical cues concomitant to observing sub-cellular adaptation of structure. These studies demonstrate the use of microscale particle image velocimetry (μ-PIV) for in situ spatiotemporal mapping of flow fields around mesenchymal stem cells, i.e. murine embryonic multipotent cell line C3H10T1/2, at the subcellular length scale, providing a tool for real time observation and analysis of stem cell adaptation to the prevailing mechanical milieu. In the absence of cells, computational fluid dynamics (CFD) predicts flow regimes within 12% of μ-PIV measures, achieving the technical specifications of the chamber and the flow rates necessary to deliver target shear stresses at a particular height from the base of the flow chamber. However, our μ-PIV studies show that the presence of cells per se as well as the density at which cells are seeded significantly influences local flow fields. Furthermore, for any given cell or cell seeding density, flow regimes vary significantly along the vertical profile of the cell. Hence, the mechanical milieu of the stem cell exposed to shape changing shear stresses, induced by fluid drag, varies with respect to proximity of surrounding cells as well as with respect to apical height. The current study addresses a previously unmet need to predict and observe both flow regimes as well as mechanoadaptation of cells in flow chambers designed to deliver precisely controlled mechanical signals to live cells. An understanding of interactions and adaptation in response to forces at the interface between the surface of the cell and its immediate local environment may be key for de novo engineering of functional tissues from stem cell templates as well as for unraveling the mechanisms underlying multiscale development, growth and adaptation of organisms. PMID:20862249

Density-dependent regulation of growth of BSC-1 cells in cell culture: control of growth by serum factors.

PubMed Central

Holley, R W; Armour, R; Baldwin, J H; Brown, K D; Yeh, Y C

1977-01-01

BSC-1 cells grow slowly, to high cell density, in medium with 0.1% calf serum. An increase in the serum concentration increases both the growth rate of the cells and the final cell density. The serum can be replaced to some extent by epidermal growth factor (EGF). Initiation of DNA synthesis in BSC-1 cells that have spread into a "wound" in a crowded cell layer requires the addition of a trace of serum or EGF, if the cells have previously been deprived of serum. The binding of 125I-labeled EGF to low-density and high-density BSC-1 cells has been studied. Binding is faster to low-density cells. Cells at low cell density also bind much more EGF per cell than cells at high cell density. The fraction of bound 125I-labeled EGF that is present on the cell surface as intact EGF is larger at low than at high cell density. The results indicate that the number of available EGF receptors per cell decreases drastically as the cell density increases. It is suggested that a decrease in the number of available EGF receptor sites per cell, and the accompanying decrease in sensitivity of the cells to EGF, contributes to density-dependent regulation of growth of these cells. Images PMID:303774

Real-time imaging of microparticles and living cells with CMOS nanocapacitor arrays

NASA Astrophysics Data System (ADS)

Laborde, C.; Pittino, F.; Verhoeven, H. A.; Lemay, S. G.; Selmi, L.; Jongsma, M. A.; Widdershoven, F. P.

2015-09-01

Platforms that offer massively parallel, label-free biosensing can, in principle, be created by combining all-electrical detection with low-cost integrated circuits. Examples include field-effect transistor arrays, which are used for mapping neuronal signals and sequencing DNA. Despite these successes, however, bioelectronics has so far failed to deliver a broadly applicable biosensing platform. This is due, in part, to the fact that d.c. or low-frequency signals cannot be used to probe beyond the electrical double layer formed by screening salt ions, which means that under physiological conditions the sensing of a target analyte located even a short distance from the sensor (∼1 nm) is severely hampered. Here, we show that high-frequency impedance spectroscopy can be used to detect and image microparticles and living cells under physiological salt conditions. Our assay employs a large-scale, high-density array of nanoelectrodes integrated with CMOS electronics on a single chip and the sensor response depends on the electrical properties of the analyte, allowing impedance-based fingerprinting. With our platform, we image the dynamic attachment and micromotion of BEAS, THP1 and MCF7 cancer cell lines in real time at submicrometre resolution in growth medium, demonstrating the potential of the platform for label/tracer-free high-throughput screening of anti-tumour drug candidates.

Real-time electrochemical detection of hydrogen peroxide secretion in live cells by Pt nanoparticles decorated graphene-carbon nanotube hybrid paper electrode.

PubMed

Sun, Yimin; He, Kui; Zhang, Zefen; Zhou, Aijun; Duan, Hongwei

2015-06-15

In this work, we develop a new type of flexible and lightweight electrode based on highly dense Pt nanoparticles decorated free-standing graphene-carbon nanotube (CNT) hybrid paper (Pt/graphene-CNT paper), and explore its practical application as flexible electrochemical biosensor for the real-time tracking hydrogen peroxide (H2O2) secretion by live cells. For the fabrication of flexible nanohybrid electrode, the incorporation of CNT in graphene paper not only improves the electrical conductivity and the mechanical strength of graphene paper, but also increases its surface roughness and provides more nucleation sites for metal nanoparticles. Ultrafine Pt nanoparticles are further decorated on graphene-CNT paper by well controlled sputter deposition method, which offers several advantages such as defined particle size and dispersion, high loading density and strong adhesion between the nanoparticles and the substrate. Consequently, the resultant flexible Pt/graphene-CNT paper electrode demonstrates a variety of desirable electrochemical properties including large electrochemical active surface area, excellent electrocatalytic activity, high stability and exceptional flexibility. When used for nonenzymatic detection of H2O2, Pt/graphene-CNT paper exhibits outstanding sensing performance such as high sensitivity, selectivity, stability and reproducibility. The sensitivity is 1.41 µA µM(-1) cm(-2) with a linear range up to 25 µM and a low detection limit of 10 nM (S/N=3), which enables the resultant biosensor for the real-time tracking H2O2 secretion by live cells macrophages. Copyright © 2015. Published by Elsevier B.V.

78 FR 49528 - Consolidation of Wound Care Products Containing Live Cells

Federal Register 2010, 2011, 2012, 2013, 2014

2013-08-14

...] Consolidation of Wound Care Products Containing Live Cells AGENCY: Food and Drug Administration, HHS. ACTION... certain wound care products containing live cells from the Center for Devices and Radiological Health... CDRH and CBER. FDA believes that as more wound care products containing live cells are developed such...

Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells

PubMed Central

Prakash, Satya; Malgorzata Urbanska, Aleksandra

2008-01-01

There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review. PMID:19707368

Evaluation of results of cell electrophoresis experiments on space shuttle STS-3 including pre-flight and post-flight laboratory experiments

NASA Technical Reports Server (NTRS)

Todd, P. W.

1985-01-01

The objectives of the red blood cell experiments were to provide a visual check on the electrophoretic process and especially electroosmotic flow in space as well as to provide test separations of non-degradable standard particles for comparison with the separations of the three viable cell types studied on the Apollo-Soyuz Test Project. Determination of the maximum concentrations of cells that can be separated in column electrophore was a significant goal. Two of the eight columns were available for red cell experiments, so two concentrations of human and rabbit RBC mixtures were used. The objectives of another experiment were to evaluate the reproducibility of microgravity electrophoretic separation of living kidney cells, to separate cells with highly viability despite two freeze-thaw cycles, and to optimize the physical conditions of cell separation. Owing to the uncertain heterogeneity of the starting material, the experimental design does not assess resolution in microgravity, but improved separability was sought in comparison to density-gradient electrophoresis or continuous-flow electrophoresis. Efforts were made to increase cell yield and cell viability and to assess reproducibility directly.

Engineering of Neuron Growth and Enhancing Cell-Chip Communication via Mixed SAMs.

PubMed

Markov, Aleksandr; Maybeck, Vanessa; Wolf, Nikolaus; Mayer, Dirk; Offenhäusser, Andreas; Wördenweber, Roger

2018-06-06

The interface between cells and inorganic surfaces represents one of the key elements for bioelectronics experiments and applications ranging from cell cultures and bioelectronics devices to medical implants. In the present paper, we describe a way to tailor the biocompatibility of substrates in terms of cell growth and to significantly improve cell-chip communication, and we also demonstrate the reusability of the substrates for cell experiments. All these improvements are achieved by coating the substrates or chips with a self-assembled monolayer (SAM) consisting of a mixture of organic molecules, (3-aminopropyl)-triethoxysilane and (3-glycidyloxypropyl)-trimethoxysilane. By varying the ratio of these molecules, we are able to tune the cell density and live/dead ratios of rat cortical neurons cultured directly on the mixed SAM as well as neurons cultured on protein-coated SAMs. Furthermore, the use of the SAM leads to a significant improvement in cell-chip communications. Action potential signals of up to 9.4 ± 0.6 mV (signal-to-noise ratio up to 47) are obtained for HL-1 cells on microelectrode arrays. Finally, we demonstrate that the SAMs facilitate a reusability of the samples for all cell experiments with little re-processing.

Hydraulics of high-yield orchard trees: a case study of three Malus domestica cultivars.

PubMed

Beikircher, Barbara; De Cesare, Chiara; Mayr, Stefan

2013-12-01

The drought tolerance of three economically important apple cultivars, Golden Delicious, Braeburn and Red Delicious, was analysed. The work offers insights into the hydraulics of these high-yield trees and indicates a possible hydraulic limitation of carbon gain. The hydraulic safety and efficiency of branch xylem and leaves were quantified, drought tolerance of living tissues was measured and stomatal regulation, turgor-loss point and osmotic potential at full turgor were analysed. Physiological measurements were correlated with anatomical parameters, such as conduit diameter, cell-wall reinforcement, stomatal density and stomatal pore length. Hydraulic safety differed considerably between the three cultivars with Golden Delicious being significantly less vulnerable to drought-induced embolism than Braeburn and Red Delicious. In Golden Delicious, leaves were less resistant than branch xylem, while in the other cultivars leaves were more resistant than branch xylem. Hydraulic efficiency and xylem anatomical measurements indicate differences in pit properties, which may also be responsible for variations in hydraulic safety. In all three cultivars, full stomatal closure occurred at water potentials where turgor had already been lost and severe loss of hydraulic conductivity as well as damage to living cells had been induced. The consequential negative safety margins pose a risk for hydraulic failure but facilitate carbon gain, which is further improved by the observed high stomatal conductance. Maximal stomatal conductance was clearly seen to be related to stomatal density and size. Based on our results, these three high-yield Malus domestica Borkh. cultivars span a wide range of drought tolerances, appear optimized for maximal carbon gain and, thus, all perform best under well-managed growing conditions.

Modular control of endothelial sheet migration

PubMed Central

Vitorino, Philip; Meyer, Tobias

2008-01-01

Growth factor-induced migration of endothelial cell monolayers enables embryonic development, wound healing, and angiogenesis. Although collective migration is widespread and therapeutically relevant, the underlying mechanism by which cell monolayers respond to growth factor, sense directional signals, induce motility, and coordinate individual cell movements is only partially understood. Here we used RNAi to identify 100 regulatory proteins that enhance or suppress endothelial sheet migration into cell-free space. We measured multiple live-cell migration parameters for all siRNA perturbations and found that each targeted protein primarily regulates one of four functional outputs: cell motility, directed migration, cell–cell coordination, or cell density. We demonstrate that cell motility regulators drive random, growth factor-independent motility in the presence or absence of open space. In contrast, directed migration regulators selectively transduce growth factor signals to direct cells along the monolayer boundary toward open space. Lastly, we found that regulators of cell–cell coordination are growth factor-independent and reorient randomly migrating cells inside the sheet when boundary cells begin to migrate. Thus, cells transition from random to collective migration through a modular control system, whereby growth factor signals convert boundary cells into pioneers, while cells inside the monolayer reorient and follow pioneers through growth factor-independent migration and cell–cell coordination. PMID:19056882

Effects of dietary live and heat-inactive baker's yeast on growth, gut health, and disease resistance of Nile tilapia under high rearing density.

PubMed

Ran, Chao; Huang, Lu; Hu, Jun; Tacon, Philippe; He, Suxu; Li, Zhimin; Wang, Yibing; Liu, Zhi; Xu, Li; Yang, Yalin; Zhou, Zhigang

2016-09-01

In this study, the effects of baker's yeast as probiotics was evaluated in Nile tilapia reared at high density. Juvenile tilapia were distributed to tanks at high density (436 fish/m(3)) and fed with basal diet (CK) or diets supplemented with live (LY) or heat-inactivated yeast (HIY). Another group of fish reared at low density (218 fish/m(3)) and fed with basal diet was also included (LowCK). After 8 weeks of feeding, growth, feed utilization, gut microvilli morphology, digestive enzymes, and expressions of hsp70 and inflammation-related cytokines in the intestine were assessed. Intestinal microbiota was investigated using 16S rRNA gene pyrosequencing. Fish were challenged with Aeromonas hydrophila to evaluate disease resistance. High rearing density significantly decreased the growth, feed utilization, microvilli length, and disease resistance of fish (CK versus LowCK). Moreover, the intestinal hsp70 expression was increased in fish reared at high density, supporting a stress condition. Compared to CK group, supplementation of live yeast significantly increased gut microvilli length and trypsin activity, decreased intestinal hsp70 expression, and enhanced resistance of fish against A. hydrophila (reflected by reduced intestinal alkaline phosphatase activity 24 h post infection). The gut microbiota was not markedly influenced by either rearing density or yeast supplementation. Heat-inactivated yeast (HIY) didn't display the beneficial effects observed in LY except an increase in gut trypsin activity, suggesting the importance of yeast viability and thus secretory metabolites of yeast. In conclusion, live baker's yeast may alleviate the negative effects induced by crowding stress, and has the potential to be used as probiotics for tilapia reared at high density. Copyright © 2016 Elsevier Ltd. All rights reserved.

Versatile quantitative phase imaging system applied to high-speed, low noise and multimodal imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Federici, Antoine; Aknoun, Sherazade; Savatier, Julien; Wattellier, Benoit F.

2017-02-01

Quadriwave lateral shearing interferometry (QWLSI) is a well-established quantitative phase imaging (QPI) technique based on the analysis of interference patterns of four diffraction orders by an optical grating set in front of an array detector [1]. As a QPI modality, this is a non-invasive imaging technique which allow to measure the optical path difference (OPD) of semi-transparent samples. We present a system enabling QWLSI with high-performance sCMOS cameras [2] and apply it to perform high-speed imaging, low noise as well as multimodal imaging. This modified QWLSI system contains a versatile optomechanical device which images the optical grating near the detector plane. Such a device is coupled with any kind of camera by varying its magnification. In this paper, we study the use of a sCMOS Zyla5.5 camera from Andor along with our modified QWLSI system. We will present high-speed live cell imaging, up to 200Hz frame rate, in order to follow intracellular fast motions while measuring the quantitative phase information. The structural and density information extracted from the OPD signal is complementary to the specific and localized fluorescence signal [2]. In addition, QPI detects cells even when the fluorophore is not expressed. This is very useful to follow a protein expression with time. The 10 µm spatial pixel resolution of our modified QWLSI associated to the high sensitivity of the Zyla5.5 enabling to perform high quality fluorescence imaging, we have carried out multimodal imaging revealing fine structures cells, like actin filaments, merged with the morphological information of the phase. References [1]. P. Bon, G. Maucort, B. Wattellier, and S. Monneret, "Quadriwave lateral shearing interferometry for quantitative phase microscopy of living cells," Opt. Express, vol. 17, pp. 13080-13094, 2009. [2] P. Bon, S. Lécart, E. Fort and S. Lévêque-Fort, "Fast label-free cytoskeletal network imaging in living mammalian cells," Biophysical journal, 106(8), pp. 1588-1595, 2014

Formation and dissolution of bacterial colonies.

PubMed

Weber, Christoph A; Lin, Yen Ting; Biais, Nicolas; Zaburdaev, Vasily

2015-09-01

Many organisms form colonies for a transient period of time to withstand environmental pressure. Bacterial biofilms are a prototypical example of such behavior. Despite significant interest across disciplines, physical mechanisms governing the formation and dissolution of bacterial colonies are still poorly understood. Starting from a kinetic description of motile and interacting cells we derive a hydrodynamic equation for their density on a surface, where most of the kinetic coefficients are estimated from experimental data for N. gonorrhoeae bacteria. We use it to describe the formation of multiple colonies with sizes consistent with experimental observations. Finally, we show how the changes in the cell-to-cell interactions lead to the dissolution of the bacterial colonies. The successful application of kinetic theory to a complex far from equilibrium system such as formation and dissolution of living bacterial colonies potentially paves the way for the physical quantification of the initial stages of biofilm formation.

A novel mechanism important for the alignment of microtubules.

PubMed

Wightman, Raymond; Turner, Simon R

2008-04-01

Using a live-cell imaging approach to study individual micro-tubules, we have compared microtubule behavior between net-like and aligned cortical arrays. In contrast to previous studies, a steep angled collision between the growing end of a microtubule and a preexisting microtubule was found to favor crossover. Frequencies of microtubule crossovers, bundling and catastrophes are similar regardless of whether the cell exhibited a net-like or aligned microtubule array. In the predominantly aligned array of petiole cells, severing occurs at the sites of microtubule crossovers and serves to remove unaligned microtubules and to increase microtubule density. Severing was observed to be rare in net-like arrays. Microtubule severing is carried out by the katanin enzyme. In this addendum, we present new insights into the possible mechanism of crossing over and preliminary data looking at organization of the array in a katanin mutant.

Experimental investigation of fire propagation in single live shrubs

Treesearch

Jing Li; Shankar Mahalingam; David R. Weise

2017-01-01

This work focuses broadly on individual, live shrubs and, more specifically, it examines bulk density in chaparral and its combined effects with wind and ignition location on the resulting fire behaviour. Empirical functions to predict bulk density as a function of height for 4-year-old chaparral were developed for two typical species of shrub fuels in southern...

Live single cell functional phenotyping in droplet nano-liter reactors

NASA Astrophysics Data System (ADS)

Konry, Tania; Golberg, Alexander; Yarmush, Martin

2013-11-01

While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surfaceand secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel.

PubMed

Bolgioni, Amanda F; Vittoria, Marc A; Ganem, Neil J

2018-05-14

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 °C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant

PubMed Central

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-01-01

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration. PMID:28772704

Fabrication of Biocompatible Potassium Sodium Niobate Piezoelectric Ceramic as an Electroactive Implant.

PubMed

Chen, Wei; Yu, Zunxiong; Pang, Jinshan; Yu, Peng; Tan, Guoxin; Ning, Chengyun

2017-03-26

The discovery of piezoelectricity in natural bone has attracted extensive research in emulating biological electricity for various tissue regeneration. Here, we carried out experiments to build biocompatible potassium sodium niobate (KNN) ceramics. Then, influence substrate surface charges on bovine serum albumin (BSA) protein adsorption and cell proliferation on KNN ceramics surfaces was investigated. KNN ceramics with piezoelectric constant of ~93 pC/N and relative density of ~93% were fabricated. The adsorption of protein on the positive surfaces (Ps) and negative surfaces (Ns) of KNN ceramics with piezoelectric constant of ~93 pC/N showed greater protein adsorption capacity than that on non-polarized surfaces (NPs). Biocompatibility of KNN ceramics was verified through cell culturing and live/dead cell staining of MC3T3. The cells experiment showed enhanced cell growth on the positive surfaces (Ps) and negative surfaces (Ns) compared to non-polarized surfaces (NPs). These results revealed that KNN ceramics had great potential to be used to understand the effect of surface potential on cells processes and would benefit future research in designing piezoelectric materials for tissue regeneration.

Effect of mungbean (Vigna radiate) living mulch on density and dry weight of weeds in corn (Zea mays) field.

PubMed

Moghadam, M Bakhtiari; Vazan, S; Darvishi, B; Golzardi, F; Farahani, M Esfini

2011-01-01

Living mulch is a suitable solution for weeds ecological management and is considered as an effective method in decreasing of weeds density and dry weight. In order to evaluate of mungbean living mulch effect on density and dry weight of weeds in corn field, an experiment was conducted as a split plot based on randomized complete block design with four blocks in Research Field of Department of Agronomy, Karaj Branch, Islamic Azad University in 2010. Main plots were time of mungbean suppression with 2,4-D herbicide in four levels (4, 6, 8 and 10 leaves stages of corn) and control without weeding and sub plots were densities of mungbean in three levels (50%, 100% and 150% more than optimum density). Density and dry weight of the weeds were measured in all plots with a quadrate (60 x 100 cm) in 10 days after tasseling. Totally, 9 species of weeds were identified in the field, which included 4 broad leave species that were existed in all plots. The results showed that the best time for suppression of mungbean is the 8 leaves stage of corn, which decreased density and dry weight of weeds, 53% and 51% in comparison with control, respectively. Increase of density of mungbean from 50% into 150% more than optimum density, decrease the density and dry weight of weeds, 27.5% and 22%, respectively. It is concluded that the best time and density for suppression mungbean was 8 leaves stage of corn, and 150% more than optimum density, which decreased density and dry weight of the weeds 69% and 63.5% in comparison with control, respectively.

Live-cell imaging combined with immunofluorescence, RNA, or DNA FISH to study the nuclear dynamics and expression of the X-inactivation center.

PubMed

Pollex, Tim; Piolot, Tristan; Heard, Edith

2013-01-01

Differentiation of embryonic stem cells is accompanied by changes of gene expression and chromatin and chromosome dynamics. One of the most impressive examples for these changes is inactivation of one of the two X chromosomes occurring upon differentiation of mouse female embryonic stem cells. With a few exceptions, these events have been mainly studied in fixed cells. In order to better understand the dynamics, kinetics, and order of events during differentiation, one needs to employ live-cell imaging techniques. Here, we describe a combination of live-cell imaging with techniques that can be used in fixed cells (e.g., RNA FISH) to correlate locus dynamics or subnuclear localization with, e.g., gene expression. To study locus dynamics in female ES cells, we generated cell lines containing TetO arrays in the X-inactivation center, the locus on the X chromosome regulating X-inactivation, which can be visualized upon expression of TetR fused to fluorescent proteins. We will use this system to elaborate on how to generate ES cell lines for live-cell imaging of locus dynamics, how to culture ES cells prior to live-cell imaging, and to describe typical live-cell imaging conditions for ES cells using different microscopes. Furthermore, we will explain how RNA, DNA FISH, or immunofluorescence can be applied following live-cell imaging to correlate gene expression with locus dynamics.

Bone mineral density, growth, pubertal development and other parameters in Brazilian children and young adults with sickle cell anaemia.

PubMed

Meeuwes, M; Souza de Carvalho, T F; Cipolotti, R; Gurgel, R Q; Ferrão, T O; Peters, M; Agyemang, C

2013-12-01

To evaluate the occurrence of low bone mineral density (BMD) and its relationship with clinical and laboratorial characteristics in children and young adults with sickle cell anaemia living in Northeast-Brazil, and to assess the role of radiography in diagnosing low BMD. Bone mineral density of lumbar spine was measured by dual energy X-ray absorptiometry (DXA) in 27 patients with Sickle cell anaemia (SCA) aged 7-28 years. Clinical history, calcium and calorie intake, laboratory measurements, anthropometrics and pubertal development were assessed, and X-rays were obtained. Z-scores and T-scores for weight, height, Body Mass Index (BMI) and BMD were calculated using age and gender matched reference data. Mean lumbar spine BMD Z-scores and T-scores were -1.81 SD in boys and -0.80 SD in girls. BMD Z-scores were below -2 SD in 33.3% of girls and in 46.7% of boys. Low BMD (<-2 SD) occurred significantly more in patients with low height-for-age (P = 0.02), low weight-for-age (P = 0.001) and low BMI-for-age (P = 0.006). No significant relationships were found between BMD and other clinical and laboratory parameters. Radiography had a sensitivity of 75% and a specificity of 36% to detect low BMD, and was considered not useful in this context. Patients with low height and/or low weight-for-age seem to be at high risk for developing low BMD. © 2013 John Wiley & Sons Ltd.

Ocean Acidification: Coccolithophore's Light Controlled Effect on Alkalinity

NASA Astrophysics Data System (ADS)

Dobbins, W.

2015-12-01

Coccolithophorids, which play a significant role in the flux of calcite and organic carbon from the photic region to deeper pelagic and benthic zones, are potentially far more useful than siliceous phytoplankton for ocean fertilization projects designed to sequester CO2. However, the production of H+ ions during calcification (HCO3 + Ca+ —> CaCO3 + H+) has resulted in localized acidification around coccolithophore blooms. It has been hypothesized that under the correct light conditions photosynthesis could proceed at a rate such that CO2 is removed in amounts equimolar or greater than the H+ produced by calcification, allowing stable or increasing alkalinity despite ongoing calcification. Previously, this effect had not been demonstrated under laboratory conditions. Fifteen Emiliania huxleyi cultures were separated into equal groups with each receiving: 0, 6, 12, 18, or 24 hours of light each day for 24 days. Daily pH, cell density, and temperature measurements revealed a strong positive correlation between light exposure and pH, and no significant decline in pH in any of the cultures. Alkalinity increases were temperature independent and not strongly correlated with cell density, implying photosynthetic removal of carbon dioxide as the root cause. The average pH across living cultures increased from 7.9 to 8.3 over the first week and changed little for the reminder of the 24-day period. The results demonstrate coccolithophorids can increase alkalinity across a broad range of cell densities, despite the acidification inherent to the calcification process. If the light-alkalinity effect reported here proves scalable to larger cultures, Emiliania huxleyi are a strong candidate for carbon sequestration via targeted ocean fertilization.

Effect of cell density on adipogenic differentiation of mesenchymal stem cells

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

Lu, Hongxu; Biomaterials Center, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044; Guo, Likun

2009-04-10

The effect of cell density on the adipogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) was investigated by using a patterning technique to induce the formation of a cell density gradient on a micropatterned surface. The adipogenic differentiation of MSCs at a density gradient from 5 x 10{sup 3} to 3 x 10{sup 4} cells/cm{sup 2} was examined. Lipid vacuoles were observed at all cell densities after 1-3 weeks of culture in adipogenic differentiation medium although the lipid vacuoles were scarce at the low cell density and abundant at the high cell density. Real-time RT-PCR analysis showed thatmore » adipogenesis marker genes encoding peroxisome proliferator-activated receptor {gamma}2 (PPAR{gamma}2), lipoprotein lipase (LPL), and fatty acid binding protein-4 (FABP4) were detected in the MSCs cultured at all cell densities. The results suggest that there was no apparent effect of cell density on the adipogenic differentiation of human MSCs.« less

Functional DNA-containing nanomaterials: cellular applications in biosensing, imaging, and targeted therapy.

PubMed

Liang, Hao; Zhang, Xiao-Bing; Lv, Yifan; Gong, Liang; Wang, Ruowen; Zhu, Xiaoyan; Yang, Ronghua; Tan, Weihong

2014-06-17

CONSPECTUS: DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell's nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer-micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand-receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA-nanomaterial complexes can enter cells more easily than free single-stranded DNA. Nanomaterials can be designed and synthesized in needed sizes and shapes, and they possess unique chemical and physical properties, which make them useful as DNA carriers or assistants, excellent signal reporters, transducers, and amplifiers. When nanomaterials are combined with functional DNAs to create novel assay platforms, highly sensitive biosensing and high-resolution imaging result. For example, gold nanoparticles and graphene oxides can quench fluorescence efficiently to achieve low background and effectively increase the signal-to-background ratio. Meanwhile, gold nanoparticles themselves can be colorimetric reporters because of their different optical absorptions between monodispersion and aggregation. DNA self-assembled nanomaterials contain several properties of both DNA and nanomaterials. Compared with DNA-nanomaterial complexes, DNA self-assembled nanomaterials more closely resemble living beings, and therefore they have lower cytotoxicity at high concentrations. Functional DNA self-assemblies also have high density of DNA for multivalent reaction and three-dimensional nanostructures for cell uptake. Now and in the future, we envision the use of DNA bases in making designer molecules for many challenging applications confronting chemists. With the further development of artificial DNA bases using smart organic synthesis, DNA macromolecules based on elegant molecular assembly approaches are expected to achieve great diversity, additional versatility, and advanced functions.

Complexity for Survival of Living Systems

NASA Technical Reports Server (NTRS)

Zak, Michail

2009-01-01

A logical connection between the survivability of living systems and the complexity of their behavior (equivalently, mental complexity) has been established. This connection is an important intermediate result of continuing research on mathematical models that could constitute a unified representation of the evolution of both living and non-living systems. Earlier results of this research were reported in several prior NASA Tech Briefs articles, the two most relevant being Characteristics of Dynamics of Intelligent Systems (NPO- 21037), NASA Tech Briefs, Vol. 26, No. 12 (December 2002), page 48; and Self-Supervised Dynamical Systems (NPO- 30634) NASA Tech Briefs, Vol. 27, No. 3 (March 2003), page 72. As used here, living systems is synonymous with active systems and intelligent systems. The quoted terms can signify artificial agents (e.g., suitably programmed computers) or natural biological systems ranging from single-cell organisms at one extreme to the whole of human society at the other extreme. One of the requirements that must be satisfied in mathematical modeling of living systems is reconciliation of evolution of life with the second law of thermodynamics. In the approach followed in this research, this reconciliation is effected by means of a model, inspired partly by quantum mechanics, in which the quantum potential is replaced with an information potential. The model captures the most fundamental property of life - the ability to evolve from disorder to order without any external interference. The model incorporates the equations of classical dynamics, including Newton s equations of motion and equations for random components caused by uncertainties in initial conditions and by Langevin forces. The equations of classical dynamics are coupled with corresponding Liouville or Fokker-Planck equations that describe the evolutions of probability densities that represent the uncertainties. The coupling is effected by fictitious information-based forces that are gradients of the information potential, which, in turn, is a function of the probability densities. The probability densities are associated with mental images both self-image and nonself images (images of external objects that can include other agents). The evolution of the probability densities represents mental dynamics. Then the interaction between the physical and metal aspects of behavior is implemented by feedback from mental to motor dynamics, as represented by the aforementioned fictitious forces. The interaction of a system with its self and nonself images affords unlimited capacity for increase of complexity. There is a biological basis for this model of mental dynamics in the discovery of mirror neurons that learn by imitation. The levels of complexity attained by use of this model match those observed in living systems. To establish a mechanism for increasing the complexity of dynamics of an active system, the model enables exploitation of a chain of reflections exemplified by questions of the form, "What do you think that I think that you think...?" Mathematically, each level of reflection is represented in the form of an attractor performing the corresponding level of abstraction with more details removed from higher levels. The model can be used to describe the behaviors, not only of biological systems, but also of ecological, social, and economics ones.

Cell-size distribution in epithelial tissue formation and homeostasis

PubMed Central

Primo, Luca; Celani, Antonio

2017-01-01

How cell growth and proliferation are orchestrated in living tissues to achieve a given biological function is a central problem in biology. During development, tissue regeneration and homeostasis, cell proliferation must be coordinated by spatial cues in order for cells to attain the correct size and shape. Biological tissues also feature a notable homogeneity of cell size, which, in specific cases, represents a physiological need. Here, we study the temporal evolution of the cell-size distribution by applying the theory of kinetic fragmentation to tissue development and homeostasis. Our theory predicts self-similar probability density function (PDF) of cell size and explains how division times and redistribution ensure cell size homogeneity across the tissue. Theoretical predictions and numerical simulations of confluent non-homeostatic tissue cultures show that cell size distribution is self-similar. Our experimental data confirm predictions and reveal that, as assumed in the theory, cell division times scale like a power-law of the cell size. We find that in homeostatic conditions there is a stationary distribution with lognormal tails, consistently with our experimental data. Our theoretical predictions and numerical simulations show that the shape of the PDF depends on how the space inherited by apoptotic cells is redistributed and that apoptotic cell rates might also depend on size. PMID:28330988

Cell-size distribution in epithelial tissue formation and homeostasis.

PubMed

Puliafito, Alberto; Primo, Luca; Celani, Antonio

2017-03-01

How cell growth and proliferation are orchestrated in living tissues to achieve a given biological function is a central problem in biology. During development, tissue regeneration and homeostasis, cell proliferation must be coordinated by spatial cues in order for cells to attain the correct size and shape. Biological tissues also feature a notable homogeneity of cell size, which, in specific cases, represents a physiological need. Here, we study the temporal evolution of the cell-size distribution by applying the theory of kinetic fragmentation to tissue development and homeostasis. Our theory predicts self-similar probability density function (PDF) of cell size and explains how division times and redistribution ensure cell size homogeneity across the tissue. Theoretical predictions and numerical simulations of confluent non-homeostatic tissue cultures show that cell size distribution is self-similar. Our experimental data confirm predictions and reveal that, as assumed in the theory, cell division times scale like a power-law of the cell size. We find that in homeostatic conditions there is a stationary distribution with lognormal tails, consistently with our experimental data. Our theoretical predictions and numerical simulations show that the shape of the PDF depends on how the space inherited by apoptotic cells is redistributed and that apoptotic cell rates might also depend on size. © 2017 The Author(s).

Live Cell Imaging and Measurements of Molecular Dynamics

PubMed Central

Frigault, M.; Lacoste, J.; Swift, J.; Brown, C.

2010-01-01

w3-2 Live cell microscopy is becoming widespread across all fields of the life sciences, as well as, many areas of the physical sciences. In order to accurately obtain live cell microscopy data, the live specimens must be properly maintained on the imaging platform. In addition, the fluorescence light path must be optimized for efficient light transmission in order to reduce the intensity of excitation light impacting the living sample. With low incident light intensities the processes under study should not be altered due to phototoxic effects from the light allowing for the long term visualization of viable living samples. Aspects for maintaining a suitable environment for the living sample, minimizing incident light and maximizing detection efficiency will be presented for various fluorescence based live cell instruments. Raster Image Correlation Spectroscopy (RICS) is a technique that uses the intensity fluctuations within laser scanning confocal images, as well as the well characterized scanning dynamics of the laser beam, to extract the dynamics, concentrations and clustering of fluorescent molecules within the cell. In addition, two color cross-correlation RICS can be used to determine protein-protein interactions in living cells without the many technical difficulties encountered in FRET based measurements. RICS is an ideal live cell technique for measuring cellular dynamics because the potentially damaging high intensity laser bursts required for photobleaching recovery measurements are not required, rather low laser powers, suitable for imaging, can be used. The RICS theory will be presented along with examples of live cell applications.

DLA based compressed sensing for high resolution MR microscopy of neuronal tissue

NASA Astrophysics Data System (ADS)

Nguyen, Khieu-Van; Li, Jing-Rebecca; Radecki, Guillaume; Ciobanu, Luisa

2015-10-01

In this work we present the implementation of compressed sensing (CS) on a high field preclinical scanner (17.2 T) using an undersampling trajectory based on the diffusion limited aggregation (DLA) random growth model. When applied to a library of images this approach performs better than the traditional undersampling based on the polynomial probability density function. In addition, we show that the method is applicable to imaging live neuronal tissues, allowing significantly shorter acquisition times while maintaining the image quality necessary for identifying the majority of neurons via an automatic cell segmentation algorithm.

The ethnic density effect on alcohol use among ethnic minority people in the UK.

PubMed

Bécares, Laia; Nazroo, James; Stafford, Mai

2011-01-01

Despite lower alcohol drinking rates of UK ethnic minority people (excluding Irish) compared with those of the white majority, events of racial discrimination expose ethnic minorities to unique stressors that elevate the risk for escapist drinking. Studies of ethnic density, the geographical concentration of ethnic minorities in an area, have found racism to be less prevalent in areas of increased ethnic density, and this study hypothesises that ethnic minority people living in areas of high ethnic density will report less alcohol use relative to their counterparts, due to decreased experienced racism and increased sociocultural norms. Multilevel logistic regressions were applied to data from the 1999 and 2004 Health Survey for England linked to ethnic density data from 2001 census. Respondents living in non-White area types and areas of higher coethnic density reported decreased odds of being current drinkers relative to their counterparts. A statistically significant reduction in the odds of exceeding sensible drinking recommendations was observed for Caribbeans in Black area types, Africans in areas of higher coethnic density and Indian people living in Indian area types. Results confirmed a protective ethnic density effect for current alcohol consumption, but showed a less consistent picture of an ethnic density effect for adherence to sensible drinking guidelines. Previous research has shown that alcohol use is increasing among ethnic minorities, and so a greater understanding of alcohol-related behaviour among UK ethnic minority people is important to establish their need for preventive care and advice on safe drinking practices.

Monte Carlo simulations of protein micropatterning in biomembranes: effects of immobile sticky obstacles

NASA Astrophysics Data System (ADS)

Arnold, Andreas M.; Sevcsik, Eva; Schütz, Gerhard J.

2016-09-01

Single molecule trajectories of lipids and proteins can yield valuable information about the nanoscopic organization of the plasma membrane itself. The interpretation of such trajectories, however, is complicated, as the mobility of molecules can be affected by the presence of immobile obstacles, and the transient binding of the tracers to these obstacles. We have previously developed a micropatterning approach that allows for immobilizing a plasma membrane protein and probing the diffusional behavior of a putative interaction partner in living cells. Here, we provide guidelines on how this micropatterning approach can be extended to quantify interaction parameters between plasma membrane constituents in their natural environment. We simulated a patterned membrane system and evaluated the effect of different surface densities of patterned immobile obstacles on the relative mobility as well as the surface density of diffusing tracers. In the case of inert obstacles, the size of the obstacle can be assessed from its surface density at the percolation threshold, which in turn can be extracted from the diffusion behavior of the tracer. For sticky obstacles, 2D dissociation constants can be determined from the tracer diffusion or surface density.

Understanding the effect of ethnic density on mental health: multi-level investigation of survey data from England.

PubMed

Das-Munshi, Jayati; Becares, Laia; Dewey, Michael E; Stansfeld, Stephen A; Prince, Martin J

2010-10-21

To determine if living in areas where higher proportions of people of the same ethnicity reside is protective for common mental disorders, and associated with a reduced exposure to discrimination and improved social support. Finally, to determine if any protective ethnic density effects are mediated by reduced exposure to racism and improved social support. Multi-level logistic regression analysis of national survey data, with area-level, own-group ethnic density modelled as the main exposure. Participants and setting 4281 participants of Irish, black Caribbean, Indian, Pakistani, Bangladeshi, and white British ethnicity, aged 16-74 years, randomly sampled from 892 "middle layer super output areas" in England. Common mental disorders (assessed via structured interviews); discrimination (assessed via structured questionnaire); and social support and social networks (assessed via structured questionnaire). Although the most ethnically dense areas were also the poorest, for each 10 percentage point increase in own-group ethnic density, there was evidence of a decreased risk of common mental disorders, for the full ethnic minority sample (odds ratio 0.94 (95% confidence interval 0.89 to 0.99); P=0.02, trend), for the Irish group (odds ratio 0.21 (0.06 to 0.74); P=0.01, trend), and for the Bangladeshi group (odds ratio 0.75 (0.62 to 0.91); P=0.005, trend), after adjusting for a priori confounders. For some groups, living in areas of higher own-group density was associated with a reduction in the reporting of discrimination and with improved social support and improved social networks. However, none of these factors mediated ethnic density effects. A protective effect of living in areas of higher own-group ethnic density was present for common mental disorders for some minority groups. People living in areas of higher own-group density may report improved social support and less discrimination, but these associations did not fully account for density effects.

Noninvasive near infrared autofluorescence imaging of retinal pigment epithelial cells in the human retina using adaptive optics.

PubMed

Liu, Tao; Jung, HaeWon; Liu, Jianfei; Droettboom, Michael; Tam, Johnny

2017-10-01

The retinal pigment epithelial (RPE) cells contain intrinsic fluorophores that can be visualized using infrared autofluorescence (IRAF). Although IRAF is routinely utilized in the clinic for visualizing retinal health and disease, currently, it is not possible to discern cellular details using IRAF due to limits in resolution. We demonstrate that the combination of adaptive optics (AO) with IRAF (AO-IRAF) enables higher-resolution imaging of the IRAF signal, revealing the RPE mosaic in the living human eye. Quantitative analysis of visualized RPE cells in 10 healthy subjects across various eccentricities demonstrates the possibility for in vivo density measurements of RPE cells, which range from 6505 to 5388 cells/mm 2 for the areas measured (peaking at the fovea). We also identified cone photoreceptors in relation to underlying RPE cells, and found that RPE cells support on average up to 18.74 cone photoreceptors in the fovea down to an average of 1.03 cone photoreceptors per RPE cell at an eccentricity of 6 mm. Clinical application of AO-IRAF to a patient with retinitis pigmentosa illustrates the potential for AO-IRAF imaging to become a valuable complementary approach to the current landscape of high resolution imaging modalities.

Sickle cell detection using a smartphone

PubMed Central

Knowlton, S. M.; Sencan, I.; Aytar, Y.; Khoory, J.; Heeney, M. M.; Ghiran, I. C.; Tasoglu, S.

2015-01-01

Sickle cell disease affects 25% of people living in Central and West Africa and, if left undiagnosed, can cause life threatening “silent” strokes and lifelong damage. However, ubiquitous testing procedures have yet to be implemented in these areas, necessitating a simple, rapid, and accurate testing platform to diagnose sickle cell disease. Here, we present a label-free, sensitive, and specific testing platform using only a small blood sample (<1 μl) based on the higher density of sickle red blood cells under deoxygenated conditions. Testing is performed with a lightweight and compact 3D-printed attachment installed on a commercial smartphone. This attachment includes an LED to illuminate the sample, an optical lens to magnify the image, and two permanent magnets for magnetic levitation of red blood cells. The sample is suspended in a paramagnetic medium with sodium metabisulfite and loaded in a microcapillary tube that is inserted between the magnets. Red blood cells are levitated in the magnetic field based on equilibrium between the magnetic and buoyancy forces acting on the cells. Using this approach, we were able to distinguish between the levitation patterns of sickle versus control red blood cells based on their degree of confinement. PMID:26492382

Sickle cell detection using a smartphone.

PubMed

Knowlton, S M; Sencan, I; Aytar, Y; Khoory, J; Heeney, M M; Ghiran, I C; Tasoglu, S

2015-10-22

Sickle cell disease affects 25% of people living in Central and West Africa and, if left undiagnosed, can cause life threatening "silent" strokes and lifelong damage. However, ubiquitous testing procedures have yet to be implemented in these areas, necessitating a simple, rapid, and accurate testing platform to diagnose sickle cell disease. Here, we present a label-free, sensitive, and specific testing platform using only a small blood sample (<1 μl) based on the higher density of sickle red blood cells under deoxygenated conditions. Testing is performed with a lightweight and compact 3D-printed attachment installed on a commercial smartphone. This attachment includes an LED to illuminate the sample, an optical lens to magnify the image, and two permanent magnets for magnetic levitation of red blood cells. The sample is suspended in a paramagnetic medium with sodium metabisulfite and loaded in a microcapillary tube that is inserted between the magnets. Red blood cells are levitated in the magnetic field based on equilibrium between the magnetic and buoyancy forces acting on the cells. Using this approach, we were able to distinguish between the levitation patterns of sickle versus control red blood cells based on their degree of confinement.

Cell-permeable nanobodies for targeted immunolabelling and antigen manipulation in living cells

NASA Astrophysics Data System (ADS)

Herce, Henry D.; Schumacher, Dominik; Schneider, Anselm F. L.; Ludwig, Anne K.; Mann, Florian A.; Fillies, Marion; Kasper, Marc-André; Reinke, Stefan; Krause, Eberhard; Leonhardt, Heinrich; Cardoso, M. Cristina; Hackenberger, Christian P. R.

2017-08-01

Functional antibody delivery in living cells would enable the labelling and manipulation of intracellular antigens, which constitutes a long-thought goal in cell biology and medicine. Here we present a modular strategy to create functional cell-permeable nanobodies capable of targeted labelling and manipulation of intracellular antigens in living cells. The cell-permeable nanobodies are formed by the site-specific attachment of intracellularly stable (or cleavable) cyclic arginine-rich cell-penetrating peptides to camelid-derived single-chain VHH antibody fragments. We used this strategy for the non-endocytic delivery of two recombinant nanobodies into living cells, which enabled the relocalization of the polymerase clamp PCNA (proliferating cell nuclear antigen) and tumour suppressor p53 to the nucleolus, and thereby allowed the detection of protein-protein interactions that involve these two proteins in living cells. Furthermore, cell-permeable nanobodies permitted the co-transport of therapeutically relevant proteins, such as Mecp2, into the cells. This technology constitutes a major step in the labelling, delivery and targeted manipulation of intracellular antigens. Ultimately, this approach opens the door towards immunostaining in living cells and the expansion of immunotherapies to intracellular antigen targets.

Quantitative imaging of glutathione in live cells using a reversible reaction-based ratiometric fluorescent probe

USDA-ARS?s Scientific Manuscript database

Glutathione (GSH) plays an important role in maintaining redox homeostasis inside cells. Currently, there are no methods available to quantitatively assess the GSH concentration in live cells. Live cell fluorescence imaging revolutionized the understanding of cell biology and has become an indispens...

Live single cell functional phenotyping in droplet nano-liter reactors.

PubMed

Konry, Tania; Golberg, Alexander; Yarmush, Martin

2013-11-11

While single cell heterogeneity is present in all biological systems, most studies cannot address it due to technical limitations. Here we describe a nano-liter droplet microfluidic-based approach for stimulation and monitoring of surface and secreted markers of live single immune dendritic cells (DCs) as well as monitoring the live T cell/DC interaction. This nano-liter in vivo simulating microenvironment allows delivering various stimuli reagents to each cell and appropriate gas exchanges which are necessary to ensure functionality and viability of encapsulated cells. Labeling bioassay and microsphere sensors were integrated into nano-liter reaction volume of the droplet to monitor live single cell surface markers and secretion analysis in the time-dependent fashion. Thus live cell stimulation, secretion and surface monitoring can be obtained simultaneously in distinct microenvironment, which previously was possible using complicated and multi-step in vitro and in vivo live-cell microscopy, together with immunological studies of the outcome secretion of cellular function.

Location and Dynamics of the Immunodominant CD8 T Cell Response to SIVΔnef Immunization and SIVmac251 Vaginal Challenge

PubMed Central

Sasikala-Appukuttan, Arun K.; Kim, Hyeon O.; Kinzel, Nikilyn J.; Hong, Jung Joo; Smith, Anthony J.; Wagstaff, Reece; Reilly, Cavan; Piatak, Michael; Lifson, Jeffrey D.; Reeves, R. Keith; Johnson, R. Paul; Haase, Ashley T.; Skinner, Pamela J.

2013-01-01

Live-attenuated SIV vaccines (LAVs) have been the most effective to date in preventing or partially controlling infection by wild-type SIV in non-human primate models of HIV-1 transmission to women acting by mechanisms of protection that are not well understood. To gain insights into mechanisms of protection by LAVs that could aid development of effective vaccines to prevent HIV-1 transmission to women, we used in situ tetramer staining to determine whether increased densities or changes in the local distribution of SIV-specific CD8 T cells correlated with the maturation of SIVΔnef vaccine-induced protection prior to and after intra-vaginal challenge with wild-type SIVmac251. We evaluated the immunodominant Mamu-A1*001:01/Gag (CM9) and Mamu-A1*001:01/Tat (SL8) epitope response in genital and lymphoid tissues, and found that tetramer+ cells were present at all time points examined. In the cervical vaginal tissues, most tetramer+ cells were distributed diffusely throughout the lamina propria or co-localized with other CD8 T cells within lymphoid aggregates. The distribution and densities of the tetramer+ cells at the portal of entry did not correlate with the maturation of protection or change after challenge. Given these findings, we discuss the possibility that changes in other aspects of the immune system, including the quality of the resident population of virus-specific effector CD8 T cells could contribute to maturation of protection, as well as the potential for vaccine strategies that further increase the size and quality of this effector population to prevent HIV-1 transmission. PMID:24349100

Laser bioprinting of human induced pluripotent stem cells-the effect of printing and biomaterials on cell survival, pluripotency, and differentiation.

PubMed

Koch, Lothar; Deiwick, Andrea; Franke, Annika; Schwanke, Kristin; Haverich, Axel; Zweigerdt, Robert; Chichkov, Boris

2018-04-25

Research on human induced pluripotent stem cells (hiPSCs) is one of the fastest growing fields in biomedicine. Generated from patient's own somatic cells, hiPSCs can be differentiated towards all functional cell types and returned to the patient without immunological concerns. 3D printing of hiPSCs could enable the generation of functional organs for replacement therapies or realization of organ-on-chip systems for individualized medicine. Printing of living cells was demonstrated with immortalized cell lines, primary cells, and adult stem cells with different printing technologies and biomaterials. However, hiPSCs are more sensitive to handling procedures, in particular, when dissociated into single cells. Both pluripotency and directed differentiation are influenced by numerous environmental factors including culture media, biomaterials, and cell density. Notably, existing literature on the effect of applied biomaterials on pluripotency is rather ambiguous. In this study, laser bioprinting of undifferentiated hiPSCs in combination with different biomaterials was performed and the impact on cells' behavior, pluripotency, and differentiation was investigated. Our findings suggest that hiPSCs are indeed more sensitive to the applied biomaterials, but not to laser printing itself. With appropriate biomaterials, such as the hyaluronic acid based solutions applied in this study, hiPSCs can be successfully laser printed without losing their pluripotency.

Fluorescence Live Cell Imaging

PubMed Central

Ettinger, Andreas

2014-01-01

Fluorescence microscopy of live cells has become an integral part of modern cell biology. Fluorescent protein tags, live cell dyes, and other methods to fluorescently label proteins of interest provide a range of tools to investigate virtually any cellular process under the microscope. The two main experimental challenges in collecting meaningful live cell microscopy data are to minimize photodamage while retaining a useful signal-to-noise ratio, and to provide a suitable environment for cells or tissues to replicate physiological cell dynamics. This chapter aims to give a general overview on microscope design choices critical for fluorescence live cell imaging that apply to most fluorescence microscopy modalities, and on environmental control with a focus on mammalian tissue culture cells. In addition, we provide guidance on how to design and evaluate fluorescent protein constructs by spinning disk confocal microscopy. PMID:24974023

Engineering Lipid Bilayer Membranes for Protein Studies

PubMed Central

Khan, Muhammad Shuja; Dosoky, Noura Sayed; Williams, John Dalton

2013-01-01

Lipid membranes regulate the flow of nutrients and communication signaling between cells and protect the sub-cellular structures. Recent attempts to fabricate artificial systems using nanostructures that mimic the physiological properties of natural lipid bilayer membranes (LBM) fused with transmembrane proteins have helped demonstrate the importance of temperature, pH, ionic strength, adsorption behavior, conformational reorientation and surface density in cellular membranes which all affect the incorporation of proteins on solid surfaces. Much of this work is performed on artificial templates made of polymer sponges or porous materials based on alumina, mica, and porous silicon (PSi) surfaces. For example, porous silicon materials have high biocompatibility, biodegradability, and photoluminescence, which allow them to be used both as a support structure for lipid bilayers or a template to measure the electrochemical functionality of living cells grown over the surface as in vivo. The variety of these media, coupled with the complex physiological conditions present in living systems, warrant a summary and prospectus detailing which artificial systems provide the most promise for different biological conditions. This study summarizes the use of electrochemical impedance spectroscopy (EIS) data on artificial biological membranes that are closely matched with previously published biological systems using both black lipid membrane and patch clamp techniques. PMID:24185908

Expansion of Endothelial Progenitor Cells in High Density Dot Culture of Rat Bone Marrow Cells

PubMed Central

Wang, Ling; Kretlow, James D.; Zhou, Guangdong; Cao, Yilin; Liu, Wei; Zhang, Wen Jie

2014-01-01

In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2×105 cells/cm2) by seeding total 9×105 cells into six high density dots or cultured in regular density (1.6×104 cells/cm2) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells. PMID:25254487

Microbial Burden Approach : New Monitoring Approach for Measuring Microbial Burden

NASA Technical Reports Server (NTRS)

Venkateswaran, Kasthuri; Vaishampayan, Parag; Barmatz, Martin

2013-01-01

Advantages of new approach for differentiating live cells/ spores from dead cells/spores. Four examples of Salmonella outbreaks leading to costly destruction of dairy products. List of possible collaboration activities between JPL and other industries (for future discussion). Limitations of traditional microbial monitoring approaches. Introduction to new approach for rapid measurement of viable (live) bacterial cells/spores and its areas of application. Detailed example for determining live spores using new approach (similar procedure for determining live cells). JPL has developed a patented approach for measuring amount of live and dead cells/spores. This novel "molecular" method takes less than 5 to 7 hrs. compared to the seven days required using conventional techniques. Conventional "molecular" techniques can not discriminate live cells/spores among dead cells/spores. The JPL-developed novel method eliminates false positive results obtained from conventional "molecular" techniques that lead to unnecessary delay in the processing and to unnecessary destruction of food products.

Nematocytes: Discovery and characterization of a novel anculeate hemocyte in Drosophila falleni and Drosophila phalerata.

PubMed

Bozler, Julianna; Kacsoh, Balint Z; Bosco, Giovanni

2017-01-01

Immune challenges, such as parasitism, can be so pervasive and deleterious that they constitute an existential threat to a species' survival. In response to these ecological pressures, organisms have developed a wide array of novel behavioral, cellular, and molecular adaptations. Research into these immune defenses in model systems has resulted in a revolutionary understanding of evolution and functional biology. As the field has expanded beyond the limited number of model organisms our appreciation of evolutionary innovation and unique biology has widened as well. With this in mind, we have surveyed the hemolymph of several non-model species of Drosophila. Here we identify and describe a novel hemocyte, type-II nematocytes, found in larval stages of numerous Drosophila species. Examined in detail in Drosophila falleni and Drosophila phalerata, we find that these remarkable cells are distinct from previously described hemocytes due to their anucleate state (lacking a nucleus) and unusual morphology. Type-II nematocytes are long, narrow cells with spindle-like projections extending from a cell body with high densities of mitochondria and microtubules, and exhibit the ability to synthesize proteins. These properties are unexpected for enucleated cells, and together with our additional characterization, we demonstrate that these type-II nematocytes represent a biological novelty. Surprisingly, despite the absence of a nucleus, we observe through live cell imaging that these cells remain motile with a highly dynamic cellular shape. Furthermore, these cells demonstrate the ability to form multicellular structures, which we suggest may be a component of the innate immune response to macro-parasites. In addition, live cell imaging points to a large nucleated hemocyte, type-I nematocyte, as the progenitor cell, leading to enucleation through a budding or asymmetrical division process rather than nuclear ejection: This study is the first to report such a process of enucleation. Here we describe these cells in detail for the first time and examine their evolutionary history in Drosophila.

Processes on the emergent landscapes of biochemical reaction networks and heterogeneous cell population dynamics: differentiation in living matters

PubMed Central

Li, Fangting

2017-01-01

The notion of an attractor has been widely employed in thinking about the nonlinear dynamics of organisms and biological phenomena as systems and as processes. The notion of a landscape with valleys and mountains encoding multiple attractors, however, has a rigorous foundation only for closed, thermodynamically non-driven, chemical systems, such as a protein. Recent advances in the theory of nonlinear stochastic dynamical systems and its applications to mesoscopic reaction networks, one reaction at a time, have provided a new basis for a landscape of open, driven biochemical reaction systems under sustained chemostat. The theory is equally applicable not only to intracellular dynamics of biochemical regulatory networks within an individual cell but also to tissue dynamics of heterogeneous interacting cell populations. The landscape for an individual cell, applicable to a population of isogenic non-interacting cells under the same environmental conditions, is defined on the counting space of intracellular chemical compositions x = (x1,x2, … ,xN) in a cell, where xℓ is the concentration of the ℓth biochemical species. Equivalently, for heterogeneous cell population dynamics xℓ is the number density of cells of the ℓth cell type. One of the insights derived from the landscape perspective is that the life history of an individual organism, which occurs on the hillsides of a landscape, is nearly deterministic and ‘programmed’, while population-wise an asynchronous non-equilibrium steady state resides mostly in the lowlands of the landscape. We argue that a dynamic ‘blue-sky’ bifurcation, as a representation of Waddington's landscape, is a more robust mechanism for a cell fate decision and subsequent differentiation than the widely pictured pitch-fork bifurcation. We revisit, in terms of the chemostatic driving forces upon active, living matter, the notions of near-equilibrium thermodynamic branches versus far-from-equilibrium states. The emergent landscape perspective permits a quantitative discussion of a wide range of biological phenomena as nonlinear, stochastic dynamics. PMID:28490602

Micromorphological effect of calcium phosphate coating on compatibility of magnesium alloy with osteoblast

PubMed Central

Hiromoto, Sachiko; Yamazaki, Tomohiko

2017-01-01

Abstract Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were developed to control the degradation speed and to improve the biocompatibility of biodegradable magnesium alloys. Osteoblast MG-63 was cultured directly on OCP- and HAp-coated Mg-3Al-1Zn (wt%, AZ31) alloy (OCP- and HAp-AZ31) to evaluate cell compatibility. Cell proliferation was remarkably improved with OCP and HAp coatings which reduced the corrosion and prevented the H2O2 generation on Mg alloy substrate. OCP-AZ31 showed sparse distribution of living cell colonies and dead cells. HAp-AZ31 showed dense and homogeneous distribution of living cells, with dead cells localized over and around corrosion pits, some of which were formed underneath the coating. These results demonstrated that cells were dead due to changes in the local environment, and it is necessary to evaluate the local biocompatibility of magnesium alloys. Cell density on HAp-AZ31 was higher than that on OCP-AZ31 although there was not a significant difference in the amount of Mg ions released in medium between OCP- and HAp-AZ31. The outer layer of OCP and HAp coatings consisted of plate-like crystal with a thickness of around 0.1 μm and rod-like crystals with a diameter of around 0.1 μm, respectively, which grew from a continuous inner layer. Osteoblasts formed focal contacts on the tips of plate-like OCP and rod-like HAp crystals, with heights of 2–5 μm. The spacing between OCP tips of 0.8–1.1 μm was wider than that between HAp tips of 0.2–0.3 μm. These results demonstrated that cell proliferation depended on the micromorphology of the coatings which governed spacing of focal contacts. Consequently, HAp coating is suitable for improving cell compatibility and bone-forming ability of the Mg alloy. PMID:28179963

Micromorphological effect of calcium phosphate coating on compatibility of magnesium alloy with osteoblast

NASA Astrophysics Data System (ADS)

Hiromoto, Sachiko; Yamazaki, Tomohiko

2017-12-01

Octacalcium phosphate (OCP) and hydroxyapatite (HAp) coatings were developed to control the degradation speed and to improve the biocompatibility of biodegradable magnesium alloys. Osteoblast MG-63 was cultured directly on OCP- and HAp-coated Mg-3Al-1Zn (wt%, AZ31) alloy (OCP- and HAp-AZ31) to evaluate cell compatibility. Cell proliferation was remarkably improved with OCP and HAp coatings which reduced the corrosion and prevented the H2O2 generation on Mg alloy substrate. OCP-AZ31 showed sparse distribution of living cell colonies and dead cells. HAp-AZ31 showed dense and homogeneous distribution of living cells, with dead cells localized over and around corrosion pits, some of which were formed underneath the coating. These results demonstrated that cells were dead due to changes in the local environment, and it is necessary to evaluate the local biocompatibility of magnesium alloys. Cell density on HAp-AZ31 was higher than that on OCP-AZ31 although there was not a significant difference in the amount of Mg ions released in medium between OCP- and HAp-AZ31. The outer layer of OCP and HAp coatings consisted of plate-like crystal with a thickness of around 0.1 μm and rod-like crystals with a diameter of around 0.1 μm, respectively, which grew from a continuous inner layer. Osteoblasts formed focal contacts on the tips of plate-like OCP and rod-like HAp crystals, with heights of 2-5 μm. The spacing between OCP tips of 0.8-1.1 μm was wider than that between HAp tips of 0.2-0.3 μm. These results demonstrated that cell proliferation depended on the micromorphology of the coatings which governed spacing of focal contacts. Consequently, HAp coating is suitable for improving cell compatibility and bone-forming ability of the Mg alloy.

Thermodynamic properties of water in confined environments: a Monte Carlo study

NASA Astrophysics Data System (ADS)

Gladovic, Martin; Bren, Urban; Urbic, Tomaž

2018-05-01

Monte Carlo simulations of Mercedes-Benz water in a crowded environment were performed. The simulated systems are representative of both composite, porous or sintered materials and living cells with typical matrix packings. We studied the influence of overall temperature as well as the density and size of matrix particles on water density, particle distributions, hydrogen bond formation and thermodynamic quantities. Interestingly, temperature and space occupancy of matrix exhibit a similar effect on water properties following the competition between the kinetic and the potential energy of the system, whereby temperature increases the kinetic and matrix packing decreases the potential contribution. A novel thermodynamic decomposition approach was applied to gain insight into individual contributions of different types of inter-particle interactions. This decomposition proved to be useful and in good agreement with the total thermodynamic quantities especially at higher temperatures and matrix packings, where higher-order potential-energy mixing terms lose their importance.

An evaluation of image quality and accuracy of eye bank measurement of donor cornea endothelial cell density in the Specular Microscopy Ancillary Study.

PubMed

Lass, Jonathan H; Gal, Robin L; Ruedy, Katrina J; Benetz, Beth Ann; Beck, Roy W; Baratz, Keith H; Holland, Edward J; Kalajian, Andrea; Kollman, Craig; Manning, Francis J; Mannis, Mark J; McCoy, Kristen; Montoya, Monty; Stulting, Doyle; Xing, Dongyuan

2005-03-01

The Specular Microscopy Ancillary Study was designed to examine donor corneal endothelial specular image quality, compare the central endothelial cell density determined by eye banks with the endothelial cell density determined by a central specular microscopy reading center, and evaluate donor factors that may have an impact on specular image quality and endothelial cell density accuracy. Nonrandomized comparative trial. Endothelial specular images of donor corneas assigned in the Cornea Donor Study. Certified readers assessed donor image quality (analyzable from fair to excellent vs. unanalyzable) and determined the central endothelial cell density. Independent adjudication was performed if there was a difference in the quality of grading or if the endothelial cell density varied by > or =5.0% between readers. Average reading center-determined endothelial cell density was compared with the endothelial cell density determined by each eye bank. Evaluation of image quality and accuracy of endothelial cell density. Of 688 donor endothelial images submitted by 23 eye banks, 663 (96%) were analyzable (excellent, 40 [6%]; good, 302 [44%]; fair, 321 [47%]), and 25 (4%) were unanalyzable by reading center standards. In situ retrieval and greater epithelial exposure correlated with a higher image quality grading. The eye bank-determined endothelial cell density of 434 of the 663 (65%) analyzable images were within 10% of the endothelial cell density determined by the reading center, whereas 185 (28%) were more than 10% higher and 44 (7%) were more than 10% lower. Greater variation in endothelial cell density between the eye banks and the reading center was observed with shorter time of death to preservation, presence of an epithelial defect, folds in Descemet's membrane, lower image quality, and the use of fixed-frame or center method endothelial cell density analysis. Overall, donor endothelial specular image quality and accuracy of endothelial cell density determination were good. However, the data suggest that factors that may affect image quality and contribute to variation in interpretation of the endothelial cell density should be addressed, because the donor endothelial cell density is an important parameter for assessing long-term corneal graft survival.

Perception of high-density living in Hong Kong

Treesearch

Lawrence H. Travers

1977-01-01

Analysis of the Hong Kong experience of adaptation to urban living can provide insights into some of the problems that can be expected to occur in the rapidly expanding cities of the Third World. Population densities in Hong Kong are among the highest in the world, exceeding 400,000 persons per square mile in parts of Kowloon. Research based upon residence in a worker...

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

PubMed

Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

2013-05-03

Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF

PubMed Central

Leontieva, Ekaterina A.; Kornilova, Elena S.

2017-01-01

Quantum dots (QDs) complexed to ligands recognizing surface receptors undergoing internalization are an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific tracking of the cells of interest. However, conjugation of quasi-multivalent large QD-particle to monovalent small growth factors like EGF that bound their tyrosine-kinase receptors may affect key endocytic events tightly bound to signaling. Here, by means of confocal microscopy we have addressed the key endocytic events of lysosomal degradative pathway stimulated by native EGF or EGF-QD bioconjugate. We have demonstrated that the decrease in endosome number, increase in mean endosome integrated density and the pattern of EEA1 co-localization with EGF-EGFR complexes at early stages of endocytosis were similar for the both native and QD-conjugated ligands. In both cases enlarged hollow endosomes appeared after wortmannin treatment. This indicates that early endosomal fusions and their maturation proceed similar for both ligands. EGF-QD and native EGF similarly accumulated in juxtanuclear region, and live cell imaging of endosome motion revealed the behavior described elsewhere for microtubule-facilitated motility. Finally, EGF-QD and the receptor were found in lysosomes. However, degradation of receptor part of QD-EGF-EGFR-complex was delayed compared to native EGF, but not inhibited, while QDs fluorescence was detected in lysosomes even after 24 hours. Importantly, in HeLa and A549 cells the both ligands behaved similarly. We conclude that during endocytosis EGF-QD behaves as a neutral marker for degradative pathway up to lysosomal stage and can also be used as a long-term cell marker. PMID:28574831

The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

PubMed Central

Kerr, Niall; Holmes, Fiona E.; Hobson, Sally-Ann; Vanderplank, Penny; Leard, Alan; Balthasar, Nina; Wynick, David

2015-01-01

The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). PMID:26292267

Molecular Medicine II: Hormone Dependent Cancers

DTIC Science & Technology

2005-04-01

multimode, live-cell videomicroscopy - we are analyzing the cycles of centrosome amplification in living S-phase arrested cells. Finally, we will directly...living cells by time-lapse multi-mode videomicroscopy . The key construct for our work is the generation of a mammalian somatic cell line stably expressing

Live imaging of apoptotic cells in zebrafish

PubMed Central

van Ham, Tjakko J.; Mapes, James; Kokel, David; Peterson, Randall T.

2010-01-01

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.—Van Ham, T. J., Mapes, J., Kokel, D., Peterson, R. T. Live imaging of apoptotic cells in zebrafish. PMID:20601526

Compartmental genomics in living cells revealed by single-cell nanobiopsy.

PubMed

Actis, Paolo; Maalouf, Michelle M; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R Adam; Pourmand, Nader

2014-01-28

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

High-resolution Identification and Separation of Living Cell Types by Multiple microRNA-responsive Synthetic mRNAs.

PubMed

Endo, Kei; Hayashi, Karin; Saito, Hirohide

2016-02-23

The precise identification and separation of living cell types is critical to both study cell function and prepare cells for medical applications. However, intracellular information to distinguish live cells remains largely inaccessible. Here, we develop a method for high-resolution identification and separation of cell types by quantifying multiple microRNA (miRNA) activities in live cell populations. We found that a set of miRNA-responsive, in vitro synthesized mRNAs identify a specific cell population as a sharp peak and clearly separate different cell types based on less than two-fold differences in miRNA activities. Increasing the number of miRNA-responsive mRNAs enhanced the capability for cell identification and separation, as we precisely and simultaneously distinguished different cell types with similar miRNA profiles. In addition, the set of synthetic mRNAs separated HeLa cells into subgroups, uncovering heterogeneity of the cells and the level of resolution achievable. Our method could identify target live cells and improve the efficiency of cell purification from heterogeneous populations.

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C.

PubMed

Moreau, David; Lefort, Claire; Pas, Jolien; Bardet, Sylvia M; Leveque, Philippe; O'Connor, Rodney P

2018-02-01

The influence of infrared laser pulses on intracellular Ca 2+ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo-4 was used to measure Ca 2+ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo-4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca 2+ transient with similar dose-response characteristics in hippocampal neurons and glioblastoma cells (half-maximal effective energy density EC 50 of around 58 J.cm -2 ). For both type of cells, the source of the infrared-induced Ca 2+ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP 3 -induced Ca 2+ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP 3 cell signaling cascade has many potential applications including the development of optoceutical therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bioink properties before, during and after 3D bioprinting.

PubMed

Hölzl, Katja; Lin, Shengmao; Tytgat, Liesbeth; Van Vlierberghe, Sandra; Gu, Linxia; Ovsianikov, Aleksandr

2016-09-23

Bioprinting is a process based on additive manufacturing from materials containing living cells. These materials, often referred to as bioink, are based on cytocompatible hydrogel precursor formulations, which gel in a manner compatible with different bioprinting approaches. The bioink properties before, during and after gelation are essential for its printability, comprising such features as achievable structural resolution, shape fidelity and cell survival. However, it is the final properties of the matured bioprinted tissue construct that are crucial for the end application. During tissue formation these properties are influenced by the amount of cells present in the construct, their proliferation, migration and interaction with the material. A calibrated computational framework is able to predict the tissue development and maturation and to optimize the bioprinting input parameters such as the starting material, the initial cell loading and the construct geometry. In this contribution relevant bioink properties are reviewed and discussed on the example of most popular bioprinting approaches. The effect of cells on hydrogel processing and vice versa is highlighted. Furthermore, numerical approaches were reviewed and implemented for depicting the cellular mechanics within the hydrogel as well as for prediction of mechanical properties to achieve the desired hydrogel construct considering cell density, distribution and material-cell interaction.

Retinal specialization through spatially varying cell densities and opsin coexpression in cichlid fish.

PubMed

Dalton, Brian E; de Busserolles, Fanny; Marshall, N Justin; Carleton, Karen L

2017-01-15

The distinct behaviours of animals and the varied habitats in which animals live place different requirements on their visual systems. A trade-off exists between resolution and sensitivity, with these properties varying across the retina. Spectral sensitivity, which affects both achromatic and chromatic (colour) vision, also varies across the retina, though the function of this inhomogeneity is less clear. We previously demonstrated spatially varying spectral sensitivity of double cones in the cichlid fish Metriaclima zebra owing to coexpression of different opsins. Here, we map the distributions of ganglion cells and cone cells and quantify opsin coexpression in single cones to show these also vary across the retina. We identify an area centralis with peak acuity and infrequent coexpression, which may be suited for tasks such as foraging and detecting male signals. The peripheral retina has reduced ganglion cell densities and increased opsin coexpression. Modeling of cichlid visual tasks indicates that coexpression might hinder colour discrimination of foraging targets and some fish colours. But, coexpression might improve contrast detection of dark objects against bright backgrounds, which might be useful for detecting predators or zooplankton. This suggests a trade-off between acuity and colour discrimination in the central retina versus lower resolution but more sensitive contrast detection in the peripheral retina. Significant variation in the pattern of coexpression among individuals, however, raises interesting questions about the selective forces at work. © 2017. Published by The Company of Biologists Ltd.

Layer-by-layer assembly of small interfering RNA and poly(ethyleneimine) for substrate-mediated electroporation with high efficiency.

PubMed

Fujimoto, Hiroyuki; Kato, Koichi; Iwata, Hiroo

2010-05-01

Electroporation microarrays have been developed for the high-throughput transfection of expression constructs and small interfering RNAs (siRNAs) into living mammalian cells. These techniques have potential to provide a platform for the cell-based analysis of gene functions. One of the key issues associated with microarray technology is the efficiency of transfection. The capability of attaining reasonably high transfection efficiency is the basis for obtaining functional data without false negatives. In this study, we aimed at improving the transfection efficiency in the system that siRNA loaded on an electrode is electroporated into cells cultured directly on the electrode. The strategy we adopted here is to increase the surface density of siRNA loaded onto electrodes. For this purpose, the layer-by-layer assembly of siRNA and cationic polymers, branched or linear form of poly(ethyleneimine), was performed. The multilayer thus obtained was characterized by infrared reflection-adsorption spectroscopy and surface plasmon resonance analysis. Transfection efficiency was evaluated in a system that siRNA specific for enhanced green fluorescent protein (EGFP) was electroporated on the electrode into human embryonic kidney cells stably transformed with the EGFP gene. The suppression of EGFP expression was assessed by fluorescence microscopy and flow cytometry. Our data showed that the layer-by-layer assembly of siRNA with branched poly(ethyleneimine) facilitated to increase the surface density of loaded siRNA. As a result, the expression of EGFP gene in the electroporated cells was suppressed much more on the electrodes with the multilayer of siRNA than that with the monolayer.

Is ethnic density associated with risk of child pedestrian injury? A comparison of inter-census changes in ethnic populations and injury rates.

PubMed

Steinbach, Rebecca; Green, Judith; Kenward, Michael G; Edwards, Phil

2016-01-01

Research on inequalities in child pedestrian injury risk has identified some puzzling trends: although, in general, living in more affluent areas protects children from injury, this is not true for those in some minority ethnic groups. This study aimed to identify whether 'group density' effects are associated with injury risk, and whether taking these into account alters the relationship between area deprivation and injury risk. 'Group density' effects exist when ethnic minorities living in an area with a higher proportion of people from a similar ethnic group enjoy better health than those who live in areas with a lower proportion, even though areas with dense minority ethnic populations can be relatively more materially disadvantaged. This study utilised variation in minority ethnic densities in London between two census periods to identify any associations between group density and injury risk. Using police data on road traffic injury and population census data from 2001 to 2011, the numbers of 'White,' 'Asian' and 'Black' child pedestrian injuries in an area were modelled as a function of the percentage of the population in that area that are 'White,' 'Asian' and 'Black,' controlling for socio-economic disadvantage and characteristics of the road environment. There was strong evidence (p < 0.001) of a negative association between 'Black' population density and 'Black' child pedestrian injury risk [incidence (of injury) rate ratios (IRR) 0.575, 95% CI 0.515-0.642]. There was weak evidence (p = 0.083) of a negative association between 'Asian' density and 'Asian' child pedestrian injury risk (IRR 0.901, 95% CI 0.801-1.014) and no evidence (p = 0.412) of an association between 'White' density and 'White' child pedestrian injury risk (IRR 1.075, 95% CI 0.904-1.279). When group density effects are taken into account, area deprivation is associated with injury risk of all ethnic groups. Group density appears to protect 'Black' children living in London against pedestrian injury risk. These findings suggest that future research should focus on structural properties of societies to explain the relationships between minority ethnicity and risk.

Direct Force Measurements of Receptor-Ligand Interactions on Living Cells

NASA Astrophysics Data System (ADS)

Eibl, Robert H.

The characterization of cell adhesion between two living cells at the level of single receptor-ligand bonds is an experimental challenge. This chapter describes how the extremely sensitive method of atomic force microscopy (AFM) based force spectroscopy can be applied to living cells in order to probe for cell-to-cell or cell-to-substrate interactions mediated by single pairs of adhesion receptors. In addition, it is outlined how single-molecule AFM force spectroscopy can be used to detect physiologic changes of an adhesion receptor in a living cell. This force spectroscopy allows us to detect in living cells rapidly changing, chemokine SDF-1 triggered activation states of single VLA-4 receptors. This recently developed AFM application will allow for the detailed investigation of the integrin-chemokine crosstalk of integrin activation mechanisms and on how other adhesion receptors are modulated in health and disease. As adhesion molecules, living cells and even bacteria can be studied by single-molecule AFM force spectroscopy, this method is set to become a powerful tool that can not only be used in biophysics, but in cell biology as well as in immunology and cancer research.

Clinical relevance of voltage-gated potassium channel–complex antibodies in children.

PubMed

Hacohen, Yael; Singh, Rahul; Rossi, Meghan; Lang, Bethan; Hemingway, Cheryl; Lim, Ming; Vincent, Angela

2015-09-15

To assess the clinical and immunologic findings in children with voltage-gated potassium channel (VGKC)-complex antibodies (Abs). Thirty-nine of 363 sera, referred from 2 pediatric centers from 2007 to 2013, had been reported positive (.100 pM) for VGKC-complex Abs. Medical records were reviewed retrospectively and the patients’ condition was independently classified as inflammatory (n 5 159) or noninflammatory (n 5 204). Positive sera (.100 pM) were tested/retested for the VGKC complex Ab–positive complex proteins LGI1 and CASPR2, screened for binding to live hippocampal neurons, and 12 high-titer sera (.400 pM) tested by radioimmunoassay for binding to VGKC Kv1 subunits with or without intracellular postsynaptic density proteins. VGKC-complex Abs were found in 39 children, including 20% of encephalopathies and 7.6% of other conditions (p 5 0.001). Thirty children had inflammatory conditions and 9 had noninflammatory etiologies but titers.400 pM (n512) were found only in inflammatory diseases (p , 0.0001). Four sera, including from 2 children with coexisting NMDA receptor Abs and one with Guillain-Barré syndrome and Abs to both LGI1 and CASPR2, bound to hippocampal neurons. None of the sera bound detectably to VGKC Kv1 subunits on live HEK cells, but 4 of 12 .400 pM sera immunoprecipitated VGKC Kv1 subunits, with or without postsynaptic densities, extracted from transfected cells. Positive VGKC-complex Abs cannot be taken to indicate a specific clinical syndrome in children, but appear to be a nonspecific biomarker of inflammatory neurologic diseases, particularly of encephalopathy. Some of the Abs may bind to intracellular epitopes on the VGKC subunits, or to the intracellular interacting proteins, but in many the targets remain undefined.

Clinical relevance of voltage-gated potassium channel–complex antibodies in children

PubMed Central

Hacohen, Yael; Singh, Rahul; Rossi, Meghan; Lang, Bethan; Hemingway, Cheryl; Lim, Ming

2015-01-01

Objective: To assess the clinical and immunologic findings in children with voltage-gated potassium channel (VGKC)-complex antibodies (Abs). Methods: Thirty-nine of 363 sera, referred from 2 pediatric centers from 2007 to 2013, had been reported positive (>100 pM) for VGKC-complex Abs. Medical records were reviewed retrospectively and the patients' condition was independently classified as inflammatory (n = 159) or noninflammatory (n = 204). Positive sera (>100 pM) were tested/retested for the VGKC-complex Ab–positive complex proteins LGI1 and CASPR2, screened for binding to live hippocampal neurons, and 12 high-titer sera (>400 pM) tested by radioimmunoassay for binding to VGKC Kv1 subunits with or without intracellular postsynaptic density proteins. Results: VGKC-complex Abs were found in 39 children, including 20% of encephalopathies and 7.6% of other conditions (p = 0.001). Thirty children had inflammatory conditions and 9 had noninflammatory etiologies but titers >400 pM (n = 12) were found only in inflammatory diseases (p < 0.0001). Four sera, including from 2 children with coexisting NMDA receptor Abs and one with Guillain-Barré syndrome and Abs to both LGI1 and CASPR2, bound to hippocampal neurons. None of the sera bound detectably to VGKC Kv1 subunits on live HEK cells, but 4 of 12 >400 pM sera immunoprecipitated VGKC Kv1 subunits, with or without postsynaptic densities, extracted from transfected cells. Conclusion: Positive VGKC-complex Abs cannot be taken to indicate a specific clinical syndrome in children, but appear to be a nonspecific biomarker of inflammatory neurologic diseases, particularly of encephalopathy. Some of the Abs may bind to intracellular epitopes on the VGKC subunits, or to the intracellular interacting proteins, but in many the targets remain undefined. PMID:26296514

Insertion of Vertically Aligned Nanowires into Living Cells by Inkjet Printing of Cells.

PubMed

Lee, Donggyu; Lee, Daehee; Won, Yulim; Hong, Hyeonaug; Kim, Yongjae; Song, Hyunwoo; Pyun, Jae-Chul; Cho, Yong Soo; Ryu, Wonhyoung; Moon, Jooho

2016-03-01

Effective insertion of vertically aligned nanowires (NWs) into cells is critical for bioelectrical and biochemical devices, biological delivery systems, and photosynthetic bioenergy harvesting. However, accurate insertion of NWs into living cells using scalable processes has not yet been achieved. Here, NWs are inserted into living Chlamydomonas reinhardtii cells (Chlamy cells) via inkjet printing of the Chlamy cells, representing a low-cost and large-scale method for inserting NWs into living cells. Jetting conditions and printable bioink composed of living Chlamy cells are optimized to achieve stable jetting and precise ink deposition of bioink for indentation of NWs into Chlamy cells. Fluorescence confocal microscopy is used to verify the viability of Chlamy cells after inkjet printing. Simple mechanical considerations of the cell membrane and droplet kinetics are developed to control the jetting force to allow penetration of the NWs into cells. The results suggest that inkjet printing is an effective, controllable tool for stable insertion of NWs into cells with economic and scale-related advantages. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Imprinting the Fate of Antigen-Reactive B Cells through the Affinity of the B Cell Receptor

PubMed Central

O'Connor, Brian P.; Vogel, Laura A.; Zhang, Weijun; Loo, William; Shnider, Danielle; Lind, Evan F.; Ratliff, Michelle; Noelle, Randolph J.; Erickson, Loren D.

2010-01-01

Long-lived plasma cells (PCs) and memory B cells (Bmem) constitute the cellular components of enduring humoral immunity, whereas short-lived PCs that rapidly produce Ig correspond to the host's need for immediate protection against pathogens. In this study we show that the innate affinity of the BCR for Ag imprints upon naive B cells their differentiation fate to become short-or long-lived PCs and Bmem. Using BCR transgenic mice with varying affinities for Ag, naive B cells with high affinity lose their capacity to form germinal centers (GCs), develop neither Bmem nor long-lived PCs, and are destined to a short-lived PC fate. Moderate affinity interactions result in hastened GC responses, and differentiation to long-lived PCs, but Bmem remain extinct. In contrast, lower affinity interactions show tempered GCs, producing Bmem and affinity-matured, long-lived PCs. Thus, a continuum of elementary to comprehensive humoral immune responses exists that is controlled by inherent BCR affinity. PMID:17114443

Intelligent data analysis to model and understand live cell time-lapse sequences.

PubMed

Paterson, Allan; Ashtari, M; Ribé, D; Stenbeck, G; Tucker, A

2012-01-01

One important aspect of cellular function, which is at the basis of tissue homeostasis, is the delivery of proteins to their correct destinations. Significant advances in live cell microscopy have allowed tracking of these pathways by following the dynamics of fluorescently labelled proteins in living cells. This paper explores intelligent data analysis techniques to model the dynamic behavior of proteins in living cells as well as to classify different experimental conditions. We use a combination of decision tree classification and hidden Markov models. In particular, we introduce a novel approach to "align" hidden Markov models so that hidden states from different models can be cross-compared. Our models capture the dynamics of two experimental conditions accurately with a stable hidden state for control data and multiple (less stable) states for the experimental data recapitulating the behaviour of particle trajectories within live cell time-lapse data. In addition to having successfully developed an automated framework for the classification of protein transport dynamics from live cell time-lapse data our model allows us to understand the dynamics of a complex trafficking pathway in living cells in culture.

qSR: a quantitative super-resolution analysis tool reveals the cell-cycle dependent organization of RNA Polymerase I in live human cells.

PubMed

Andrews, J O; Conway, W; Cho, W -K; Narayanan, A; Spille, J -H; Jayanth, N; Inoue, T; Mullen, S; Thaler, J; Cissé, I I

2018-05-09

We present qSR, an analytical tool for the quantitative analysis of single molecule based super-resolution data. The software is created as an open-source platform integrating multiple algorithms for rigorous spatial and temporal characterizations of protein clusters in super-resolution data of living cells. First, we illustrate qSR using a sample live cell data of RNA Polymerase II (Pol II) as an example of highly dynamic sub-diffractive clusters. Then we utilize qSR to investigate the organization and dynamics of endogenous RNA Polymerase I (Pol I) in live human cells, throughout the cell cycle. Our analysis reveals a previously uncharacterized transient clustering of Pol I. Both stable and transient populations of Pol I clusters co-exist in individual living cells, and their relative fraction vary during cell cycle, in a manner correlating with global gene expression. Thus, qSR serves to facilitate the study of protein organization and dynamics with very high spatial and temporal resolutions directly in live cell.

Use of diluted urine for cultivation of Chlorella vulgaris.

PubMed

Jaatinen, Sanna; Lakaniemi, Aino-Maija; Rintala, Jukka

2016-01-01

Our aim was to study the biomass growth of microalga Chlorella vulgaris using diluted human urine as a sole nutrient source. Batch cultivations (21 days) were conducted in five different urine dilutions (1:25-1:300), in 1:100-diluted urine as such and with added trace elements, and as a reference, in artificial growth medium. The highest biomass density was obtained in 1:100-diluted urine with and without additional trace elements (0.73 and 0.60 g L(-1), respectively). Similar biomass growth trends and densities were obtained with 1:25- and 1:300-diluted urine (0.52 vs. 0.48 gVSS L(-1)) indicating that urine at dilution 1:25 can be used to cultivate microalgal based biomass. Interestingly, even 1:300-diluted urine contained sufficiently nutrients and trace elements to support biomass growth. Biomass production was similar despite pH-variation from < 5 to 9 in different incubations indicating robustness of the biomass growth. Ammonium formation did not inhibit overall biomass growth. At the beginning of cultivation, the majority of the biomass consisted of living algal cells, while towards the end, their share decreased and the estimated share of bacteria and cell debris increased.

Transcriptomic Profiling of High-Density Giardia Foci Encysting in the Murine Proximal Intestine.

PubMed

Pham, Jonathan K; Nosala, Christopher; Scott, Erica Y; Nguyen, Kristofer F; Hagen, Kari D; Starcevich, Hannah N; Dawson, Scott C

2017-01-01

Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci. Encystation is initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (>10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of nutrient limitation, and also impact local metabolism and physiology.

Transcriptomic Profiling of High-Density Giardia Foci Encysting in the Murine Proximal Intestine

PubMed Central

Pham, Jonathan K.; Nosala, Christopher; Scott, Erica Y.; Nguyen, Kristofer F.; Hagen, Kari D.; Starcevich, Hannah N.; Dawson, Scott C.

2017-01-01

Giardia is a highly prevalent, understudied protistan parasite causing significant diarrheal disease worldwide. Its life cycle consists of two stages: infectious cysts ingested from contaminated food or water sources, and motile trophozoites that colonize and attach to the gut epithelium, later encysting to form new cysts that are excreted into the environment. Current understanding of parasite physiology in the host is largely inferred from transcriptomic studies using Giardia grown axenically or in co-culture with mammalian cell lines. The dearth of information about the diversity of host-parasite interactions occurring within distinct regions of the gastrointestinal tract has been exacerbated by a lack of methods to directly and non-invasively interrogate disease progression and parasite physiology in live animal hosts. By visualizing Giardia infections in the mouse gastrointestinal tract using bioluminescent imaging (BLI) of tagged parasites, we recently showed that parasites colonize the gut in high-density foci. Encystation is initiated in these foci throughout the entire course of infection, yet how the physiology of parasites within high-density foci in the host gut differs from that of cells in laboratory culture is unclear. Here we use BLI to precisely select parasite samples from high-density foci in the proximal intestine to interrogate in vivo Giardia gene expression in the host. Relative to axenic culture, we noted significantly higher expression (>10-fold) of oxidative stress, membrane transporter, and metabolic and structural genes associated with encystation in the high-density foci. These differences in gene expression within parasite foci in the host may reflect physiological changes associated with high-density growth in localized regions of the gut. We also identified and verified six novel cyst-specific proteins, including new components of the cyst wall that were highly expressed in these foci. Our in vivo transcriptome data support an emerging view that parasites encyst early in localized regions in the gut, possibly as a consequence of nutrient limitation, and also impact local metabolism and physiology. PMID:28620589

First report of important causal relationship between the Adamkiewicz artery vasospasm and dorsal root ganglion cell degeneration in spinal subarachnoid hemorrhage: An experimental study using a rabbit model.

PubMed

Turkmenoglu, Osman N; Kanat, Ayhan; Yolas, Coskun; Aydin, Mehmet Dumlu; Ezirmik, Naci; Gundogdu, Cemal

2017-01-01

The blood supply of the lower spinal cord is heavily dependent on the artery of Adamkiewicz. The goal of this study was to elucidate the effects of lumbar subarachnoid hemorrhage (SAH) on the lumbar 4 dorsal root ganglion (L4DRG) cells secondary to Adamkiewicz artery (AKA) vasospasm. This study was conducted on 20 rabbits, which were randomly divided into three groups: Spinal SAH ( n = 8), serum saline (SS) (SS; n = 6) and control ( n = 6) groups. Experimental spinal SAH was performed. After 20 days, volume values of AKA and neuron density of L4DRG were analyzed. The mean alive neuron density of the L4DRG was 15420 ± 1240/mm 3 and degenerated neuron density was 1045 ± 260/mm 3 in the control group. Whereas, the density of living and degenerated neurons density were 12930 ± 1060/mm 3 and 1365 ± 480/mm 3 in serum saline (SS), 9845 ± 1028/mm 3 and 4560 ± 1340/mm 3 in the SAH group. The mean volume of imaginary AKAs was estimated as 1,250 ± 0,310 mm 3 in the control group and 1,030 ± 0,240 mm 3 in the SF group and 0,910 ± 0,170 mm 3 in SAH group. Volume reduction of the AKAs and neuron density L4DRG were significantly different between the SAH and other two groups ( P < 0.05). Decreased volume of the lumen of the artery of Adamkiewicz was observed in animals with SAH compared with controls. Increased degeneration the L4 dorsal root ganglion in animals with SAH was also noted. Our findings will aid in the planning of future experimental studies and determining the clinical relevance on such studies.

First report of important causal relationship between the Adamkiewicz artery vasospasm and dorsal root ganglion cell degeneration in spinal subarachnoid hemorrhage: An experimental study using a rabbit model

PubMed Central

Turkmenoglu, Osman N.; Kanat, Ayhan; Yolas, Coskun; Aydin, Mehmet Dumlu; Ezirmik, Naci; Gundogdu, Cemal

2017-01-01

Background: The blood supply of the lower spinal cord is heavily dependent on the artery of Adamkiewicz. The goal of this study was to elucidate the effects of lumbar subarachnoid hemorrhage (SAH) on the lumbar 4 dorsal root ganglion (L4DRG) cells secondary to Adamkiewicz artery (AKA) vasospasm. Materials and Methods: This study was conducted on 20 rabbits, which were randomly divided into three groups: Spinal SAH (n = 8), serum saline (SS) (SS; n = 6) and control (n = 6) groups. Experimental spinal SAH was performed. After 20 days, volume values of AKA and neuron density of L4DRG were analyzed. Results: The mean alive neuron density of the L4DRG was 15420 ± 1240/mm3 and degenerated neuron density was 1045 ± 260/mm3 in the control group. Whereas, the density of living and degenerated neurons density were 12930 ± 1060/mm3 and 1365 ± 480/mm3 in serum saline (SS), 9845 ± 1028/mm3 and 4560 ± 1340/mm3 in the SAH group. The mean volume of imaginary AKAs was estimated as 1,250 ± 0,310 mm3 in the control group and 1,030 ± 0,240 mm3 in the SF group and 0,910 ± 0,170 mm3 in SAH group. Volume reduction of the AKAs and neuron density L4DRG were significantly different between the SAH and other two groups (P < 0.05). Conclusion: Decreased volume of the lumen of the artery of Adamkiewicz was observed in animals with SAH compared with controls. Increased degeneration the L4 dorsal root ganglion in animals with SAH was also noted. Our findings will aid in the planning of future experimental studies and determining the clinical relevance on such studies. PMID:28413527

Morphogen and community effects determine cell fates in response to BMP4 signaling in human embryonic stem cells.

PubMed

Nemashkalo, Anastasiia; Ruzo, Albert; Heemskerk, Idse; Warmflash, Aryeh

2017-09-01

Paracrine signals maintain developmental states and create cell fate patterns in vivo and influence differentiation outcomes in human embryonic stem cells (hESCs) in vitro Systematic investigation of morphogen signaling is hampered by the difficulty of disentangling endogenous signaling from experimentally applied ligands. Here, we grow hESCs in micropatterned colonies of 1-8 cells ('µColonies') to quantitatively investigate paracrine signaling and the response to external stimuli. We examine BMP4-mediated differentiation in µColonies and standard culture conditions and find that in µColonies, above a threshold concentration, BMP4 gives rise to only a single cell fate, contrary to its role as a morphogen in other developmental systems. Under standard culture conditions BMP4 acts as a morphogen but this requires secondary signals and particular cell densities. We find that a 'community effect' enforces a common fate within µColonies, both in the state of pluripotency and when cells are differentiated, and that this effect allows a more precise response to external signals. Using live cell imaging to correlate signaling histories with cell fates, we demonstrate that interactions between neighbors result in sustained, homogenous signaling necessary for differentiation. © 2017. Published by The Company of Biologists Ltd.

Multiple factors affect a population of Agassiz's desert tortoise (Gopherus agassizii) in the Northwestern Mojave Desert

USGS Publications Warehouse

Berry, Kristin H.; Yee, Julie L.; Coble, Ashley A.; Perry, William M.; Shields, Timothy A.

2013-01-01

Numerous factors have contributed to declines in populations of the federally threatened Agassiz's Desert Tortoise (Gopherus agassizii) and continue to limit recovery. In 2010, we surveyed a low-density population on a military test facility in the northwestern Mojave Desert of California, USA, to evaluate population status and identify potential factors contributing to distribution and low densities. Estimated densities of live tortoises ranged spatially from 1.2/km2 to 15.1/km2. Although only one death of a breeding-age tortoise was recorded for the 4-yr period prior to the survey, remains of 16 juvenile and immature tortoises were found, and most showed signs of predation by Common Ravens (Corvus corax) and mammals. Predation may have limited recruitment of young tortoises into the adult size classes. To evaluate the relative importance of different types of impacts to tortoises, we developed predictive models for spatially explicit densities of tortoise sign and live tortoises using topography (i.e., slope), predators (Common Raven, signs of mammalian predators), and anthropogenic impacts (distances from paved road and denuded areas, density of ordnance fragments) as covariates. Models suggest that densities of tortoise sign increased with slope and signs of mammalian predators and decreased with Common Ravens, while also varying based on interaction effects involving these predictors as well as distances from paved roads, denuded areas, and ordnance. Similarly, densities of live tortoises varied by interaction effects among distances to denuded areas and paved roads, density of ordnance fragments, and slope. Thus multiple factors predict the densities and distribution of this population.

Diatom-associated bacteria are required for aggregation of Thalassiosira weissflogii

PubMed Central

Gärdes, Astrid; Iversen, Morten H; Grossart, Hans-Peter; Passow, Uta; Ullrich, Matthias S

2011-01-01

Aggregation of algae, mainly diatoms, is an important process in marine systems leading to the settling of particulate organic carbon predominantly in the form of marine snow. Exudation products of phytoplankton form transparent exopolymer particles (TEP), which acts as the glue for particle aggregation. Heterotrophic bacteria interacting with phytoplankton may influence TEP formation and phytoplankton aggregation. This bacterial impact has not been explored in detail. We hypothesized that bacteria attaching to Thalassiosira weissflogii might interact in a yet-to-be determined manner, which could impact TEP formation and aggregate abundance. The role of individual T. weissflogii-attaching and free-living new bacterial isolates for TEP production and diatom aggregation was investigated in vitro. T. weissflogii did not aggregate in axenic culture, and striking differences in aggregation dynamics and TEP abundance were observed when diatom cultures were inoculated with either diatom-attaching or free-living bacteria. The data indicated that free-living bacteria might not influence aggregation whereas bacteria attaching to diatom cells may increase aggregate formation. Interestingly, photosynthetically inactivated T. weissflogii cells did not aggregate regardless of the presence of bacteria. Comparison of aggregate formation, TEP production, aggregate sinking velocity and solid hydrated density revealed remarkable differences. Both, photosynthetically active T. weissflogii and specific diatom-attaching bacteria were required for aggregation. It was concluded that interactions between heterotrophic bacteria and diatoms increased aggregate formation and particle sinking and thus may enhance the efficiency of the biological pump. PMID:20827289

Quantitative Analysis, Design, and Fabrication of Biosensing and Bioprocessing Devices in Living Cells

DTIC Science & Technology

2015-03-10

AFRL-OSR-VA-TR-2015-0080 Biosensing and Bioprocessing Devices in Living Cells Domitilla Del Vecchio MASSACHUSETTS INSTITUTE OF TECHNOLOGY Final...Of Biosensing And Bioprocessing Devices In Living Cells FA9550-12-1-0129 D. Del Vecchio Massachusetts Institute of Technology -- 77 Massachusetts...research is to develop quantitative techniques for the de novo design and fabrication of biosensing devices in living cells . Such devices will be entirely

Testing a dual-fluorescence assay to monitor the viability of filamentous cyanobacteria.

PubMed

Johnson, Tylor J; Hildreth, Michael B; Gu, Liping; Zhou, Ruanbao; Gibbons, William R

2015-06-01

Filamentous cyanobacteria are currently being engineered to produce long-chain organic compounds, including 3rd generation biofuels. Because of their filamentous morphology, standard methods to quantify viability (e.g., plate counts) are not possible. This study investigated a dual-fluorescence assay based upon the LIVE/DEAD® BacLight™ Bacterial Viability Kit to quantify the percent viability of filamentous cyanobacteria using a microplate reader in a high throughput 96-well plate format. The manufacturer's protocol calls for an optical density normalization step to equalize the numbers of viable and non-viable cells used to generate calibration curves. Unfortunately, the isopropanol treatment used to generate non-viable cells released a blue pigment that altered absorbance readings of the non-viable cell solution, resulting in an inaccurate calibration curve. Thus we omitted this optical density normalization step, and carefully divided cell cultures into two equal fractions before the isopropanol treatment. While the resulting calibration curves had relatively high correlation coefficients, their use in various experiments resulted in viability estimates ranging from below 0% to far above 100%. We traced this to the apparent inaccuracy of the propidium iodide (PI) dye that was to stain only non-viable cells. Through further analysis via microplate reader, as well as confocal and wide-field epi-fluorescence microscopy, we observed non-specific binding of PI in viable filamentous cyanobacteria. While PI will not work for filamentous cyanobacteria, it is possible that other fluorochrome dyes could be used to selectively stain non-viable cells. This will be essential in future studies for screening mutants and optimizing photobioreactor system performance for filamentous cyanobacteria. Copyright © 2015 Elsevier B.V. All rights reserved.

Time-Domain Ab Initio Analysis of Excitation Dynamics in a Quantum Dot/Polymer Hybrid: Atomistic Description Rationalizes Experiment.

PubMed

Long, Run; Prezhdo, Oleg V

2015-07-08

Hybrid organic/inorganic polymer/quantum dot (QD) solar cells are an attractive alternative to the traditional cells. The original, simple models postulate that one-dimensional polymers have continuous energy levels, while zero-dimensional QDs exhibit atom-like electronic structure. A realistic, atomistic viewpoint provides an alternative description. Electronic states in polymers are molecule-like: finite in size and discrete in energy. QDs are composed of many atoms and have high, bulk-like densities of states. We employ ab initio time-domain simulation to model the experimentally observed ultrafast photoinduced dynamics in a QD/polymer hybrid and show that an atomistic description is essential for understanding the time-resolved experimental data. Both electron and hole transfers across the interface exhibit subpicosecond time scales. The interfacial processes are fast due to strong electronic donor-acceptor, as evidenced by the densities of the photoexcited states which are delocalized between the donor and the acceptor. The nonadiabatic charge-phonon coupling is also strong, especially in the polymer, resulting in rapid energy losses. The electron transfer from the polymer is notably faster than the hole transfer from the QD, due to a significantly higher density of acceptor states. The stronger molecule-like electronic and charge-phonon coupling in the polymer rationalizes why the electron-hole recombination inside the polymer is several orders of magnitude faster than in the QD. As a result, experiments exhibit multiple transfer times for the long-lived hole inside the QD, ranging from subpicoseconds to nanoseconds. In contrast, transfer of the short-lived electron inside the polymer does not occur beyond the first picosecond. The energy lost by the hole on its transit into the polymer is accommodated by polymer's high-frequency vibrations. The energy lost by the electron injected into the QD is accommodated primarily by much lower-frequency collective and QD modes. The electron dynamics is exponential, whereas evolution of the injected hole through the low density manifold of states of the polymer is highly nonexponential. The time scale of the electron-hole recombination at the interface is intermediate between those in pristine polymer and QD and is closer to that in the polymer. The detailed atomistic insights into the photoinduced charge and energy dynamics at the polymer/QD interface provide valuable guidelines for optimization of solar light harvesting and photovoltaic efficiency in modern nanoscale materials.

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

PubMed Central

Actis, Paolo; Maalouf, Michelle; Kim, Hyunsung John; Lohith, Akshar; Vilozny, Boaz; Seger, R. Adam; Pourmand, Nader

2014-01-01

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis. PMID:24279711

A Novel Electrophototrophic Bacterium Rhodopseudomonas palustris Strain RP2, Exhibits Hydrocarbonoclastic Potential in Anaerobic Environments

PubMed Central

Venkidusamy, Krishnaveni; Megharaj, Mallavarapu

2016-01-01

An electrophototrophic, hydrocarbonoclastic bacterium Rhodopseudomonas palustris stain RP2 was isolated from the anodic biofilms of hydrocarbon fed microbial electrochemical remediation systems (MERS). Salient properties of the strain RP2 were direct electrode respiration, dissimilatory metal oxide reduction, spore formation, anaerobic nitrate reduction, free living diazotrophy and the ability to degrade n-alkane components of petroleum hydrocarbons (PH) in anoxic, photic environments. In acetate fed microbial electrochemical cells, a maximum current density of 305 ± 10 mA/m2 (1000Ω) was generated (power density 131.65 ± 10 mW/m2) by strain RP2 with a coulombic efficiency of 46.7 ± 1.3%. Cyclic voltammetry studies showed that anaerobically grown cells of strain RP2 is electrochemically active and likely to transfer electrons extracellularly to solid electron acceptors through membrane bound compounds, however, aerobically grown cells lacked the electrochemical activity. The ability of strain RP2 to produce current (maximum current density 21 ± 3 mA/m2; power density 720 ± 7 μW/m2, 1000 Ω) using PH as a sole energy source was also examined using an initial concentration of 800 mg l-1 of diesel range hydrocarbons (C9-C36) with a concomitant removal of 47.4 ± 2.7% hydrocarbons in MERS. Here, we also report the first study that shows an initial evidence for the existence of a hydrocarbonoclastic behavior in the strain RP2 when grown in different electron accepting and illuminated conditions (anaerobic and MERS degradation). Such observations reveal the importance of photoorganotrophic growth in the utilization of hydrocarbons from contaminated environments. Identification of such novel petrochemical hydrocarbon degrading electricigens, not only expands the knowledge on the range of bacteria known for the hydrocarbon bioremediation but also shows a biotechnological potential that goes well beyond its applications to MERS. PMID:27462307

Tobacco outlet density near home and school: Associations with smoking and norms among US teens

PubMed Central

Schleicher, Nina C.; Johnson, Trent O.; Fortmann, Stephen P.; Henriksen, Lisa

2016-01-01

This study examined whether living or going to school in neighborhoods with higher tobacco outlet density is associated with higher odds of cigarette smoking among teens, and with perceptions of greater smoking prevalence and peer approval. Using an Internet panel that is representative of US households, we matched data from teen-parent pairs (n=2,771, surveyed June 2011– December 2012) with environmental data about home and school neighborhoods. Density was measured as the number of tobacco outlets per square mile for a ½-mile roadway service area around each participant’s home and school. Logistic regressions tested relationships between tobacco outlet density near home and schools with ever smoking. Linear regressions tested relationships between density, perceived prevalence and peer approval. Models were adjusted for teen, parent/household and neighborhood characteristics. In total, 41.0% of US teens (ages 13–16) lived within ½ mile of a tobacco outlet, and 44.4% attended school within 1,000 feet of a tobacco outlet. Higher tobacco outlet density near home was associated with higher odds of ever smoking, although the relationship was small, OR=1.01, 95% CI (1.00,1.02). Higher tobacco outlet density near home was also associated with perceptions that more adults smoked, coef.=0.09, 95% CI (0.01,0.17). Higher tobacco outlet density near schools was not associated with any outcomes. Living in neighborhoods with higher tobacco outlet density may contribute to teen smoking by increasing access to tobacco products and by cultivating perceptions that smoking is more prevalent. Policy interventions to restrict tobacco outlet density should not be limited to school environments. PMID:27569829

Tobacco outlet density near home and school: Associations with smoking and norms among US teens.

PubMed

Schleicher, Nina C; Johnson, Trent O; Fortmann, Stephen P; Henriksen, Lisa

2016-10-01

This study examined whether living or going to school in neighborhoods with higher tobacco outlet density is associated with higher odds of cigarette smoking among teens, and with perceptions of greater smoking prevalence and peer approval. Using an Internet panel that is representative of US households, we matched data from teen-parent pairs (n=2771, surveyed June 2011-December 2012) with environmental data about home and school neighborhoods. Density was measured as the number of tobacco outlets per square mile for a ½-mile roadway service area around each participant's home and school. Logistic regressions tested relationships between tobacco outlet density near home and schools with ever smoking. Linear regressions tested relationships between density, perceived prevalence and peer approval. Models were adjusted for teen, parent/household and neighborhood characteristics. In total, 41.0% of US teens (ages 13-16) lived within ½ mile of a tobacco outlet, and 44.4% attended school within 1000ft of a tobacco outlet. Higher tobacco outlet density near home was associated with higher odds of ever smoking, although the relationship was small, OR=1.01, 95% CI (1.00, 1.02). Higher tobacco outlet density near home was also associated with perceptions that more adults smoked, coef.=0.09, 95% CI (0.01, 0.17). Higher tobacco outlet density near schools was not associated with any outcomes. Living in neighborhoods with higher tobacco outlet density may contribute to teen smoking by increasing access to tobacco products and by cultivating perceptions that smoking is more prevalent. Policy interventions to restrict tobacco outlet density should not be limited to school environments. Copyright © 2016 Elsevier Inc. All rights reserved.

Relation of initial spacing and relative stand density indices to stand characteristics in a Douglas-fir plantation spacing trial

USGS Publications Warehouse

Curtis, Robert O.; Bansal, Sheel; Harrington, Constance A.

2016-01-01

This report presents updated information on a 1981 Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) plantation spacing trial at 33 years from planting. Stand statistics at the most recent measurement were compared for initial spacing of 1 through 6 meters and associated relative densities. There was no clear relationship of spacing to top height. Diameter, live crown ratio, and percent survival increased with spacing; basal area and relative density decreased with increase in spacing. Volume in trees ≥ 4 cm diameter was greatest at 2 m spacing, while utilizable volume (trees ≥20 cm dbh) was greatest at 4 m spacing. Live crown ratio decreased and total crown projectional area increased with increasing relative density indices. Total crown projectional area was more closely related to relative density than to basal area.

Real-time Raman spectroscopy of optically trapped living cells and organelles

NASA Astrophysics Data System (ADS)

Xie, Changan; Goodman, Charles; Dinno, Mumtaz A.; Li, Yong-Qing

2004-12-01

We report on real-time Raman spectroscopic studies of optically trapped living cells and organelles using an inverted confocal laser-tweezers-Raman-spectroscopy (LTRS) system. The LTRS system was used to hold a single living cell in a physiological solution or to hold a functional organelle within a living cell and consequently measured its Raman spectra. We have measured the changes in Raman spectra of a trapped yeast cell as the function of the temperature of the bathing solution and studied the irreversible cell degeneration during the heat denaturation. In addition, we measured the in-vitro Raman spectra of the nuclei within living pine cells and B. sporeformer, Strep. salivarius, and E. coli bacteria suspended in solution and showed the possibility of using LTRS system as a sensor for rapid identification of microbes in a fluid.

Morphological Analysis of Live Undifferentiated Cells Derived from Induced Pluripotent Stem Cells.

PubMed

Osawa, Yukihiko; Miyamoto, Tomoyuki; Ohno, Setsuyo; Ohno, Eiji

2018-01-01

Induced pluripotent stem (iPS) cells possess pluripotency and self-renewal ability. Therefore, iPS cells are expected to be useful in regenerative medicine. However, iPS cells form malignant immature teratomas after transplantation into animals, even after differentiation induction. It has been suggested that undifferentiated cells expressing Nanog that remain after differentiation induction are responsible for teratoma formation. Various methods of removing these undifferentiated cells have therefore been investigated, but few methods involve morphological approaches, which may induce less cell damage. In addition, for cells derived from iPS cells to be applied in regenerative medicine, they must be alive. However, detailed morphological analysis of live undifferentiated cells has not been performed. For the above reasons, we assessed the morphological features of live undifferentiated cells remaining after differentiation induction as a basic investigation into the clinical application of iPS cells. As a result, live undifferentiated cells remaining after differentiation induction exhibited a round or oval cytoplasm about 12 μm in diameter and a nucleus. They exhibited nucleo-cytoplasmic (N/C) ratio of about 60% and eccentric nuclei, and they possessed partially granule-like structures in the cytoplasm and prominent nucleoli. Although they were similar to iPS cells, they were smaller than live iPS cells. Furthermore, very small cells were present among undifferentiated cells after differentiation induction. These results suggest that the removal of undifferentiated cells may be possible using the morphological features of live iPS cells and undifferentiated cells after differentiation induction. In addition, this study supports safe regenerative medicine using iPS cells.

Learning DMEK From YouTube.

PubMed

McKee, Hamish D; Jhanji, Vishal

2017-12-01

To evaluate the outcomes of the first cases of Descemet membrane endothelial keratoplasty (DMEK) performed by an anterior segment surgeon, learning the procedure, including graft preparation, primarily from watching YouTube videos. DMEK surgery was not learned during fellowship training; there was no attendance at DMEK courses, no witnessing of live surgery, and no supervision by an experienced DMEK surgeon. All graft tissue was prepared by the surgeon on the day of surgery. This is a retrospective review of the 3-month postoperative results of the first 40 consecutive cases. The success rate of graft preparation, intraoperative and postoperative complications, spectacle-corrected visual acuity, endothelial cell density, and central corneal thickness were evaluated. Grafts were successfully prepared in all cases with no loss of donor tissue. DMEK surgery was successful in 39 of 40 eyes with the one failure occurring in a vitrectomized eye without an intact iris-lens diaphragm. Spectacle-corrected visual acuity was ≥6/6 in 23 of the 25 eyes without comorbidity. Mean endothelial cell density was 1515 (±474) cells/mm. Mean central corneal thickness decreased from 624 (±40) μm preoperatively to 513 (±34) μm postoperatively. Although formal training is desirable, good results can be obtained by an anterior segment surgeon learning DMEK, including graft preparation, without it. DMEK should no longer be considered a procedure with a long learning curve in routine cases.

Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains

PubMed Central

Burkholder, JoAnn M.; Gordon, Andrew S.; Moeller, Peter D.; Law, J. Mac; Coyne, Kathryn J.; Lewitus, Alan J.; Ramsdell, John S.; Marshall, Harold G.; Deamer, Nora J.; Cary, S. Craig; Kempton, Jason W.; Morton, Steven L.; Rublee, Parke A.

2005-01-01

Toxicity and its detection in the dinoflagellate fish predators Pfiesteria piscicida and Pfiesteria shumwayae depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic Pfiesteria. The comparison included strains with confirmed toxicity, negative controls (noninducible Pfiesteria strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and P. shumwayae strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic Pfiesteria (coupled with tests confirming Pfiesteria toxin) and conditions conducive to toxicity expression, reliably detected actively toxic Pfiesteria, but FMAs did not. Pfiesteria toxin was found in fish- and algae-fed clonal Pfiesteria cultures, including CCMP2089, but not in controls. In contrast, noninducible Pfiesteria and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of Pfiesteria spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified Pfiesteria toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays. PMID:15728353

Evaluating the efficacy of subcellular fractionation of blast cells using live cell labeling and 2D DIGE.

PubMed

Ho, Yin Ying; Penno, Megan; Perugini, Michelle; Lewis, Ian; Hoffmann, Peter

2012-01-01

Labeling of exposed cell surface proteins of live cells using CyDye DIGE fluor minimal dyes is an efficient strategy for cell surface proteome profiling and quantifying differentially expressed proteins in diseases. Here we describe a strategy to evaluate a two-step detergent-based protein fractionation method using live cell labeling followed by visualization of the fluorescently labeled cell surface proteins and fractionated proteins within a single 2D gel.

Kinase Activity Studied in Living Cells Using an Immunoassay

ERIC Educational Resources Information Center

Bavec, Aljos?a

2014-01-01

This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

Ocular anatomy, ganglion cell distribution and retinal resolution of a killer whale (Orcinus orca).

PubMed

Mass, Alla M; Supin, Alexander Y; Abramov, Andrey V; Mukhametov, Lev M; Rozanova, Elena I

2013-01-01

Retinal topography, cell density and sizes of ganglion cells in the killer whale (Orcinus orca) were analyzed in retinal whole mounts stained with cresyl violet. A distinctive feature of the killer whale's retina is the large size of ganglion cells and low cell density compared to terrestrial mammals. The ganglion cell diameter ranged from 8 to 100 µm, with the majority of cells within a range of 20-40 µm. The topographic distribution of ganglion cells displayed two spots of high cell density located in the temporal and nasal quadrants, 20 mm from the optic disk. The high-density areas were connected by a horizontal belt-like area passing below the optic disk of the retina. Peak cell densities in these areas were evaluated. Mean peak cell densities were 334 and 288 cells/mm(2) in the temporal and nasal high-density areas, respectively. With a posterior nodal distance of 19.5 mm, these high-density data predict a retinal resolution of 9.6' (3.1 cycles/deg.) and 12.6' (2.4 cycles/deg.) in the temporal and nasal areas, respectively, in water. Copyright © 2012 S. Karger AG, Basel.

Functional DNA-Containing Nanomaterials: Cellular Applications in Biosensing, Imaging, and Targeted Therapy

PubMed Central

2015-01-01

Conspectus DNA performs a vital function as a carrier of genetic code, but in the field of nanotechnology, DNA molecules can catalyze chemical reactions in the cell, that is, DNAzymes, or bind with target-specific ligands, that is, aptamers. These functional DNAs with different modifications have been developed for sensing, imaging, and therapeutic systems. Thus, functional DNAs hold great promise for future applications in nanotechnology and bioanalysis. However, these functional DNAs face challenges, especially in the field of biomedicine. For example, functional DNAs typically require the use of cationic transfection reagents to realize cellular uptake. Such reagents enter the cells, increasing the difficulty of performing bioassays in vivo and potentially damaging the cell’s nucleus. To address this obstacle, nanomaterials, such as metallic, carbon, silica, or magnetic materials, have been utilized as DNA carriers or assistants. In this Account, we describe selected examples of functional DNA-containing nanomaterials and their applications from our recent research and those of others. As models, we have chosen to highlight DNA/nanomaterial complexes consisting of gold nanoparticles, graphene oxides, and aptamer–micelles, and we illustrate the potential of such complexes in biosensing, imaging, and medical diagnostics. Under proper conditions, multiple ligand–receptor interactions, decreased steric hindrance, and increased surface roughness can be achieved from a high density of DNA that is bound to the surface of nanomaterials, resulting in a higher affinity for complementary DNA and other targets. In addition, this high density of DNA causes a high local salt concentration and negative charge density, which can prevent DNA degradation. For example, DNAzymes assembled on gold nanoparticles can effectively catalyze chemical reactions even in living cells. And it has been confirmed that DNA–nanomaterial complexes can enter cells more easily than free single-stranded DNA. Nanomaterials can be designed and synthesized in needed sizes and shapes, and they possess unique chemical and physical properties, which make them useful as DNA carriers or assistants, excellent signal reporters, transducers, and amplifiers. When nanomaterials are combined with functional DNAs to create novel assay platforms, highly sensitive biosensing and high-resolution imaging result. For example, gold nanoparticles and graphene oxides can quench fluorescence efficiently to achieve low background and effectively increase the signal-to-background ratio. Meanwhile, gold nanoparticles themselves can be colorimetric reporters because of their different optical absorptions between monodispersion and aggregation. DNA self-assembled nanomaterials contain several properties of both DNA and nanomaterials. Compared with DNA–nanomaterial complexes, DNA self-assembled nanomaterials more closely resemble living beings, and therefore they have lower cytotoxicity at high concentrations. Functional DNA self-assemblies also have high density of DNA for multivalent reaction and three-dimensional nanostructures for cell uptake. Now and in the future, we envision the use of DNA bases in making designer molecules for many challenging applications confronting chemists. With the further development of artificial DNA bases using smart organic synthesis, DNA macromolecules based on elegant molecular assembly approaches are expected to achieve great diversity, additional versatility, and advanced functions. PMID:24780000

The relative density of forests in the United States

Treesearch

Christopher W. Woodall; Charles H. Perry; Patrick D. Miles

2006-01-01

A relative stand density assessment technique, using the mean specific gravity of all trees in a stand to predict its maximum stand density index (SDI) and subsequently its relative stand density (current SDI divided by maximum SDI), was used to estimate the relative density of forests across the United States using a national-scale forest inventory. Live tree biomass...

Multi-casting approach for vascular networks in cellularized hydrogels.

PubMed

Justin, Alexander W; Brooks, Roger A; Markaki, Athina E

2016-12-01

Vascularization is essential for living tissue and remains a major challenge in the field of tissue engineering. A lack of a perfusable channel network within a large and densely populated tissue engineered construct leads to necrotic core formation, preventing fabrication of functional tissues and organs. We report a new method for producing a hierarchical, three-dimensional (3D) and perfusable vasculature in a large, cellularized fibrin hydrogel. Bifurcating channels, varying in size from 1 mm to 200-250 µm, are formed using a novel process in which we convert a 3D printed thermoplastic material into a gelatin network template, by way of an intermediate alginate hydrogel. This enables a CAD-based model design, which is highly customizable, reproducible, and which can yield highly complex architectures, to be made into a removable material, which can be used in cellular environments. Our approach yields constructs with a uniform and high density of cells in the bulk, made from bioactive collagen and fibrin hydrogels. Using standard cell staining and immuno-histochemistry techniques, we showed good cell seeding and the presence of tight junctions between channel endothelial cells, and high cell viability and cell spreading in the bulk hydrogel. © 2016 The Authors.

Premixed calcium phosphate cements: Synthesis, physical properties, and cell cytotoxicity

PubMed Central

Xu, Hockin H.K.; Carey, Lisa E.; Simon, Carl G.; Takagi, Shozo; Chow, Laurence C.

2009-01-01

Objectives Calcium phosphate cement (CPC) is a promising material for dental, periodontal, and craniofacial repairs. However, its use requires on-site powder–liquid mixing that increases the surgical placement time and raises concerns of insufficient and inhomogeneous mixing. The objective of this study was to determine a formulation of premixed CPC (PCPC) with rapid setting, high strength, and good in vitro cell viability. Methods PCPCs were formulated from CPC powder + non-aqueous liquid + gelling agent + hardening accelerator. Five PCPCs were thus developed: PCPC-Tartaric, PCPC-Malonic, PCPC-Citric, PCPC-Glycolic, and PCPC-Malic. Formulations and controls were compared for setting time, diametral tensile strength, and osteoblast cell compatibility. Results Setting time (mean ± S.D.; n = 4) for PCPC-Tartaric was 8.2 ± 0.8 min, significantly less than the 61.7 ± 1.5 min for the Premixed Control developed previously (p < 0.001). On 7th day immersion, the diametral tensile strength of PCPC-Tartaric reached 6.5 ± 0.8 MPa, higher than 4.5 ± 0.8 MPa of Premixed Control (p = 0.036). Osteoblast cells displayed a polygonal morphology and attached to the nano-hydroxyapatite crystals in the PCPCs. All cements had similar live cell density values (p = 0.126), indicating that the new PCPCs were as cell compatible as a non-premixed CPC control known to be biocompatible. Each of the new PCPCs had a cell viability that was not significantly different (p > 0.1) from that of the non-premixed CPC control. Significance PCPCs will eliminate the powder–liquid mixing during surgery and may also improve the cement performance. The new PCPCs supported cell attachment and yielded a high cell density and viability. Their mechanical strengths approached the reported strengths of sintered porous hydroxyapatite implants and cancellous bone. These nano-crystalline hydroxyapatite cements may be useful in dental, periodontal, and craniofacial repairs. PMID:16678895

Curative potential of GM-CSF-secreting tumor cell vaccines on established orthotopic liver tumors: mechanisms for the superior antitumor activity of live tumor cell vaccines.

PubMed

Tai, Kuo-Feng; Chen, Ding-Shinn; Hwang, Lih-Hwa

2004-01-01

In preclinical studies, tumor cells genetically engineered to secrete cytokines, hereafter referred to as tumor cell vaccines, can often generate systemic antitumor immunity. This study investigated the therapeutic effects of live or irradiated tumor cell vaccines that secrete granulocyte-macrophage colony-stimulating factor (GM-CSF) on established orthotopic liver tumors. Experimental results indicated that two doses (3 x 10(7) cells per dose) of irradiated tumor cell vaccines were therapeutically ineffective, whereas one dose (3 x 10(6) cells) of live tumor cell vaccines caused complete tumor regression. In vivo depletion of CD8+ T cells, but not natural killer cells, restored tumor formation in the live vaccine-treated animals. Additionally, the treatment of cells with live vaccine induced markedly higher levels of cytotoxic T lymphocyte activity than the irradiated vaccines in the draining lymph nodes. The higher levels of cytokine and antigen loads could partly explain the superior antitumor activity of live tumor cell vaccines, but other unidentified mechanisms could also play a role in the early T cell activation in the lymph nodes. A protocol using multiple and higher dosages of irradiated tumor cell vaccines also caused significant regression of liver tumors. These results suggest that the GM-CSF-secreting tumor cell vaccines are highly promising for orthotopic liver tumors if higher levels of immune responses are elicited during early tumor development. Copyright 2004 National Science Council, ROC and S. Karger AG, Basel

Site-Specific Bioorthogonal Labeling for Fluorescence Imaging of Intracellular Proteins in Living Cells.

PubMed

Peng, Tao; Hang, Howard C

2016-11-02

Over the past years, fluorescent proteins (e.g., green fluorescent proteins) have been widely utilized to visualize recombinant protein expression and localization in live cells. Although powerful, fluorescent protein tags are limited by their relatively large sizes and potential perturbation to protein function. Alternatively, site-specific labeling of proteins with small-molecule organic fluorophores using bioorthogonal chemistry may provide a more precise and less perturbing method. This approach involves site-specific incorporation of unnatural amino acids (UAAs) into proteins via genetic code expansion, followed by bioorthogonal chemical labeling with small organic fluorophores in living cells. While this approach has been used to label extracellular proteins for live cell imaging studies, site-specific bioorthogonal labeling and fluorescence imaging of intracellular proteins in live cells is still challenging. Herein, we systematically evaluate site-specific incorporation of diastereomerically pure bioorthogonal UAAs bearing stained alkynes or alkenes into intracellular proteins for inverse-electron-demand Diels-Alder cycloaddition reactions with tetrazine-functionalized fluorophores for live cell labeling and imaging in mammalian cells. Our studies show that site-specific incorporation of axial diastereomer of trans-cyclooct-2-ene-lysine robustly affords highly efficient and specific bioorthogonal labeling with monosubstituted tetrazine fluorophores in live mammalian cells, which enabled us to image the intracellular localization and real-time dynamic trafficking of IFITM3, a small membrane-associated protein with only 137 amino acids, for the first time. Our optimized UAA incorporation and bioorthogonal labeling conditions also enabled efficient site-specific fluorescence labeling of other intracellular proteins for live cell imaging studies in mammalian cells.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion.

PubMed

Laget, Sophie; Broncy, Lucile; Hormigos, Katia; Dhingra, Dalia M; BenMohamed, Fatima; Capiod, Thierry; Osteras, Magne; Farinelli, Laurent; Jackson, Stephen; Paterlini-Bréchot, Patrizia

2017-01-01

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion.

Photo- and electropatterning of hydrogel-encapsulated living cell arrays.

PubMed

Albrecht, Dirk R; Tsang, Valerie Liu; Sah, Robert L; Bhatia, Sangeeta N

2005-01-01

Living cells have the potential to serve as sensors, naturally integrating the response to stimuli to generate predictions about cell fate (e.g., differentiation, migration, proliferation, apoptosis). Miniaturized arrays of living cells further offer the capability to interrogate many cells in parallel and thereby enable high-throughput and/or combinatorial assays. However, the interface between living cells and synthetic chip platforms is a critical one wherein the cellular phenotype must be preserved to generate useful signals. While some cell types retain tissue-specific features on a flat (2-D) surface, it has become increasingly apparent that a 3-D physical environment will be required for others. In this paper, we present two independent methods for creating living cell arrays that are encapsulated within a poly(ethylene glycol)-based hydrogel to create a local 3-D microenvironment. First, 'photopatterning' selectively crosslinks hydrogel microstructures containing living cells with approximately 100 microm feature size. Second, 'electropatterning' utilizes dielectrophoretic forces to position cells within a prepolymer solution prior to crosslinking, forming cell patterns with micron resolution. We further combine these methods to obtain hierarchical control of cell positioning over length scales ranging from microns to centimeters. This level of microenvironmental control should enable the fabrication of next-generation cellular microarrays in which robust 3-D cultures of cells are presented with appropriate physical and chemical cues and, consequently, report on cellular responses that resemble in vivo behavior.

Polyvalent Display of Biomolecules on Live Cells.

PubMed

Shi, Peng; Zhao, Nan; Lai, Jinping; Coyne, James; Gaddes, Erin R; Wang, Yong

2018-06-04

Surface display of biomolecules on live cells offers new opportunities to treat human diseases and perform basic studies. Existing methods are primarily focused on monovalent functionalization, that is, the display of single biomolecules across the cell surface. Here we show that the surface of live cells can be functionalized to display polyvalent biomolecular structures through two-step reactions under physiological conditions. This polyvalent functionalization enables the cell surface to recognize the microenvironment one order of magnitude more effectively than with monovalent functionalization. Thus, polyvalent display of biomolecules on live cells holds great potential for various biological and biomedical applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

DLA based compressed sensing for high resolution MR microscopy of neuronal tissue.

PubMed

Nguyen, Khieu-Van; Li, Jing-Rebecca; Radecki, Guillaume; Ciobanu, Luisa

2015-10-01

In this work we present the implementation of compressed sensing (CS) on a high field preclinical scanner (17.2 T) using an undersampling trajectory based on the diffusion limited aggregation (DLA) random growth model. When applied to a library of images this approach performs better than the traditional undersampling based on the polynomial probability density function. In addition, we show that the method is applicable to imaging live neuronal tissues, allowing significantly shorter acquisition times while maintaining the image quality necessary for identifying the majority of neurons via an automatic cell segmentation algorithm. Copyright © 2015 Elsevier Inc. All rights reserved.

Drosophila Regulate Yeast Density and Increase Yeast Community Similarity in a Natural Substrate

PubMed Central

Stamps, Judy A.; Yang, Louie H.; Morales, Vanessa M.; Boundy-Mills, Kyria L.

2012-01-01

Drosophila melanogaster adults and larvae, but especially larvae, had profound effects on the densities and community structure of yeasts that developed in banana fruits. Pieces of fruit exposed to adult female flies previously fed fly-conditioned bananas developed higher yeast densities than pieces of the same fruits that were not exposed to flies, supporting previous suggestions that adult Drosophila vector yeasts to new substrates. However, larvae alone had dramatic effects on yeast density and species composition. When yeast densities were compared in pieces of the same fruits assigned to different treatments, fruits that developed low yeast densities in the absence of flies developed significantly higher yeast densities when exposed to larvae. Across all of the fruits, larvae regulated yeast densities within narrow limits, as compared to a much wider range of yeast densities that developed in pieces of the same fruits not exposed to flies. Larvae also affected yeast species composition, dramatically reducing species diversity across fruits, reducing variation in yeast communities from one fruit to the next (beta diversity), and encouraging the consistent development of a yeast community composed of three species of yeast (Candida californica, C. zemplinina, and Pichia kluvyeri), all of which were palatable to larvae. Larvae excreted viable cells of these three yeast species in their fecal pools, and discouraged the growth of filamentous fungi, processes which may have contributed to their effects on the yeast communities in banana fruits. These and other findings suggest that D. melanogaster adults and their larval offspring together engage in ‘niche construction’, facilitating a predictable microbial environment in the fruit substrates in which the larvae live and develop. PMID:22860093

Hypsographic demography: The distribution of human population by altitude

PubMed Central

Cohen, Joel E.; Small, Christopher

1998-01-01

The global distribution of the human population by elevation is quantified here. As of 1994, an estimated 1.88 × 109 people, or 33.5% of the world’s population, lived within 100 vertical meters of sea level, but only 15.6% of all inhabited land lies below 100 m elevation. The median person lived at an elevation of 194 m above sea level. Numbers of people decreased faster than exponentially with increasing elevation. The integrated population density (IPD, the number of people divided by the land area) within 100 vertical meters of sea level was significantly larger than that of any other range of elevations and represented far more people. A significant percentage of the low-elevation population lived at moderate population densities rather than at the highest densities of central large cities. Assessments of coastal hazards that focus only on large cities may substantially underestimate the number of people who could be affected. PMID:9826643

A Novel Technique to Follow Consequences of Exogenous Factors, Including Therapeutic Drugs, on Living Human Breast Epithelial Cells

DTIC Science & Technology

1999-07-01

and lipid vectors, are being tested. Concurrent with the development of procedures for live - cell imaging , we are examining the distribution of proteins...dimensional matrix. These studies have not yet begun. There are a number of procedures that must be developed and perfected in the live - cell imaging , as...components of the Wnt signaling pathway are too preliminary and require additional research prior to publication. (9) CONCLUSIONS Live cell imaging of

Scavenging of long-lived radicals by (-)-epigallocatechin-3- O-gallate and simultaneous suppression of mutation in irradiated mammalian cells

NASA Astrophysics Data System (ADS)

Kumagai, Jun; Nakama, Mitsuo; Miyazaki, Tetsuo; Ise, Tamaki; Kodama, Seiji; Watanabe, Masami

2002-07-01

Effect of (-)-epigallocatechin-3- O-gallate (EGCg) on scavenging long-lived radicals and its biological significance were investigated using electron-spin-resonance spectroscopy and mutation assay in cultured human embryo cells. EGCg scavenged long-lived radicals in irradiated golden hamster embryo cells and albumin solution, and simultaneously reduced mutation frequency in the irradiated human embryo cells. These results indicate that long-lived radials are involved in the induction of mutation by radiation.

Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient.

PubMed

Ren, Xiang; Wang, Fuyou; Chen, Cheng; Gong, Xiaoyuan; Yin, Li; Yang, Liu

2016-07-20

Cartilage tissue engineering is a promising approach for repairing and regenerating cartilage tissue. To date, attempts have been made to construct zonal cartilage that mimics the cartilaginous matrix in different zones. However, little attention has been paid to the chondrocyte density gradient within the articular cartilage. We hypothesized that the chondrocyte density gradient plays an important role in forming the zonal distribution of extracellular matrix (ECM). In this study, collagen type II hydrogel/chondrocyte constructs were fabricated using a bioprinter. Three groups were created according to the total cell seeding density in collagen type II pre-gel: Group A, 2 × 10(7) cells/mL; Group B, 1 × 10(7) cells/mL; and Group C, 0.5 × 10(7) cells/mL. Each group included two types of construct: one with a biomimetic chondrocyte density gradient and the other with a single cell density. The constructs were cultured in vitro and harvested at 0, 1, 2, and 3 weeks for cell viability testing, reverse-transcription quantitative PCR (RT-qPCR), biochemical assays, and histological analysis. We found that total ECM production was positively correlated with the total cell density in the early culture stage, that the cell density gradient distribution resulted in a gradient distribution of ECM, and that the chondrocytes' biosynthetic ability was affected by both the total cell density and the cell distribution pattern. Our results suggested that zonal engineered cartilage could be fabricated by bioprinting collagen type II hydrogel constructs with a biomimetic cell density gradient. Both the total cell density and the cell distribution pattern should be optimized to achieve synergistic biological effects.

Cuticular antifungals in spiders: density- and condition dependence.

PubMed

González-Tokman, Daniel; Ruch, Jasmin; Pulpitel, Tamara; Ponton, Fleur

2014-01-01

Animals living in groups face a high risk of disease contagion. In many arthropod species, cuticular antimicrobials constitute the first protective barrier that prevents infections. Here we report that group-living spiders produce cuticular chemicals which inhibit fungal growth. Given that cuticular antifungals may be costly to produce, we explored whether they can be modulated according to the risk of contagion (i.e. under high densities). For this purpose, we quantified cuticular antifungal activity in the subsocial crab spider Diaea ergandros in both natural nests and experimentally manipulated nests of varying density. We quantified the body-condition of spiders to test whether antifungal activity is condition dependent, as well as the effect of spider density on body-condition. We predicted cuticular antifungal activity to increase and body-condition to decrease with high spider densities, and that antifungal activity would be inversely related to body-condition. Contrary to our predictions, antifungal activity was neither density- nor condition-dependent. However, body-condition decreased with density in natural nests, but increased in experimental nests. We suggest that pathogen pressure is so important in nature that it maintains high levels of cuticular antifungal activity in spiders, impacting negatively on individual energetic condition. Future studies should identify the chemical structure of the isolated antifungal compounds in order to understand the physiological basis of a trade-off between disease prevention and energetic condition caused by group living, and its consequences in the evolution of sociality in spiders.

Cuticular Antifungals in Spiders: Density- and Condition Dependence

PubMed Central

González-Tokman, Daniel; Ruch, Jasmin; Pulpitel, Tamara; Ponton, Fleur

2014-01-01

Animals living in groups face a high risk of disease contagion. In many arthropod species, cuticular antimicrobials constitute the first protective barrier that prevents infections. Here we report that group-living spiders produce cuticular chemicals which inhibit fungal growth. Given that cuticular antifungals may be costly to produce, we explored whether they can be modulated according to the risk of contagion (i.e. under high densities). For this purpose, we quantified cuticular antifungal activity in the subsocial crab spider Diaea ergandros in both natural nests and experimentally manipulated nests of varying density. We quantified the body-condition of spiders to test whether antifungal activity is condition dependent, as well as the effect of spider density on body-condition. We predicted cuticular antifungal activity to increase and body-condition to decrease with high spider densities, and that antifungal activity would be inversely related to body-condition. Contrary to our predictions, antifungal activity was neither density- nor condition-dependent. However, body-condition decreased with density in natural nests, but increased in experimental nests. We suggest that pathogen pressure is so important in nature that it maintains high levels of cuticular antifungal activity in spiders, impacting negatively on individual energetic condition. Future studies should identify the chemical structure of the isolated antifungal compounds in order to understand the physiological basis of a trade-off between disease prevention and energetic condition caused by group living, and its consequences in the evolution of sociality in spiders. PMID:24637563

Cellular Prion Protein and Caveolin-1 Interaction in a Neuronal Cell Line Precedes Fyn/Erk 1/2 Signal Transduction

PubMed Central

Toni, Mattia; Spisni, Enzo; Griffoni, Cristiana; Santi, Spartaco; Riccio, Massimo; Lenaz, Patrizia; Tomasi, Vittorio

2006-01-01

It has been reported that cellular prion protein (PrPc) is enriched in caveolae or caveolae-like domains with caveolin-1 (Cav-1) participating to signal transduction events by Fyn kinase recruitment. By using the Glutathione-S-transferase (GST)-fusion proteins assay, we observed that PrPc strongly interacts in vitro with Cav-1. Thus, we ascertained the PrPc caveolar localization in a hypothalamic neuronal cell line (GN11), by confocal microscopy analysis, flotation on density gradient, and coimmunoprecipitation experiments. Following the anti-PrPc antibody-mediated stimulation of live GN11 cells, we observed that PrPc clustered on plasma membrane domains rich in Cav-1 in which Fyn kinase converged to be activated. After these events, a signaling cascade through p42/44 MAP kinase (Erk 1/2) was triggered, suggesting that following translocations from rafts to caveolae or caveolaelike domains PrPc could interact with Cav-1 and induce signal transduction events. PMID:17489019

New turn-on fluorescent and colorimetric probe for cyanide detection based on BODIPY-salicylaldehyde and its application in cell imaging.

PubMed

Sukato, Rangsarit; Sangpetch, Nuanphan; Palaga, Tanapat; Jantra, Suthikorn; Vchirawongkwin, Viwat; Jongwohan, Chanantida; Sukwattanasinitt, Mongkol; Wacharasindhu, Sumrit

2016-08-15

Development of cyanide sensor is important as the anion is harmful to human health and the environment. Herein, a new colorimetric and fluorescent probe GSB based on boron dipyrrole-methene (BODIPY) containing salicylaldehyde group for cyanide detection has been reported. GSB undergoes exclusive colorimetric change from orange to colorless and exhibits selective fluorescence turn-on at 504nm upon the addition of cyanide. Other 13 anions give almost no interference under physiological condition. Detection limit of the new cyanide-sensing GSB is 0.88μM, which is below World Health Organization (WHO) recommended level in drinking water. A calculation by density functional theory (DFT) shows suppression of photoinduced electron transfer (PET) mechanism along with the interruption of π-conjugation between salicylaldehyde and BODIPY core by cyanide anion. Cell imaging studies demonstrated that GSB is compatible and capable of sensing cyanide anion in living cells. Copyright © 2016 Elsevier B.V. All rights reserved.

A new mode of pancreatic islet innervation revealed by live imaging in zebrafish.

PubMed

Yang, Yu Hsuan Carol; Kawakami, Koichi; Stainier, Didier Yr

2018-06-19

Pancreatic islets are innervated by autonomic and sensory nerves that influence their function. Analyzing the innervation process should provide insight into the nerve-endocrine interactions and their roles in development and disease. Here, using in vivo time-lapse imaging and genetic analyses in zebrafish, we determined the events leading to islet innervation. Comparable neural density in the absence of vasculature indicates that it is dispensable for early pancreatic innervation. Neural crest cells are in close contact with endocrine cells early in development. We find these cells give rise to neurons that extend axons towards the islet as they surprisingly migrate away. Specific ablation of these neurons partly prevents other neurons from migrating away from the islet resulting in diminished innervation. Thus, our studies establish the zebrafish as a model to interrogate mechanisms of organ innervation, and reveal a novel mode of innervation whereby neurons establish connections with their targets before migrating away. © 2018, Yang et al.

A turn-on supramolecular fluorescent probe for sensing benzimidazole fungicides and its application in living cell imaging

NASA Astrophysics Data System (ADS)

Tang, Qing; Zhang, Jing; Sun, Tao; Wang, Cheng-Hui; Huang, Ying; Zhou, Qingdi; Wei, Gang

2018-02-01

A cucurbit[8]uril-based turn-on supramolecular fluorescent probe between cucurbit[8]uril (Q[8]) and pyronine Y (PyY) (designated 2PyY@Q[8]) in acidic aqueous solution showed a remarkable fluorescence 'turn-on' response to benzimidazole fungicides such as thiabendazole, fuberidazole and carbendazim. The 2PyY@Q[8] fluorescent probe can be used to detect benzimidazole fungicides with high sensitivity and selectivity with a detection limit of 10- 8 mol/L. A good linear relationship of emission intensity at 580 nm for benzimidazole fungicides at concentrations of 0.4-5.0 μmol/L was observed. The proposed sensing mechanism was investigated using 1H NMR spectroscopy combined with density functional theory calculations at the B3LYP/6-31G(d) level. The cell imaging study showed that the 2PyY@Q[8] complex could be used to image benzimidazole fungicide in prostate cancer (PC3) cells, which may help to elucidate relevant biological processes at the molecular level.

Classification of phytoplankton cells as live or dead using the vital stains fluorescein diacetate and 5-chloromethylfluorescein diacetate.

PubMed

MacIntyre, Hugh L; Cullen, John J

2016-08-01

Regulations for ballast water treatment specify limits on the concentrations of living cells in discharge water. The vital stains fluorescein diacetate (FDA) and 5-chloromethylfluorescein diacetate (CMFDA) in combination have been recommended for use in verification of ballast water treatment technology. We tested the effectiveness of FDA and CMFDA, singly and in combination, in discriminating between living and heat-killed populations of 24 species of phytoplankton from seven divisions, verifying with quantitative growth assays that uniformly live and dead populations were compared. The diagnostic signal, per-cell fluorescence intensity, was measured by flow cytometry and alternate discriminatory thresholds were defined statistically from the frequency distributions of the dead or living cells. Species were clustered by staining patterns: for four species, the staining of live versus dead cells was distinct, and live-dead classification was essentially error free. But overlap between the frequency distributions of living and heat-killed cells in the other taxa led to unavoidable errors, well in excess of 20% in many. In 4 very weakly staining taxa, the mean fluorescence intensity in the heat-killed cells was higher than that of the living cells, which is inconsistent with the assumptions of the method. Applying the criteria of ≤5% false negative plus ≤5% false positive errors, and no significant loss of cells due to staining, FDA and FDA+CMFDA gave acceptably accurate results for only 8-10 of 24 species (i.e., 33%-42%). CMFDA was the least effective stain and its addition to FDA did not improve the performance of FDA alone. © 2016 The Authors. Journal of Phycology published by Wiley Periodicals, Inc. on behalf of Phycological Society of America.

Separation of active and inactive fractions from starved culture of Vibrio parahaemolyticus by density dependent cell sorting.

PubMed

Nayak, Binaya Bhusan; Kamiya, Eriko; Nishino, Tomohiko; Wada, Minoru; Nishimura, Masahiko; Kogure, Kazuhiro

2005-01-01

The co-existence of physiologically different cells in bacterial cultures is a general phenomenon. We have examined the applicability of the density dependent cell sorting (DDCS) method to separate subpopulations from a long-term starvation culture of Vibrio parahaemolyticus. The cells were subjected to Percoll density gradient and separated into 12 fractions of different buoyant densities, followed by measuring the cell numbers, culturability, respiratory activity and leucine incorporation activity. While more than 78% of cells were in lighter fractions, about 95% of culturable cells were present in heavier fractions. The high-density subpopulations also had high proportion of cells capable of forming formazan granules. Although this was accompanied by the cell specific INT-reduction rate, both leucine incorporation rates and INT-reduction rates per cell had a peak at mid-density fraction. The present results indicated that DDCS could be used to separate subpopulations of different physiological conditions.

Affinity of antigen encounter and other early B-cell signals determine B-cell fate

PubMed Central

Benson, Micah J; Erickson, Loren D; Gleeson, Michael W; Noelle, Randolph J

2010-01-01

Three possible effector fates await the naïve follicular B cell following antigen stimulation in thymus-dependent reactions. Short-lived plasma cells produce an initial burst of germline-encoded protective antibodies, and long-lived plasma cells and memory B cells arise from the germinal center and function to enhance and sustain the humoral immune response. The inherent B-cell receptor affinity of naïve follicular B cells and the contribution of other early B-cell signals pre-determines the pattern of transcription factor expression and the differentiation path taken by these cells. High initial B-cell receptor affinity shunts naïve follicular B-cell clones towards the short-lived plasma cell fate, whereas modest-affinity clones are skewed towards a plasma cell fate and low-affinity clones are recruited into the germinal center and are selected for both long-lived plasma cells and memory B cell pathways. In the germinal center reaction, increased levels of the transcription factor interferon regulatory factor-4 drive the molecular program that dictates differentiation into the long-lived plasma cell phenotype but has no impact on the memory B cell compartment. We hypothesize that graded interferon regulatory factor-4 levels driven by signals to B cells, including B-cell receptor signal strength, are responsible for this branch point in the B-cell terminal differentiation pathway. PMID:17433651

Investigating the Functional Role of Prostate-Specific Membrane Antigen and its Enzymatic Activity in Prostate Cancer Metastasis

DTIC Science & Technology

2008-02-01

fluorescent probes for live cell imaging . PSMA distribution of cells grown on different extracellular matrices will be characterized to provide guidance...PCa migration, using in vitro cell model systems and live - cell imaging methods, we characterized the role of PSMA in cell motility and adhesion. Using...Generated fluorescently conjugated anti-PSMA antibodies for live cell imaging . 2. Optimized the siRNA-PSMA transfection and achieved an approximately

Changes of buoyant density during the S-phase of the cell cycle. Direct evidence demonstrated in acute myeloid leukemia by flowcytometry.

PubMed

Daenen, S; Huiges, W; Modderman, E; Halie, M R

1993-01-01

Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows that the cellular buoyant density increases slightly with up to 0.008 g/ml during the S-phase, at least in cryo-preserved cells used in this study. This contrasts with the generally accepted belief that S-phase cells have a lower or constant buoyant density. A practical implication is that separation of cell (sub)populations based on differences in buoyant density could be flawed to the extent that these populations contain S-phase cells.

Correlating measured transient temperature rises with damage rate processes in cultured cells

NASA Astrophysics Data System (ADS)

Denton, Michael L.; Tijerina, Amanda J.; Gonzalez, Cherry C.; Gamboa, B. Giovana; Noojin, Gary D.; Ahmed, Elharith M.; Rickman, John M.; Dyer, Phillip H.; Rockwell, Benjamin A.

2017-02-01

Thermal damage rate processes in biological tissues are usually characterized by a kinetics approach. This stems from experimental data that show how the transformation of a specified biological property of cells or biomolecule (plating efficiency for viability, change in birefringence, tensile strength, etc.) is dependent upon both time and temperature. Here, two disparate approaches were used to study thermal damage rate processes in cultured retinal pigment epithelial cells. Laser exposure (photothermal) parameters included 2-μm laser exposure of non-pigmented cells and 532-nm exposures of cells possessing a variety of melanosome particle densities. Photothermal experiments used a mid-IR camera to record temperature histories with spatial resolution of about 8 μm, while fluorescence microscopy of the cell monolayers identified threshold damage at the boundary between live and dead cells. Photothermal exposure durations ranged from 0.05-20 s, and the effects of varying ambient temperature were investigated. Temperature during heat transfer using a water-jacketed cuvette was recorded with a fast microthermister, while damage and viability of the suspended cells were determined as percentages. Exposure durations for the heat transfer experiments ranged from 50- 60 s. Empirically-determined kinetic parameters for the two heating methods were compared with each other, and with values found in the literature.

Magnetization of individual yeast cells by in situ formation of iron oxide on cell surfaces

NASA Astrophysics Data System (ADS)

Choi, Jinsu; Lee, Hojae; Choi, Insung S.; Yang, Sung Ho

2017-09-01

Magnetic functionalization of living cells has intensively been investigated with the aim of various bioapplications such as selective separation, targeting, and localization of the cells by using an external magnetic field. However, the magnetism has not been introduced to individual living cells through the in situ chemical reactions because of harsh conditions required for synthesis of magnetic materials. In this work, magnetic iron oxide was formed on the surface of living cells by optimizing reactions conditions to be mild sufficiently enough to sustain cell viability. Specifically, the reactive LbL strategy led to formation of magnetically responsive yeast cells with iron oxide shells. This facile and direct post-magnetization method would be a useful tool for remote manipulation of living cells with magnetic interactions, which is an important technique for the integration of cell-based circuits and the isolation of cell in microfluidic devices.

Live-Cell Imaging of Filoviruses.

PubMed

Schudt, Gordian; Dolnik, Olga; Becker, Stephan

2017-01-01

Observation of molecular processes inside living cells is fundamental to a deeper understanding of virus-host interactions in filoviral-infected cells. These observations can provide spatiotemporal insights into protein synthesis, protein-protein interaction dynamics, and transport processes of these highly pathogenic viruses. Thus, live-cell imaging provides the possibility for antiviral screening in real time and gives mechanistic insights into understanding filovirus assembly steps that are dependent on cellular factors, which then represent potential targets against this highly fatal disease. Here we describe analysis of living filovirus-infected cells under maximum biosafety (i.e., BSL4) conditions using plasmid-driven expression of fluorescently labeled viral and cellular proteins and/or viral genome-encoded expression of fluorescently labeled proteins. Such multiple-color and multidimensional time-lapse live-cell imaging analyses are a powerful method to gain a better understanding of the filovirus infection cycle.

Therapeutic administration of enrofloxacin in mice does not select for fluoroquinolone resistance in Campylobacter jejuni.

PubMed

Inglis, G Douglas; Zaytsoff, S J M; Selinger, L Brent; Taboada, Eduardo N; Uwiera, R R E

2018-05-11

Enrofloxacin is registered for therapeutic use in beef cattle to treat bovine respiratory disease in Canada. A murine model was used to experimentally examine the impact of therapeutic administration of enrofloxacin on fluoroquinolone resistance development in Campylobacter jejuni. Administration of enrofloxacin to mice via subcutaneous injection or per os routes resulted in equivalent levels of bioactive enrofloxacin within the intestine, but bioactivity was short-lived (<48 hr after cessation). Enrofloxacin administration did not affect densities of total bacteria, Firmicutes, or Bacteroidetes in digesta, and had modest impacts on densities of Enterobacteriaceae. All mice inoculated with C. jejuni NCTC 11168 became persistently colonized by the bacterium. Enrofloxacin reduced C. jejuni cell densities within the cecal and colonic digesta for all treatments, and densities shed in feces as a function of antibiotic duration. None of the C. jejuni isolates recovered from mice after administration of enrofloxacin (n=260) developed resistance to ciprofloxacin regardless of method or duration of administration. Furthermore, only modest shifts in the minimum inhibitory concentration of the isolates by treatment were noted. The study findings indicate that the risk posed by short-term subcutaneous administration of enrofloxacin for the development of fluoroquinolone resistance in mammals is low.

High-speed atomic force microscopy imaging of live mammalian cells

PubMed Central

Shibata, Mikihiro; Watanabe, Hiroki; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

2017-01-01

Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons. PMID:28900590

Detection and quantification of subtle changes in red blood cell density using a cell phone.

PubMed

Felton, Edward J; Velasquez, Anthony; Lu, Shulin; Murphy, Ryann O; ElKhal, Abdala; Mazor, Ofer; Gorelik, Pavel; Sharda, Anish; Ghiran, Ionita C

2016-08-16

Magnetic levitation has emerged as a technique that offers the ability to differentiate between cells with different densities. We have developed a magnetic levitation system for this purpose that distinguishes not only different cell types but also density differences in cells of the same type. This small-scale system suspends cells in a paramagnetic medium in a capillary placed between two rare earth magnets, and cells levitate to an equilibrium position determined solely by their density. Uniform reference beads of known density are used in conjunction with the cells as a means to quantify their levitation positions. In one implementation images of the levitating cells are acquired with a microscope, but here we also introduce a cell phone-based device that integrates the magnets, capillary, and a lens into a compact and portable unit that acquires images with the phone's camera. To demonstrate the effectiveness of magnetic levitation in cell density analysis we carried out levitation experiments using red blood cells with artificially altered densities, and also levitated those from donors. We observed that we can distinguish red blood cells of an anemic donor from those that are healthy. Since a plethora of disease states are characterized by changes in cell density magnetic cell levitation promises to be an effective tool in identifying and analyzing pathologic states. Furthermore, the low cost, portability, and ease of use of the cell phone-based system may potentially lead to its deployment in low-resource environments.

Increased production of outer membrane vesicles by cultured freshwater bacteria in response to ultraviolet radiation.

PubMed

Gamalier, Juliana P; Silva, Thiago P; Zarantonello, Victor; Dias, Felipe F; Melo, Rossana C N

2017-01-01

Secretion of membrane vesicles is an important biological process of both eukaryotic and prokaryotic cells. This process has been characterized in pathogenic bacteria, but is less clear in non-pathogenic bacteria from aquatic ecosystems. Here, we investigated, for the first time, the process of formation of outer membranes vesicles (OMVs), nanoscale vesicles extruded from the outer membrane (OM) of gram-negative bacteria, in cultures of freshwater bacteria after exposure or not to ultraviolet radiation (UVR) as an environmental stressor. Non-axenic cultures of freshwater bacteria isolated from a Brazilian aquatic ecosystem (Funil reservoir) were exposed or not to UVR (UVA+UVB) over a 3h period, during which cell density, viability and ultrastructure were analyzed. First, we showed that UVR induce bacterial death. UVR triggered significant negative effect on cell density after 3h of UVR treatment. This decrease was directly associated with cell death as revealed by a cell viability fluorescent probe that enables the distinction of live/dead bacteria. Transmission electron microscopy (TEM) revealed changes indicative of cell death after 3h of UVR exposure, with significant increase of damaged cells compared to the control group. Second, we demonstrated that gram-negative bacteria release OMVs during normal growth and after UVR exposure. OMVs were clearly identified as round, membrane-bound vesicles budding off from the bacterial OM as isolated or clustered vesicles or free in the extracellular medium. Remarkably, quantitative TEM analyses showed that bacteria respond to UVR with increased formation of OMVs. Moreover, while OMVs numbers per intact or damaged cell did not differ in the untreated group, UVR led to a higher vesiculation by bacteria in process of death. This means that degenerating bacteria release OMVs before lysis and that this secretion might be an adaptive/protective response to rapid changes in environmental conditions such as UV radiation. Copyright © 2016 Elsevier GmbH. All rights reserved.

Rapid determination of cell mass and density using digitally controlled electric field in a microfluidic chip.

PubMed

Zhao, Yuliang; Lai, Hok Sum Sam; Zhang, Guanglie; Lee, Gwo-Bin; Li, Wen Jung

2014-11-21

The density of a single cell is a fundamental property of cells. Cells in the same cycle phase have similar volume, but the differences in their mass and density could elucidate each cell's physiological state. Here we report a novel technique to rapidly measure the density and mass of a single cell using an optically induced electrokinetics (OEK) microfluidic platform. Presently, single cellular mass and density measurement devices require a complicated fabrication process and their output is not scalable, i.e., it is extremely difficult to measure the mass and density of a large quantity of cells rapidly. The technique reported here operates on a principle combining sedimentation theory, computer vision, and microparticle manipulation techniques in an OEK microfluidic platform. We will show in this paper that this technique enables the measurement of single-cell volume, density, and mass rapidly and accurately in a repeatable manner. The technique is also scalable - it allows simultaneous measurement of volume, density, and mass of multiple cells. Essentially, a simple time-controlled projected light pattern is used to illuminate the selected area on the OEK microfluidic chip that contains cells to lift the cells to a particular height above the chip's surface. Then, the cells are allowed to "free fall" to the chip's surface, with competing buoyancy, gravitational, and fluidic drag forces acting on the cells. By using a computer vision algorithm to accurately track the motion of the cells and then relate the cells' motion trajectory to sedimentation theory, the volume, mass, and density of each cell can be rapidly determined. A theoretical model of micro-sized spheres settling towards an infinite plane in a microfluidic environment is first derived and validated experimentally using standard micropolystyrene beads to demonstrate the viability and accuracy of this new technique. Next, we show that the yeast cell volume, mass, and density could be rapidly determined using this technology, with results comparable to those using the existing method suspended microchannel resonator.

The density of apical cells of dark-grown protonemata of the moss Ceratodon purpureus

NASA Technical Reports Server (NTRS)

Schwuchow, J. M.; Kern, V. D.; Wagner, T.; Sack, F. D.

2000-01-01

Determinations of plant or algal cell density (cell mass divided by volume) have rarely accounted for the extracellular matrix or shrinkage during isolation. Three techniques were used to indirectly estimate the density of intact apical cells from protonemata of the moss Ceratodon purpureus. First, the volume fraction of each cell component was determined by stereology, and published values for component density were used to extrapolate to the entire cell. Second, protonemal tips were immersed in bovine serum albumin solutions of different densities, and then the equilibrium density was corrected for the mass of the cell wall. Third, apical cell protoplasts were centrifuged in low-osmolarity gradients, and values were corrected for shrinkage during protoplast isolation. Values from centrifugation (1.004 to 1.015 g/cm3) were considerably lower than from other methods (1.046 to 1.085 g/cm3). This work appears to provide the first corrected estimates of the density of any plant cell. It also documents a method for the isolation of protoplasts specifically from apical cells of protonemal filaments.

NaK-ATPase pump sites in cultured bovine corneal endothelium of varying cell density at confluence.

PubMed

Crawford, K M; Ernst, S A; Meyer, R F; MacCallum, D K

1995-06-01

The driving force for ion and water flow necessary for efficient deturgesence of the corneal stroma resides in the ouabain-sensitive sodium (Na) pump of corneal endothelial cells. Using a cell culture model of corneal endothelial cell hypertrophy, the authors examined the expression of Na pumps at the cell surface to see how this central element of the endothelial pump changed as corneal endothelial cell density decreased to a level associated with corneal decompensation in vivo. 3H-ouabain binding to NaK-ATPase at saturating conditions was used to quantitate the number of Na pump sites on cultured bovine corneal endothelial cells as the confluent density decreased from approximately 2750 cells/mm2 to approximately 275 cells/mm2. The mean number of Na pump sites per cell at confluence (1.92 +/- 0.07 x 10(6)) did not change as the cell density decreased 2.7-fold from 2763 cells/mm2 to 1000 cells/mm2. However, pump site expression doubled to approximately 4 x 10(6) sites/cell as the cell density decreased from 1000 cells/mm2 to 275 cells/mm2. Despite the incremental increase in Na pump site expression that occurred as the cells hypertrophied below a density of 1000/mm2 to achieve confluence, this increase was insufficient to prevent a decrease in Na pump site density of the intact monolayer, expressed as pump sites/mm2. The confluent cell density of cultured bovine corneal endothelial cells can be varied from that found in the normal native cornea to that associated with corneal decompensation. In confluent cultures with cell densities ranging from 2750 cells/mm2 to 1000 cells/mm2, the number of pump sites per cell remains relatively unchanged. Below cell densities of 1000 cells/mm2, the number of pump sites per cell progressively increases. The increased Na pump site abundance in markedly hypertrophied endothelial cells cannot adequately compensate for the progressive reduction in the number of transporting cells per unit area within the intact monolayer. Even when considered with the decrease in the size of the paracellular ion conductive pathway that is a consequence of progressive endothelial hypertrophy, the overall pumping capacity of the intact endothelial monolayer declines.

Optimizing temperament through litter size in short-lived, iteroparous mammals in seasonal environments.

PubMed

Eccard, Jana A; Rödel, Heiko G

2011-09-01

A number of short-lived, iteroparous animal species have small broods in the early breeding season and larger broods in later breeding season. Brood size affects not only offspring size, but as recent results suggest, may also affect offspring's temperament, hormonal status, and aggression as adults. Most populations of short-lived, iteroparous mammals fluctuate predictably over the season, with low densities in winter, increasing densities in summer and a population peak in late summer followed by a population breakdown. If animals live only through parts of the season, possibly such differences in density and hence also in social environments among seasons require different personality types to increase individual fitness. We present data on behavior of European rabbits from a field enclosure study. These data clearly show that aggressiveness is higher in young from smaller litters than in young from larger litters, and smaller litters are usually born during the early breeding season. Moreover, our data suggest that behavioral types of the young rabbits are stable over time, at least during their subadult life. We suggest, that changes in mean litter size over the course of the breeding season may not only be a product of mothers' age or food availability, but may also have an adaptive function by preparing offspring characteristics for adulthood in a social environment undergoing predictable density changes within the season. Copyright © 2011 Wiley Periodicals, Inc.

Discovery of a New Cellular Motion and Its Relevance to Breast Cancer and Involution

DTIC Science & Technology

2014-02-01

motion (CAMo), live cell imaging , confocal microscopy Overall Project Summary: During this first year of funding we have concentrated our work to...cell types in 3D cultures and in vivo. Subtask 1.1a: Real time live cell imaging using confocal microscopy will be used to image cellular movement...exciting as they are important steps in understanding behavior of normal myoepithelial cells using live cell imaging in physiologically

Socializing makes thick-skinned individuals: on the density of epidermal alarm substance cells in cyprinid fish, the crucian carp (Carassius carassius).

PubMed

Stabell, Ole B; Vegusdal, Anne

2010-09-01

In cyprinid fish, density of epidermal club cells (i.e. alarm substance cells) has been found to vary between lakes with different predator fauna. Because predators can be labelled with chemical cues from prey, we questioned if club cell density could be controlled indirectly by predators releasing prey cues. In particular, we suspected a possible feedback mechanism between chemical alarm signals and their cellular source. We raised crucian carp singly and in groups of four. For both rearing types, fish were exposed to skin extracts of either conspecifics or brown trout (without club cells), and provided either low or high food rations. Independent of rearing type, condition factor and club cell density increased with food ration size, but no change was found in club cell density following exposure to conspecific alarm signals. However, the density of club cells was found significantly higher for fish raised in groups than for fish raised alone. We conclude that an increased condition factor results in more club cells, but crucian carp may also possess an awareness of conspecific presence, given by higher club cell densities when raised in groups. This increase in club cell density may be induced by unknown chemical factors released by conspecifics.

Intersections between nativity, ethnic density, and neighborhood SES: using an ethnic enclave framework to explore variation in Puerto Ricans' physical health.

PubMed

Roy, Amanda Leigh; Hughes, Diane; Yoshikawa, Hirokazu

2013-06-01

Although past research has demonstrated a "health disadvantage" for Puerto Rican adults, very little is known about correlates of health among this group. Given Puerto Ricans' unique experiences of migration and settlement, an ethnic enclave framework that integrates nativity, ethnic density, and neighborhood SES may offer insight into factors influencing Puerto Ricans' health. This study uses a sample of 449 adult mainland- and island-born Puerto Ricans living in New York City and Chicago. The data, collected as a part of the MIDUS Survey of Minority Groups, are stratified by neighborhood ethnic density and neighborhood SES, allowing for the examination of the individual and joint influences of neighborhood characteristics on physical health. Results revealed that ethnic density and neighborhood SES were not independently or interactively related to physical health for mainland-born Puerto Ricans. However, the interaction between ethnic density and neighborhood SES was related to self-reported health, functional limitations, and health symptoms for island-born Puerto Ricans. Island-born Puerto Ricans living in ethnically dense, low SES neighborhoods reported worse health than island-born Puerto Ricans living in other types of neighborhoods. This may be a result of isolation from resources both within and outside the neighborhood.

Noncontact Measurement of the Local Mechanical Properties of Living Cells Using Pressure Applied via a Pipette

PubMed Central

Sánchez, Daniel; Johnson, Nick; Li, Chao; Novak, Pavel; Rheinlaender, Johannes; Zhang, Yanjun; Anand, Uma; Anand, Praveen; Gorelik, Julia; Frolenkov, Gregory I.; Benham, Christopher; Lab, Max; Ostanin, Victor P.; Schäffer, Tilman E.; Klenerman, David; Korchev, Yuri E.

2008-01-01

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1–150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells. PMID:18515369

Opportunities for Live Cell FT-Infrared Imaging: Macromolecule Identification with 2D and 3D Localization

PubMed Central

Mattson, Eric C.; Aboualizadeh, Ebrahim; Barabas, Marie E.; Stucky, Cheryl L.; Hirschmugl, Carol J.

2013-01-01

Infrared (IR) spectromicroscopy, or chemical imaging, is an evolving technique that is poised to make significant contributions in the fields of biology and medicine. Recent developments in sources, detectors, measurement techniques and speciman holders have now made diffraction-limited Fourier transform infrared (FTIR) imaging of cellular chemistry in living cells a reality. The availability of bright, broadband IR sources and large area, pixelated detectors facilitate live cell imaging, which requires rapid measurements using non-destructive probes. In this work, we review advances in the field of FTIR spectromicroscopy that have contributed to live-cell two and three-dimensional IR imaging, and discuss several key examples that highlight the utility of this technique for studying the structure and chemistry of living cells. PMID:24256815

Enteric coated spheres produced by extrusion/spheronization provide effective gastric protection and efficient release of live therapeutic bacteria.

PubMed

de Barros, João M S; Lechner, Tabea; Charalampopoulos, Dimitrios; Khutoryanskiy, Vitaliy V; Edwards, Alexander D

2015-09-30

We present a novel but simple enteric coated sphere formulation containing probiotic bacteria (Lactobacillus casei). Oral delivery of live bacterial cells (LBC) requires live cells to survive firstly manufacturing processes and secondly GI microbicidal defenses including gastric acid. We incorporated live L. casei directly in the granulation liquid, followed by granulation, extrusion, spheronization, drying and spray coating to produce dried live probiotic spheres. A blend of MCC, calcium-crosslinked alginate, and lactose was developed that gave improved live cell survival during manufacturing, and gave excellent protection from gastric acid plus rapid release in intestinal conditions. No significant loss of viability was observed in all steps except drying, which resulted in approximately 1 log loss of viable cells. Eudragit coating was used to protect dried live cells from acid, and microcrystalline cellulose (MCC) was combined with sodium alginate to achieve efficient sphere disintegration leading to rapid and complete bacterial cell release in intestinal conditions. Viability and release of L. casei was evaluated in vitro in simulated GI conditions. Uncoated spheres gave partial acid protection, but enteric coated spheres effectively protected dried probiotic LBC from acid for 2h, and subsequently released all viable cells within 1h of transfer into simulated intestinal fluid. Copyright © 2015 Elsevier B.V. All rights reserved.

Live cell isolation by laser microdissection with gravity transfer

NASA Astrophysics Data System (ADS)

Podgorny, Oleg V.

2013-05-01

Laser microdissection by pulsing ultraviolet laser allows the isolation and recultivation of live cells based on morphological features or/and fluorescent labelling from adherent cell cultures. Previous investigations described only the use of the laser microdissection and pressure catapulting (LMPC) for live cell isolation. But LMPC requires complex manipulations and some skill. Furthermore, single-cell cloning using laser microdissection has not yet been demonstrated. The first evidence of successful application of laser microdissection with gravity transfer (LMDGT) for capturing and recultivation of live cells is presented. A new strategy for LMDGT is presented because of the failure to reproduce the manufacturer's protocol. Using the new strategy, successful capturing and recultivation of circle-shaped samples from confluent monolayer of HeLa cells was demonstrated. It was found that LMDGT is easier than LMPC because it doesn't require personal participation of investigator in transferring of isolated samples to final culture dishes. Moreover, for the first time, the generation of clonal colonies from single live cells isolated by laser microdissection was demonstrated. Data obtained in this study confirm that LMDGT is a reliable and high-yield method allowing isolation and expansion of both cell clusters and single cells from adherent cell cultures.

Single-cell manipulation and DNA delivery technology using atomic force microscopy and nanoneedle.

PubMed

Han, Sung-Woong; Nakamura, Chikashi; Miyake, Jun; Chang, Sang-Mok; Adachi, Taiji

2014-01-01

The recent single-cell manipulation technology using atomic force microscopy (AFM) not only allows high-resolution visualization and probing of biomolecules and cells but also provides spatial and temporal access to the interior of living cells via the nanoneedle technology. Here we review the development and application of single-cell manipulations and the DNA delivery technology using a nanoneedle. We briefly describe various DNA delivery methods and discuss their advantages and disadvantages. Fabrication of the nanoneedle, visualization of nanoneedle insertion into living cells, DNA modification on the nanoneedle surface, and the invasiveness of nanoneedle insertion into living cells are described. Different methods of DNA delivery into a living cell, such as lipofection, microinjection, and nanoneedles, are then compared. Finally, single-cell diagnostics using the nanoneedle and the perspectives of the nanoneedle technology are outlined. The nanoneedle-based DNA delivery technology provides new opportunities for efficient and specific introduction of DNA and other biomolecules into precious living cells with a high spatial resolution within a desired time frame. This technology has the potential to be applied for many basic cellular studies and for clinical studies such as single-cell diagnostics.

Texture of cellulose microfibrils of root hair cell walls of Arabidopsis thaliana, Medicago truncatula, and Vicia sativa.

PubMed

Akkerman, M; Franssen-Verheijen, M A W; Immerzeel, P; Hollander, L D E N; Schel, J H N; Emons, A M C

2012-07-01

Cellulose is the most abundant biopolymer on earth, and has qualities that make it suitable for biofuel. There are new tools for the visualisation of the cellulose synthase complexes in living cells, but those do not show their product, the cellulose microfibrils (CMFs). In this study we report the characteristics of cell wall textures, i.e. the architectures of the CMFs in the wall, of root hairs of Arabidopsis thaliana, Medicago truncatula and Vicia sativa and compare the different techniques we used to study them. Root hairs of these species have a random primary cell wall deposited at the root hair tip, which covers the outside of the growing and fully grown hair. The secondary wall starts between 10 (Arabidopsis) and 40 (Vicia) μm from the hair tip and the CMFs make a small angle, Z as well as S direction, with the long axis of the root hair. CMFs are 3-4 nm wide in thin sections, indicating that single cellulose synthase complexes make them. Thin sections after extraction of cell wall matrix, leaving only the CMFs, reveal the type of wall texture and the orientation and width of CMFs, but CMF density within a lamella cannot be quantified, and CMF length is always underestimated by this technique. Field emission scanning electron microscopy and surface preparations for transmission electron microscopy reveal the type of wall texture and the orientation of individual CMFs. Only when the orientation of CMFs in subsequent deposited lamellae is different, their density per lamella can be determined. It is impossible to measure CMF length with any of the EM techniques. © 2012 The Authors Journal of Microscopy © 2012 Royal Microscopical Society.

Synthesis and Characterization of Functional Nanofilm-Coated Live Immune Cells.

PubMed

Hwang, Jangsun; Choi, Daheui; Choi, Moonhyun; Seo, Youngmin; Son, Jaewoo; Hong, Jinkee; Choi, Jonghoon

2018-05-30

Layer-by-layer (LbL) assembly techniques have been extensively studied in cell biology because of their simplicity of preparation and versatility. The applications of the LbL platform technology using polysaccharides, silicon, and graphene have been investigated. However, the applications of the above-mentioned technology using living cells remain to be fully understood. This study demonstrates a living cell-based LbL platform using various types of living cells. In addition, it confirms that the surplus charge on the outer surface of the coated cells can be used to bind the target protein. We develop a living cell-based LbL platform technology by stacking layers of hyaluronic acid (HA) and poly-l-lysine (PLL). The HA/PLL stacking results in three bilayers with a thickness of 4 ± 1 nm on the cell surface. Furthermore, the multilayer nanofilms on the cells are completely degraded after 3 days of the application of the LbL method. We also evaluate and visualize three bilayers of the nanofilm on adherent (AML-12 cells)-, nonadherent (trypsin-treated AML-12 cells)-, and circulation type [peripheral blood mononuclear cells (PBMCs)] cells by analyzing the zeta potential, cell viability, and imaging via scanning electron microscopy and confocal microscopy. Finally, we study the cytotoxicity of the nanofilm and characteristic functions of the immune cells after the nanofilm coating. The multilayer nanofilms are not acutely cytotoxic and did not inhibit the immune response of the PBMCs against stimulant. We conclude that a two bilayer nanofilm would be ideal for further study in any cell type. The living cell-based LbL platform is expected to be useful for a variety of applications in cell biology.

The Effects of Density in a High-Rise Residence Hall on Helping Behavior and Social Interaction

ERIC Educational Resources Information Center

Stoner, Kenneth L.; Thurman, Christopher W.

1978-01-01

Focuses on the effects of high-density housing on the helping behaviors and social interactions of the residents living within such buildings. Concludes by recommending ways to overcome some of the negative effects of high-density housing. (Author)

Far-field photostable optical nanoscopy (PHOTON) for real-time super-resolution single-molecular imaging of signaling pathways of single live cells

NASA Astrophysics Data System (ADS)

Huang, Tao; Browning, Lauren M.; Xu, Xiao-Hong Nancy

2012-04-01

Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions.Cellular signaling pathways play crucial roles in cellular functions and design of effective therapies. Unfortunately, study of cellular signaling pathways remains formidably challenging because sophisticated cascades are involved, and a few molecules are sufficient to trigger signaling responses of a single cell. Here we report the development of far-field photostable-optical-nanoscopy (PHOTON) with photostable single-molecule-nanoparticle-optical-biosensors (SMNOBS) for mapping dynamic cascades of apoptotic signaling pathways of single live cells in real-time at single-molecule (SM) and nanometer (nm) resolutions. We have quantitatively imaged single ligand molecules (tumor necrosis factor α, TNFα) and their binding kinetics with their receptors (TNFR1) on single live cells; tracked formation and internalization of their clusters and their initiation of intracellular signaling pathways in real-time; and studied apoptotic signaling dynamics and mechanisms of single live cells with sufficient temporal and spatial resolutions. This study provides new insights into complex real-time dynamic cascades and molecular mechanisms of apoptotic signaling pathways of single live cells. PHOTON provides superior imaging and sensing capabilities and SMNOBS offer unrivaled biocompatibility and photostability, which enable probing of signaling pathways of single live cells in real-time at SM and nm resolutions. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr11739h

Enrichment of human bone marrow aspirates for low-density mononuclear cells using a haemonetics discontinuous blood cell separator.

PubMed

Raijmakers, R; de Witte, T; Koekman, E; Wessels, J; Haanen, C

1986-01-01

Isopycnic density floatation centrifugation has been proven to be a suitable technique to enrich bone marrow aspirates for clonogenic cells on a small scale. We have tested a Haemonetics semicontinuous blood cell separator in order to process large volumes of bone marrow with minimal bone marrow manipulation. The efficacy of isopycnic density floatation was tested in a one and a two-step procedure. Both procedures showed a recovery of about 20% of the nucleated cells and 1-2% of the erythrocytes. The enrichment of clonogenic cells in the one-step procedure appeared superior to the two-step enrichment, first separating buffy coat cells. The recovery of clonogenic cells was 70 and 50%, respectively. Repopulation capacity of the low-density cell fraction containing the clonogenic cells was excellent after autologous reinfusion (6 cases) and allogeneic bone marrow transplantation (3 cases). Fast enrichment of large volumes of bone marrow aspirates with low-density cells containing the clonogenic cells by isopycnic density floatation centrifugation can be done safely using a Haemonetics blood cell separator.

Physiological Monitoring of Optically Trapped Cells: Studying the Effects of Confinement by 1064 NM Lazer Tweezers Using Microfluorometry

NASA Astrophysics Data System (ADS)

Liu, Yagang

A novel technique that combines microfluorometric detection and optical laser trapping has been developed for in-situ assessing the physiological state of an optically trapped biological sample. This optical diagnostic technique achieves high sensitivity (>30 dB signal -to-noise ratio) and high spatial resolution (~ 1 μm) over a broad spectral range (>400 nm). The fluorescence spectra derived from exogenous fluorescent probes, including laurdan, acridine orange, propidium iodide and Snarf, are used to assess the effects of optical confinement with respect to temperature, DNA structure, cell viability, and intracellular pH, respectively. In the latter three cases, fluorescence is excited via a two-photon absorption process, using the cw laser trap itself as the fluorescence excitation source. This enables the cw near infrared laser trapping beam to be used simultaneously as an optical diagnostic probe as well as an optical micromanipulator. Using microfluorometry, a temperature increase of less than several degrees centigrade was measured for test samples, including liposomes, Chinese hamster ovary (CHO) cells and human sperm cells that were held stationary by 1064 nm optical tweezers having a power density of ~10^7 W/cm^2. Additional physiological monitoring experiments indicated that there is no observable denaturation of DNA, or change of intracellular pH under typical continuous wave laser trapping conditions (P <= 400 mW). Under some circumstances, however, it was possible to achieve a decrease in cell viability with cw trapping, as monitored by a live/dead vital stain. In comparison, significant DNA denaturation and cellular physiological changes (e.g. cell death) were observed when a Q-switched pulsed laser at a threshold of ~30mu J/pulse was used as trapping source. These results generally support the conclusion that cw laser trapping at 1064 nm wavelength is a safe, non-invasive process and should prove to be of great value for understanding the mechanisms of laser microirradiation effects on living cells held stationary in a near-infrared trapping beam.

Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

PubMed

Yudowski, Guillermo A; Olsen, Olav; Adesnik, Hillel; Marek, Kurt W; Bredt, David S

2013-01-01

Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

Nuclear pore assembly proceeds by an inside-out extrusion of the nuclear envelope

PubMed Central

Otsuka, Shotaro; Bui, Khanh Huy; Schorb, Martin; Hossain, M Julius; Politi, Antonio Z; Koch, Birgit; Eltsov, Mikhail; Beck, Martin; Ellenberg, Jan

2016-01-01

The nuclear pore complex (NPC) mediates nucleocytoplasmic transport through the nuclear envelope. How the NPC assembles into this double membrane boundary has remained enigmatic. Here, we captured temporally staged assembly intermediates by correlating live cell imaging with high-resolution electron tomography and super-resolution microscopy. Intermediates were dome-shaped evaginations of the inner nuclear membrane (INM), that grew in diameter and depth until they fused with the flat outer nuclear membrane. Live and super-resolved fluorescence microscopy revealed the molecular maturation of the intermediates, which initially contained the nuclear and cytoplasmic ring component Nup107, and only later the cytoplasmic filament component Nup358. EM particle averaging showed that the evagination base was surrounded by an 8-fold rotationally symmetric ring structure from the beginning and that a growing mushroom-shaped density was continuously associated with the deforming membrane. Quantitative structural analysis revealed that interphase NPC assembly proceeds by an asymmetric inside-out extrusion of the INM. DOI: http://dx.doi.org/10.7554/eLife.19071.001 PMID:27630123

Evaluating the B-cell density with various activation functions using White Noise Path Integral Approach

NASA Astrophysics Data System (ADS)

Aban, C. J. G.; Bacolod, R. O.; Confesor, M. N. P.

2015-06-01

A The White Noise Path Integral Approach is used in evaluating the B-cell density or the number of B-cell per unit volume for a basic type of immune system response based on the modeling done by Perelson and Wiegel. From the scaling principles of Perelson [1], the B- cell density is obtained where antigens and antibodies mutates and activation function f(|S-SA|) is defined describing the interaction between a specific antigen and a B-cell. If the activation function f(|S-SA|) is held constant, the major form of the B-cell density evaluated using white noise analysis is similar to the form of the B-cell density obtained by Perelson and Wiegel using a differential approach.A piecewise linear functionis also used to describe the activation f(|S-SA|). If f(|S-SA|) is zero, the density decreases exponentially. If f(|S-SA|) = S-SA-SB, the B- cell density increases exponentially until it reaches a certain maximum value. For f(|S-SA|) = 2SA-SB-S, the behavior of B-cell density is oscillating and remains to be in small values.

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells.

PubMed

Zhang, Huijie; Oellers, Tobias; Feng, Wenqian; Abdulazim, Tarik; Saw, En Ning; Ludwig, Alfred; Levkin, Pavel A; Plumeré, Nicolas

2017-06-06

Microarray technology has shown great potential for various types of high-throughput screening applications. The main read-out methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability to not only detect but also address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections an array of three-electrode systems for precise control of the applied potential, which enables direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern, forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the nonwettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cell screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Combining bio-electrospraying with gene therapy: a novel biotechnique for the delivery of genetic material via living cells.

PubMed

Ward, Eliot; Chan, Emma; Gustafsson, Kenth; Jayasinghe, Suwan N

2010-05-01

The investigations reported in this article demonstrate the ability of bio-electrosprays and cell electrospinning to deliver a genetic construct in association with living cells. Previous studies on both bio-electrosprays and cell electrospinning demonstrated great promise for tissue engineering and regenerative biology/medicine. The investigations described herein widen the applicability of these biotechniques by combining gene therapy protocols, resulting in a novel drug delivery methodology previously unexplored. In these studies a human cell line was transduced with recombinant self-inactivating lentiviral particles. These particles incorporated a green fluorescent protein fused to an endosomal targeting construct. This construct encodes a peptide, which can subsequently be detected on the surface of cells by specific T-cells. The transduced cell line was subsequently manipulated in association with either bio-electrospraying or cell electrospinning. Hence this demonstrates (i) the ability to safely handle genetically modified living cells and (ii) the ability to directly form pre-determined architectures bearing living therapeutic cells. This merged technology demonstrates a unique approach for directly forming living therapeutic architectures for controlled and targeted release of experimental cells/genes, as well as medical cell/gene therapeutics for a plethora of biological and medical applications. Hence, such developments could be applied to personalised medicine.

Low Temperature Atmospheric Argon Plasma: Diagnostics and Medical Applications

NASA Astrophysics Data System (ADS)

Ermolaeva, Svetlana; Petrov, Oleg; Zigangirova, Nailya; Vasiliev, Mikhail; Sysolyatina, Elena; Antipov, Sergei; Alyapyshev, Maxim; Kolkova, Natalia; Mukhachev, Andrei; Naroditsky, Boris; Shimizu, Tetsuji; Grigoriev, Anatoly; Morfill, Gregor; Fortov, Vladimir; Gintsburg, Alexander

This study was devoted to diagnostic of low temperature plasma produced by microwave generator and investigation of its bactericidal effect against bacteria in biofilms and within eukaryotic cells. The profile of gas temperature near the torch outlet was measured. The spectrum in a wide range of wavelengths was derived by the method of optical emission spec-troscopy. Probe measurements of the floating potential of plasma were car-ried out. The estimation and adaptation of parameters of plasma flow (tem-perature, velocity, ion number density) according to medico-technical requirements were produced. The model of immersed surface-associated biofilms formed by Gram-negative bacteria, Pseudomonas aeruginosa and Burkholderia cenocepacia, and Gram-positive bacteria, Staphylococcus aureus, was used to assess bactericidal effects of plasma treatment. Reduction in the concentration of live bacteria in biofilms treated with plasma for 5 min was demonstrated by measuring Live/Dead fluorescent labeling and using direct plating. The intracellular infection model with the pathogenic bacterium, Chlamydia trachomatis, was used to study the efficacy of microwave argon plasma against intracellular parasites. A 2 min plasma treatment of mouse cells infected with C. trachomatis reduced infectious bacteria by a factor of 2×106. Plasma treatment diminished the number of viable host cells by about 20%. When the samples were covered with MgF2 glass to obstruct active particles and UV alone was applied, the bactericidal effect was re-duced by 5×104 fold compared to the whole plasma.

Quantitative Analysis of Statics and Dynamics of Actin Cables in Fission Yeast

NASA Astrophysics Data System (ADS)

Yusuf, Eddy; Wu, Jian-Qiu; Vavylonis, Dimitrios

2010-03-01

The assembly of actin and tubulin proteins into long filaments and bundles, i.e. closely-packed filaments, underlies important cellular processes such as cell motility, intracellular transport, and cell division. Recent theoretical and experimental work has addressed the nonequilibrium dynamics of single microtubules within live cells [1]. Actin filaments usually form dense networks that prevents microscopic imaging of individual filaments or bundles. Here, we studied actin dynamics using fission yeast that has low-density actin cytoskeleton consisting of actin cables (actin bundles aligned along the long axis of the cell) and ``actin patches.'' Yeast cells expressing GFP-CHD were imaged by 3D confocal microscopy. Stretching open active contours [2] were used to segment and track individual actin cables. We analyzed their curvature distribution, the tangent correlation, and the temporal bending amplitude fluctuations. We contrast our findings to equilibrium fluctuating semiflexible polymers and to microtubules in cells. We calculate the important time and length scales for the actin cables. We also discuss our findings within the broad context of understanding actin assembly in cells. [1] C. P. Brangwynne et. al., Phys. Rev. Lett. 100, 118104 (2008) [2] H. Li et. al., Proc. of the IEEE Int'l Symposium on Biomedical Imaging: From Nano to Macro, ISBI'09

Fixed-Cell Imaging of Schizosaccharomyces pombe.

PubMed

Hagan, Iain M; Bagley, Steven

2016-07-01

The acknowledged genetic malleability of fission yeast has been matched by impressive cytology to drive major advances in our understanding of basic molecular cell biological processes. In many of the more recent studies, traditional approaches of fixation followed by processing to accommodate classical staining procedures have been superseded by live-cell imaging approaches that monitor the distribution of fusion proteins between a molecule of interest and a fluorescent protein. Although such live-cell imaging is uniquely informative for many questions, fixed-cell imaging remains the better option for others and is an important-sometimes critical-complement to the analysis of fluorescent fusion proteins by live-cell imaging. Here, we discuss the merits of fixed- and live-cell imaging as well as specific issues for fluorescence microscopy imaging of fission yeast. © 2016 Cold Spring Harbor Laboratory Press.

Label-Free, High Resolution, Multi-Modal Light Microscopy for Discrimination of Live Stem Cell Differentiation Status.

PubMed

Zhang, Jing; Moradi, Emilia; Somekh, Michael G; Mather, Melissa L

2018-01-15

A label-free microscopy method for assessing the differentiation status of stem cells is presented with potential application for characterization of therapeutic stem cell populations. The microscopy system is capable of characterizing live cells based on the use of evanescent wave microscopy and quantitative phase contrast (QPC) microscopy. The capability of the microscopy system is demonstrated by studying the differentiation of live immortalised neonatal mouse neural stem cells over a 15 day time course. Metrics extracted from microscope images are assessed and images compared with results from endpoint immuno-staining studies to illustrate the system's performance. Results demonstrate the potential of the microscopy system as a valuable tool for cell biologists to readily identify the differentiation status of unlabelled live cells.

A cancer cell-specific fluorescent probe for imaging Cu2 + in living cancer cells

NASA Astrophysics Data System (ADS)

Wang, Chao; Dong, Baoli; Kong, Xiuqi; Song, Xuezhen; Zhang, Nan; Lin, Weiying

2017-07-01

Monitoring copper level in cancer cells is important for the further understanding of its roles in the cell proliferation, and also could afford novel copper-based strategy for the cancer therapy. Herein, we have developed a novel cancer cell-specific fluorescent probe for the detecting Cu2 + in living cancer cells. The probe employed biotin as the cancer cell-specific group. Before the treatment of Cu2 +, the probe showed nearly no fluorescence. However, the probe can display strong fluorescence at 581 nm in response to Cu2 +. The probe exhibited excellent sensitivity and high selectivity for Cu2 + over the other relative species. Under the guidance of biotin group, could be successfully used for detecting Cu2 + in living cancer cells. We expect that this design strategy could be further applied for detection of the other important biomolecules in living cancer cells.

Artificial Cell Therapy: New Strategies for the Therapeutic Delivery of Live Bacteria

PubMed Central

2005-01-01

There has been rapid growth in research regarding the use of live bacterial cells for therapeutic purposes. The recognition that these cells can be genetically engineered to synthesize products that have therapeutic potential has generated considerable interest and excitement among clinicians and health professionals. It is expected that a wide range of disease modifying substrates such as enzymes, hormones, antibodies, vaccines, and other genetic products will be used successfully and will impact upon health care substantially. However, a major limitation in the use of these bacterial cells is the complexity of delivering them to the correct target tissues. Oral delivery of live cells, lyophilized cells, and immobilized cells has been attempted but with limited success. Primarily, this is because bacterial cells are incapable of surviving passage through the gastrointestinal tract. In many occasions, when given orally, these cells have been found to provoke immunogenic responses that are undesirable. Recent studies show that these problems can be overcome by delivering live bacterial cells, such as genetically engineered cells, using artificial cell microcapsules. This review summarizes recent advances in the therapeutic use of live bacterial cells for therapy, discusses the principles of using artificial cells for the oral delivery of bacterial cells, outlines methods for preparing suitable artificial cells for this purpose, addresses potentials and limitations for their application in therapy, and provides insight for the future direction of this emergent and highly prospective technology. PMID:15689638

Aberration-free FTIR spectroscopic imaging of live cells in microfluidic devices.

PubMed

Chan, K L Andrew; Kazarian, Sergei G

2013-07-21

The label-free, non-destructive chemical analysis offered by FTIR spectroscopic imaging is a very attractive and potentially powerful tool for studies of live biological cells. FTIR imaging of live cells is a challenging task, due to the fact that cells are cultured in an aqueous environment. While the synchrotron facility has proven to be a valuable tool for FTIR microspectroscopic studies of single live cells, we have demonstrated that high quality infrared spectra of single live cells using an ordinary Globar source can also be obtained by adding a pair of lenses to a common transmission liquid cell. The lenses, when placed on the transmission cell window, form pseudo hemispheres which removes the refraction of light and hence improve the imaging and spectral quality of the obtained data. This study demonstrates that infrared spectra of single live cells can be obtained without the focus shifting effect at different wavenumbers, caused by the chromatic aberration. Spectra of the single cells have confirmed that the measured spectral region remains in focus across the whole range, while spectra of the single cells measured without the lenses have shown some erroneous features as a result of the shift of focus. It has also been demonstrated that the addition of lenses can be applied to the imaging of cells in microfabricated devices. We have shown that it was not possible to obtain a focused image of an isolated cell in a droplet of DPBS in oil unless the lenses are applied. The use of the approach described herein allows for well focused images of single cells in DPBS droplets to be obtained.

Live cell imaging of in vitro human trophoblast syncytialization.

PubMed

Wang, Rui; Dang, Yan-Li; Zheng, Ru; Li, Yue; Li, Weiwei; Lu, Xiaoyin; Wang, Li-Juan; Zhu, Cheng; Lin, Hai-Yan; Wang, Hongmei

2014-06-01

Human trophoblast syncytialization, a process of cell-cell fusion, is one of the most important yet least understood events during placental development. Investigating the fusion process in a placenta in vivo is very challenging given the complexity of this process. Application of primary cultured cytotrophoblast cells isolated from term placentas and BeWo cells derived from human choriocarcinoma formulates a biphasic strategy to achieve the mechanism of trophoblast cell fusion, as the former can spontaneously fuse to form the multinucleated syncytium and the latter is capable of fusing under the treatment of forskolin (FSK). Live-cell imaging is a powerful tool that is widely used to investigate many physiological or pathological processes in various animal models or humans; however, to our knowledge, the mechanism of trophoblast cell fusion has not been reported using a live- cell imaging manner. In this study, a live-cell imaging system was used to delineate the fusion process of primary term cytotrophoblast cells and BeWo cells. By using live staining with Hoechst 33342 or cytoplasmic dyes or by stably transfecting enhanced green fluorescent protein (EGFP) and DsRed2-Nuc reporter plasmids, we observed finger-like protrusions on the cell membranes of fusion partners before fusion and the exchange of cytoplasmic contents during fusion. In summary, this study provides the first video recording of the process of trophoblast syncytialization. Furthermore, the various live-cell imaging systems used in this study will help to yield molecular insights into the syncytialization process during placental development. © 2014 by the Society for the Study of Reproduction, Inc.

Membrane tension controls the assembly of curvature-generating proteins

PubMed Central

Simunovic, Mijo; Voth, Gregory A.

2015-01-01

Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell. PMID:26008710

From immobilized cells to motile cells on a bed-of-nails: effects of vertical nanowire array density on cell behaviour

PubMed Central

Persson, Henrik; Li, Zhen; Tegenfeldt, Jonas O.; Oredsson, Stina; Prinz, Christelle N.

2015-01-01

The field of vertical nanowire array-based applications in cell biology is growing rapidly and an increasing number of applications are being explored. These applications almost invariably rely on the physical properties of the nanowire arrays, creating a need for a better understanding of how their physical properties affect cell behaviour. Here, we investigate the effects of nanowire density on cell migration, division and morphology for murine fibroblasts. Our results show that few nanowires are sufficient to immobilize cells, while a high nanowire spatial density enables a ”bed-of-nails” regime, where cells reside on top of the nanowires and are fully motile. The presence of nanowires decreases the cell proliferation rate, even in the “bed-of-nails” regime. We show that the cell morphology strongly depends on the nanowire density. Cells cultured on low (0.1 μm−2) and medium (1 μm−2) density substrates exhibit an increased number of multi-nucleated cells and micronuclei. These were not observed in cells cultured on high nanowire density substrates (4 μm−2). The results offer important guidelines to minimize cell-function perturbations on nanowire arrays. Moreover, these findings offer the possibility to tune cell proliferation and migration independently by adjusting the nanowire density, which may have applications in drug testing. PMID:26691936

Welfare of organic laying hens kept at different indoor stocking densities in a multi-tier aviary system. II: live weight, health measures and perching.

PubMed

Steenfeldt, S; Nielsen, B L

2015-09-01

Multi-tier aviary systems, where conveyor belts below the tiers remove the manure at regular intervals, are becoming more common in organic egg production. The area on the tiers can be included in the net area available to the hens (also referred to as usable area) when calculating maximum indoor stocking densities in organic systems within the EU. In this article, results on live weight, health measures and perching are reported for organic laying hens housed in a multi-tier system with permanent access to a veranda and kept at stocking densities (D) of 6, 9 and 12 hens/m2 available floor area, with concomitant increases in the number of hens per trough, drinker, perch and nest space. In a fourth treatment, access to the top tier was blocked reducing vertical, trough, and perch access at the lowest stocking density (D6x). In all other aspects than stocking density, the experiment followed the EU regulations on the keeping of organic laying hens. Hen live weight, mortality and foot health were not affected by the stocking densities used in the present study. Other variables (plumage condition, presence of breast redness and blisters, pecked tail feathers, and perch use) were indirectly affected by the increase in stocking density through the simultaneous reduction in access to other resources, mainly perches and troughs. The welfare of the hens was mostly affected by these associated constraints, despite all of them being within the allowed minimum requirements for organic production in the EU. Although the welfare consequences reported here were assessed to be moderate to minor, it is important to take into account concurrent constraints on access to other resources when higher stocking densities are used in organic production.

Technical Insights into Highly Sensitive Isolation and Molecular Characterization of Fixed and Live Circulating Tumor Cells for Early Detection of Tumor Invasion

PubMed Central

Laget, Sophie; Dhingra, Dalia M.; BenMohamed, Fatima; Capiod, Thierry; Osteras, Magne; Farinelli, Laurent; Jackson, Stephen; Paterlini-Bréchot, Patrizia

2017-01-01

Circulating Tumor Cells (CTC) and Circulating Tumor Microemboli (CTM) are Circulating Rare Cells (CRC) which herald tumor invasion and are expected to provide an opportunity to improve the management of cancer patients. An unsolved technical issue in the CTC field is how to obtain highly sensitive and unbiased collection of these fragile and heterogeneous cells, in both live and fixed form, for their molecular study when they are extremely rare, particularly at the beginning of the invasion process. We report on a new protocol to enrich from blood live CTC using ISET® (Isolation by SizE of Tumor/Trophoblastic Cells), an open system originally developed for marker-independent isolation of fixed tumor cells. We have assessed the impact of our new enrichment method on live tumor cells antigen expression, cytoskeleton structure, cell viability and ability to expand in culture. We have also explored the ISET® in vitro performance to collect intact fixed and live cancer cells by using spiking analyses with extremely low number of fluorescent cultured cells. We describe results consistently showing the feasibility of isolating fixed and live tumor cells with a Lower Limit of Detection (LLOD) of one cancer cell per 10 mL of blood and a sensitivity at LLOD ranging from 83 to 100%. This very high sensitivity threshold can be maintained when plasma is collected before tumor cells isolation. Finally, we have performed a comparative next generation sequencing (NGS) analysis of tumor cells before and after isolation from blood and culture. We established the feasibility of NGS analysis of single live and fixed tumor cells enriched from blood by our system. This study provides new protocols for detection and characterization of CTC collected from blood at the very early steps of tumor invasion. PMID:28060956

Sociocultural Contexts of Time to First Sex among Hispanic Adolescents.

ERIC Educational Resources Information Center

Upchurch, Dawn M.; Aneshensel, Carol S.; Mudgal, Jyoti; McNeely, Clea Sucoff

2001-01-01

Examines the sociocultural influences on risk of first sex among Hispanic teens living in Los Angeles County. Hispanic teens living in low-density Hispanic neighborhoods have significantly higher risk of sex than do teens living in neighborhoods with higher levels of ambient hazards. Results highlight the importance of characterizing sociocultural…

Analysis of live cell images: Methods, tools and opportunities.

PubMed

Nketia, Thomas A; Sailem, Heba; Rohde, Gustavo; Machiraju, Raghu; Rittscher, Jens

2017-02-15

Advances in optical microscopy, biosensors and cell culturing technologies have transformed live cell imaging. Thanks to these advances live cell imaging plays an increasingly important role in basic biology research as well as at all stages of drug development. Image analysis methods are needed to extract quantitative information from these vast and complex data sets. The aim of this review is to provide an overview of available image analysis methods for live cell imaging, in particular required preprocessing image segmentation, cell tracking and data visualisation methods. The potential opportunities recent advances in machine learning, especially deep learning, and computer vision provide are being discussed. This review includes overview of the different available software packages and toolkits. Copyright © 2017. Published by Elsevier Inc.

Direct detection of RNAs in living cells using peptide-inserted Renilla luciferase.

PubMed

Andou, Takashi; Endoh, Tamaki; Mie, Masayasu; Kobatake, Eiry

2011-06-21

In this study, non-engineered RNAs were detected in living cells using bioluminescence. Two types of probe were utilized: a peptide inserted RLuc (PI-RLuc) probe and a split-RNA probe. Incorporation of the PI-RLuc and split-RNA probes enabled the direct detection of RNA introduced into living cells.

Glucagon-Secreting Alpha Cell Selective Two-Photon Fluorescent Probe TP-α: For Live Pancreatic Islet Imaging.

PubMed

Agrawalla, Bikram Keshari; Chandran, Yogeswari; Phue, Wut-Hmone; Lee, Sung-Chan; Jeong, Yun-Mi; Wan, Si Yan Diana; Kang, Nam-Young; Chang, Young-Tae

2015-04-29

Two-photon (TP) microscopy has an advantage for live tissue imaging which allows a deeper tissue penetration up to 1 mm comparing to one-photon (OP) microscopy. While there are several OP fluorescence probes in use for pancreatic islet imaging, TP imaging of selective cells in live islet still remains a challenge. Herein, we report the discovery of first TP live pancreatic islet imaging probe; TP-α (Two Photon-alpha) which can selectively stain glucagon secreting alpha cells. Through fluorescent image based screening using three pancreatic cell lines, we discovered TP-α from a TP fluorescent dye library TPG (TP-Green). In vitro fluorescence test showed that TP-α have direct interaction and appear glucagon with a significant fluorescence increase, but not with insulin or other hormones/analytes. Finally, TP-α was successfully applied for 3D imaging of live islets by staining alpha cell directly. The newly developed TP-α can be a practical tool to evaluate and identify live alpha cells in terms of localization, distribution and availability in the intact islets.

Live Cell in Vitro and in Vivo Imaging Applications: Accelerating Drug Discovery

PubMed Central

Isherwood, Beverley; Timpson, Paul; McGhee, Ewan J; Anderson, Kurt I; Canel, Marta; Serrels, Alan; Brunton, Valerie G; Carragher, Neil O

2011-01-01

Dynamic regulation of specific molecular processes and cellular phenotypes in live cell systems reveal unique insights into cell fate and drug pharmacology that are not gained from traditional fixed endpoint assays. Recent advances in microscopic imaging platform technology combined with the development of novel optical biosensors and sophisticated image analysis solutions have increased the scope of live cell imaging applications in drug discovery. We highlight recent literature examples where live cell imaging has uncovered novel insight into biological mechanism or drug mode-of-action. We survey distinct types of optical biosensors and associated analytical methods for monitoring molecular dynamics, in vitro and in vivo. We describe the recent expansion of live cell imaging into automated target validation and drug screening activities through the development of dedicated brightfield and fluorescence kinetic imaging platforms. We provide specific examples of how temporal profiling of phenotypic response signatures using such kinetic imaging platforms can increase the value of in vitro high-content screening. Finally, we offer a prospective view of how further application and development of live cell imaging technology and reagents can accelerate preclinical lead optimization cycles and enhance the in vitro to in vivo translation of drug candidates. PMID:24310493

Incomplete inhibition of HIV infection results in more HIV infected lymph node cells by reducing cell death

PubMed Central

Cele, Sandile; Ferreira, Isabella Markham; Young, Andrew C; Karim, Farina; Madansein, Rajhmun; Dullabh, Kaylesh J; Chen, Chih-Yuan; Buckels, Noel J; Ganga, Yashica; Khan, Khadija; Boulle, Mikael; Lustig, Gila; Neher, Richard A

2018-01-01

HIV has been reported to be cytotoxic in vitro and in lymph node infection models. Using a computational approach, we found that partial inhibition of transmissions of multiple virions per cell could lead to increased numbers of live infected cells. If the number of viral DNA copies remains above one after inhibition, then eliminating the surplus viral copies reduces cell death. Using a cell line, we observed increased numbers of live infected cells when infection was partially inhibited with the antiretroviral efavirenz or neutralizing antibody. We then used efavirenz at concentrations reported in lymph nodes to inhibit lymph node infection by partially resistant HIV mutants. We observed more live infected lymph node cells, but with fewer HIV DNA copies per cell, relative to no drug. Hence, counterintuitively, limited attenuation of HIV transmission per cell may increase live infected cell numbers in environments where the force of infection is high. PMID:29555018

Living shorelines enhanced the resilience of saltmarshes to Hurricane Matthew (2016).

PubMed

Smith, Carter S; Puckett, Brandon; Gittman, Rachel K; Peterson, Charles H

2018-06-01

Nature-based solutions, such as living shorelines, have the potential to restore critical ecosystems, enhance coastal sustainability, and increase resilience to natural disasters; however, their efficacy during storm events compared to traditional hardened shorelines is largely untested. This is a major impediment to their implementation and promotion to policy-makers and homeowners. To address this knowledge gap, we evaluated rock sill living shorelines as compared to natural marshes and hardened shorelines (i.e., bulkheads) in North Carolina, USA for changes in surface elevation, Spartina alterniflora stem density, and structural damage from 2015 to 2017, including before and after Hurricane Matthew (2016). Our results show that living shorelines exhibited better resistance to landward erosion during Hurricane Matthew than bulkheads and natural marshes. Additionally, living shorelines were more resilient than hardened shorelines, as they maintained landward elevation over the two-year study period without requiring any repair. Finally, rock sill living shorelines were able to enhance S. alterniflora stem densities over time when compared to natural marshes. Our results suggest that living shorelines have the potential to improve coastal resilience while supporting important coastal ecosystems. © 2018 by the Ecological Society of America.

Microfluidic devices, systems, and methods for quantifying particles using centrifugal force

DOEpatents

Schaff, Ulrich Y.; Sommer, Gregory J.; Singh, Anup K.

2015-11-17

Embodiments of the present invention are directed toward microfluidic systems, apparatus, and methods for measuring a quantity of cells in a fluid. Examples include a differential white blood cell measurement using a centrifugal microfluidic system. A method may include introducing a fluid sample containing a quantity of cells into a microfluidic channel defined in part by a substrate. The quantity of cells may be transported toward a detection region defined in part by the substrate, wherein the detection region contains a density media, and wherein the density media has a density lower than a density of the cells and higher than a density of the fluid sample. The substrate may be spun such that at least a portion of the quantity of cells are transported through the density media. Signals may be detected from label moieties affixed to the cells.

Cell surface expression of beta 2-microglobulin (beta 2m) correlates with stages of differentiation in B cell tumours.

PubMed Central

Jones, R A; Scott, C S; Norfolk, D R; Stark, A N; Child, J A

1987-01-01

Cell surface beta 2-microglobulin (beta 2m) densities of malignant B cells were determined by enzyme immunoassay in 97 cases of immunologically defined lymphoproliferative disease. Absolute beta 2m densities were found to depend on disease category with the lowest levels found on cells from chronic lymphocytic leukaemia (mean = 5.6 ng/10(6) cells, n = 27); atypical chronic lymphocytic leukaemia (mean = 5.9 ng/10(6) cells, n = 8); and prolymphocytoid chronic lymphocytic leukaemia variant (mean = 6.0 ng/10(6) cells, n = 16). beta 2m densities for B non-Hodgkin's lymphoma (n = 14) and B prolymphocytic leukaemia (n = 17) cases were 8.1 and 10.0 ng/10(6) cells, respectively, and the highest densities were found on cells from "late-B cell" tumours (mean = 14.3 ng/10(6) cells). Plasma cells from cases of Ig secreting tumours expressed unexpectedly low beta 2m densities (mean = 9.3 ng/10(6) cells; n = 6). PMID:3108331

Optical computed tomography for spatially isotropic four-dimensional imaging of live single cells

PubMed Central

Kelbauskas, Laimonas; Shetty, Rishabh; Cao, Bin; Wang, Kuo-Chen; Smith, Dean; Wang, Hong; Chao, Shi-Hui; Gangaraju, Sandhya; Ashcroft, Brian; Kritzer, Margaret; Glenn, Honor; Johnson, Roger H.; Meldrum, Deirdre R.

2017-01-01

Quantitative three-dimensional (3D) computed tomography (CT) imaging of living single cells enables orientation-independent morphometric analysis of the intricacies of cellular physiology. Since its invention, x-ray CT has become indispensable in the clinic for diagnostic and prognostic purposes due to its quantitative absorption-based imaging in true 3D that allows objects of interest to be viewed and measured from any orientation. However, x-ray CT has not been useful at the level of single cells because there is insufficient contrast to form an image. Recently, optical CT has been developed successfully for fixed cells, but this technology called Cell-CT is incompatible with live-cell imaging due to the use of stains, such as hematoxylin, that are not compatible with cell viability. We present a novel development of optical CT for quantitative, multispectral functional 4D (three spatial + one spectral dimension) imaging of living single cells. The method applied to immune system cells offers truly isotropic 3D spatial resolution and enables time-resolved imaging studies of cells suspended in aqueous medium. Using live-cell optical CT, we found a heterogeneous response to mitochondrial fission inhibition in mouse macrophages and differential basal remodeling of small (0.1 to 1 fl) and large (1 to 20 fl) nuclear and mitochondrial structures on a 20- to 30-s time scale in human myelogenous leukemia cells. Because of its robust 3D measurement capabilities, live-cell optical CT represents a powerful new tool in the biomedical research field. PMID:29226240

Sorting cells by their density

PubMed Central

Norouzi, Nazila; Bhakta, Heran C.

2017-01-01

Sorting cells by their type is an important capability in biological research and medical diagnostics. However, most cell sorting techniques rely on labels or tags, which may have limited availability and specificity. Sorting different cell types by their different physical properties is an attractive alternative to labels because all cells intrinsically have these physical properties. But some physical properties, like cell size, vary significantly from cell to cell within a cell type; this makes it difficult to identify and sort cells based on their sizes alone. In this work we continuously sort different cells types by their density, a physical property with much lower cell-to-cell variation within a cell type (and therefore greater potential to discriminate different cell types) than other physical properties. We accomplish this using a 3D-printed microfluidic chip containing a horizontal flowing micron-scale density gradient. As cells flow through the chip, Earth’s gravity makes each cell move vertically to the point where the cell’s density matches the surrounding fluid’s density. When the horizontal channel then splits, cells with different densities are routed to different outlets. As a proof of concept, we use our density sorter chip to sort polymer microbeads by their material (polyethylene and polystyrene) and blood cells by their type (white blood cells and red blood cells). The chip enriches the fraction of white blood cells in a blood sample from 0.1% (in whole blood) to nearly 98% (in the output of the chip), a 1000x enrichment. Any researcher with access to a 3D printer can easily replicate our density sorter chip and use it in their own research using the design files provided as online Supporting Information. Additionally, researchers can simulate the performance of a density sorter chip in their own applications using the Python-based simulation software that accompanies this work. The simplicity, resolution, and throughput of this technique make it suitable for isolating even rare cell types in complex biological samples, in a wide variety of different research and clinical applications. PMID:28723908

PGC1α is required for the induction of contact inhibition by suppressing ROS.

PubMed

Yang, Seungyeon; Hwang, Sunsook; Jang, Jiho; Kim, Minjoong; Gwak, Jihye; Jeong, Seung Min

2018-05-16

Contact inhibition (CI) is an important tumor-suppressive mechanism that arrests cell cycle when cells reach high density. Indeed, CI is aberrantly absent in cancer cells and the dysregulation of this can contribute to tumorigenesis. Previously, it has been shown that reactive oxygen species (ROS) levels are repressed at high cell density, which is required for CI, but no molecular mechanism of this ROS regulation has been reported. Here, we show that PGC1α regulates cell density-dependent CI. PGC1α is markedly induced in response to high cell density and suppresses ROS production. Although cellular ROS levels are progressively decreased with increasing cell density, knockdown of PGC1α results in a defect of density-dependent ROS suppression. Importantly, PGC1α knockdown cells become less sensitive to high cell density and exhibit loss of CI. Mechanistically, PGC1α represses ROS production by inducing mitochondrial SIRT3, and thus SIRT3 overexpression rescues the defects of CI by PGC1α knockdown. These results demonstrate that mitochondrial ROS production is a crucial regulator of cell proliferation and identify a new role of PGC1α in CI. Copyright © 2018 Elsevier Inc. All rights reserved.

Basics for the preparation of quantum dots and their interactions with living cells.

PubMed

Jiang, Xiue; Bai, Jing; Wang, Tiantian

2014-01-01

A study of the interactions between nanoparticles and living cells is invaluable in understanding the nano-biological effect and the mechanism of cellular endocytosis. Here we describe two methods for the preparation of semiconductor quantum dots with different physiochemical properties. Furthermore, we describe how to study the interaction of the two quantum dots with living HeLa cells and red blood cells with confocal microscopy.

Collagen Matrix Density Drives the Metabolic Shift in Breast Cancer Cells.

PubMed

Morris, Brett A; Burkel, Brian; Ponik, Suzanne M; Fan, Jing; Condeelis, John S; Aguirre-Ghiso, Julio A; Castracane, James; Denu, John M; Keely, Patricia J

2016-11-01

Increased breast density attributed to collagen I deposition is associated with a 4-6 fold increased risk of developing breast cancer. Here, we assessed cellular metabolic reprogramming of mammary carcinoma cells in response to increased collagen matrix density using an in vitro 3D model. Our initial observations demonstrated changes in functional metabolism in both normal mammary epithelial cells and mammary carcinoma cells in response to changes in matrix density. Further, mammary carcinoma cells grown in high density collagen matrices displayed decreased oxygen consumption and glucose metabolism via the tricarboxylic acid (TCA) cycle compared to cells cultured in low density matrices. Despite decreased glucose entry into the TCA cycle, levels of glucose uptake, cell viability, and ROS were not different between high and low density matrices. Interestingly, under high density conditions the contribution of glutamine as a fuel source to drive the TCA cycle was significantly enhanced. These alterations in functional metabolism mirrored significant changes in the expression of metabolic genes involved in glycolysis, oxidative phosphorylation, and the serine synthesis pathway. This study highlights the broad importance of the collagen microenvironment to cellular expression profiles, and shows that changes in density of the collagen microenvironment can modulate metabolic shifts of cancer cells. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Effect of Microstructural Parameters on the Relative Densities of Metal Foams

NASA Technical Reports Server (NTRS)

Raj, S. V.; Kerr, Jacob A.

2010-01-01

A detailed quantitative microstructural analyses of primarily open cell FeCrAlY and 314 stainless steel metal foams with different relative densities and pores per inch (p.p.i.) were undertaken in the present investigation to determine the effect of microstructural parameters on the relative densities of metal foams. Several elements of the microstructure, such as longitudinal and transverse cell sizes, cell areas and perimeters, ligament dimensions, cell shapes and volume fractions of closed and open cells, were measured. The cross-sections of the foam ligaments showed a large number of shrinkage cavities, and their circularity factors and average sizes were determined. The volume fractions of closed cells increased linearly with increasing relative density. In contrast, the volume fractions of the open cells and ligaments decreased with increasing relative density. The relative densities and p.p.i. were not significantly dependent on cell size, cell perimeter and ligament dimensions within the limits of experimental scatter. A phenomenological model is proposed to rationalize the present microstructural observations.

A system and methodology for high-content visual screening of individual intact living cells in suspension

NASA Astrophysics Data System (ADS)

Renaud, Olivier; Heintzmann, Rainer; Sáez-Cirión, Asier; Schnelle, Thomas; Mueller, Torsten; Shorte, Spencer

2007-02-01

Three dimensional imaging provides high-content information from living intact biology, and can serve as a visual screening cue. In the case of single cell imaging the current state of the art uses so-called "axial through-stacking". However, three-dimensional axial through-stacking requires that the object (i.e. a living cell) be adherently stabilized on an optically transparent surface, usually glass; evidently precluding use of cells in suspension. Aiming to overcome this limitation we present here the utility of dielectric field trapping of single cells in three-dimensional electrode cages. Our approach allows gentle and precise spatial orientation and vectored rotation of living, non-adherent cells in fluid suspension. Using various modes of widefield, and confocal microscope imaging we show how so-called "microrotation" can provide a unique and powerful method for multiple point-of-view (three-dimensional) interrogation of intact living biological micro-objects (e.g. single-cells, cell aggregates, and embryos). Further, we show how visual screening by micro-rotation imaging can be combined with micro-fluidic sorting, allowing selection of rare phenotype targets from small populations of cells in suspension, and subsequent one-step single cell cloning (with high-viability). Our methodology combining high-content 3D visual screening with one-step single cell cloning, will impact diverse paradigms, for example cytological and cytogenetic analysis on haematopoietic stem cells, blood cells including lymphocytes, and cancer cells.

High cell density cultivation of probiotics and lactic acid production.

PubMed

Schiraldi, Chiara; Adduci, Vincenzo; Valli, Vivien; Maresca, Carmelina; Giuliano, Mariateresa; Lamberti, Monica; Cartenì, Maria; De Rosa, Mario

2003-04-20

The commercial interest in functional foods that contain live microorganisms, also named probiotics, is paralleled by the increasing scientific attention to their functionality in the digestive tract. This is especially true of yogurts that contain strains of lactic-acid bacteria of intestinal origin, among these, Lactobacillus delbrueckii ssp. bulgaricus is extensively used in the dairy industry and it has been demonstrated to be a probiotic strain. In this work we describe high cell density cultivations of this microorganism also focusing on the stereospecific production of lactic acid. Key parameters such as medium composition (bactocasitone concentration) and diverse aeration conditions were explored. The results showed that the final concentration of biomass in anaerobic fermentation was lower than the one obtained in microaerophilic conditions, while it gave a very high productivity of lactic acid which was present as a racemic mixture in the permeate. Fermentation experiments carried out with air sparging, even at very low flow-rate, led to the production of the sole L(+) lactic acid giving sevenfold increase in biomass yield in respect to the batch cultivation. Finally, a mathematical model was developed to describe the microfiltration bioprocess applied in this research considering an inhibition kinetic and enucleating a suitable mathematical description for the decrease of the transmembrane flux. Copyright 2003 Wiley Periodicals, Inc.

Is Urografin density gradient centrifugation suitable to separate nonculturable cells from Escherichia coli populations?

PubMed

Arana, Inés; Orruño, Maite; Seco, Carolina; Muela, Alicia; Barcina, Isabel

2008-03-01

The ability of Urografin or Percoll density gradient centrifugations to separate nonculturable subpopulations from heterogeneous Escherichia coli populations was analysed. Bacterial counts (total, active and culturable cells) and flow cytometric analyses were carried out in all recovered bands. After Urografin centrifugation, and despite the different origin of E. coli populations, a common pattern was obtained. High-density bands were formed mainly by nonculturable cells. However, the increase in cell density would not be common to all nonculturable cells, since part of this subpopulations banded in low-density zones, mixed with culturable cells. Bands obtained after Percoll centrifugation were heterogeneous and culturable and nonculturable cells were recovered along the gradient. Thus, fractionation in Urografin cannot be only attributed to changes in buoyant densities during the transition from culturable to nonculturable state. Urografin density gradients allow us to obtain enriched fractions in nonculturable subpopulations from a heterogeneous population, but working conditions should be carefully chosen to avoid Urografin toxicity.

Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

NASA Astrophysics Data System (ADS)

Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

2016-09-01

Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

The effect of macromolecular crowding on mobility of biomolecules, association kinetics and gene expression in living cells

NASA Astrophysics Data System (ADS)

Tabaka, Marcin; Kalwarczyk, Tomasz; Szymanski, Jedrzej; Hou, Sen; Hołyst, Robert

2014-09-01

We discuss a quantitative influence of macromolecular crowding on biological processes: motion, bimolecular reactions, and gene expression in prokaryotic and eukaryotic cells. We present scaling laws relating diffusion coefficient of an object moving in a cytoplasm of cells to a size of this object and degree of crowding. Such description leads to the notion of the length scale dependent viscosity characteristic for all living cells. We present an application of the length-scale dependent viscosity model to the description of motion in the cytoplasm of both eukaryotic and prokaryotic living cells. We compare the model with all recent data on diffusion of nanoscopic objects in HeLa, and E. coli cells. Additionally a description of the mobility of molecules in cell nucleus is presented. Finally we discuss the influence of crowding on the bimolecular association rates and gene expression in living cells.

[Osteogenesis of human adipose-derived mesenchymal stem cells-biomaterial mixture in vivo after 3D bio-printing].

PubMed

Song, Yang; Wang, Xiao-fei; Wang, Yu-guang; Sun, Yu-chun; Lv, Pei-jun

2016-02-18

To construct human adipose-derived mesenchymal stem cells (hASCs)-biomaterial mixture 3D bio-printing body and detect its osteogenesis in vivo, and to establish a guideline of osteogenesis in vivo by use of 3D bio-printing technology preliminarily. P4 hASCs were used as seed cells, whose osteogenic potential in vitro was tested by alkaline phosphatase (ALP) staining and alizarin red staining after 14 d of osteogenic induction. The cells were added into 20 g/L sodium alginate and 80 g/L gelatin mixture (cell density was 1 × 10(6)/mL), and the cell-sodium alginate-gelatin mixture was printed by Bioplotter 3D bio-printer (Envision company, Germany), in which the cells'survival rate was detected by live- dead cell double fluorescence staining. Next, the printing body was osteogenically induced for 1 week to gain the experimental group; and the sodium alginate-gelatin mixture without cells was also printed to gain the control group. Both the experimental group and the control group were implanted into the back of the nude mice. After 6 weeks of implantation, the samples were collected, HE staining, Masson staining, immunohistochemical staining and Inveon Micro CT test were preformed to analyze their osteogenic capability. The cells'survival rate was 89%± 2% after printing. Six weeks after implantation, the samples of the control group were mostly degraded, whose shape was irregular and gel-like; the samples of the experimental group kept their original size and their texture was tough. HE staining and Masson staining showed that the bone-like tissue and vessel in-growth could be observed in the experimental group 6 weeks after implantation, immunohistochemical staining showed that the result of osteocalcin was positive, and Micro CT results showed that samples of the experimental group had a higher density and the new bone volume was 18% ± 1%. hASCs -biomaterial mixture 3D bio-printing body has capability of ectopic bone formation in nude mice, and it is feasible to apply cells-biomaterial mixture 3D bio-printing technology in the area of bone formation in vivo.

Interactions between semiconductor nanowires and living cells.

PubMed

Prinz, Christelle N

2015-06-17

Semiconductor nanowires are increasingly used for biological applications and their small dimensions make them a promising tool for sensing and manipulating cells with minimal perturbation. In order to interface cells with nanowires in a controlled fashion, it is essential to understand the interactions between nanowires and living cells. The present paper reviews current progress in the understanding of these interactions, with knowledge gathered from studies where living cells were interfaced with vertical nanowire arrays. The effect of nanowires on cells is reported in terms of viability, cell-nanowire interface morphology, cell behavior, changes in gene expression as well as cellular stress markers. Unexplored issues and unanswered questions are discussed.

The effects of treading by two breeds of dairy cow with different live weights on soil physical properties, poaching damage and herbage production on a poorly drained clay-loam soil.

PubMed

Tuohy, P; Fenton, O; Holden, N M; Humphreys, J

2015-11-01

There is little empirical evidence to indicate that dairy cow live weight affects the extent of soil damage at the hoof-soil interface during grazing on poorly drained permanent grassland. In the present study the impact of Holstein-Friesian (HF) dairy cows with a mean (±standard deviation) live weight of 570 (±61) kg were compared with Jersey × Holstein-Friesian (JX) with a mean live weight of 499 (±52) kg each at two stocking densities: mean 2·42 ± (0·062) and 2·66 (±0·079) cows/ha. Soil physical properties (bulk density, macroporosity, gravimetric water content, air-filled porosity, penetration resistance and shear strength), poaching damage (post-grazing soil surface deformation and hoof-print depth), herbage yield and milk production were measured throughout 2011 and 2012. Soil physical properties, post-grazing soil surface deformation and herbage production were not affected by dairy cow breed or by interactions between breed and stocking density. Hoof-print depth was higher in the HF treatments (39 v. 37 mm, s.e. 0·5 mm). Loading pressure imposed at the soil surface was the same for both breeds due to a direct correlation between live weight and hoof size. Poaching damage was greater at higher stocking density. Using the lighter JX cow offered little advantage in terms of lowering the negative impact of treading on soil physical properties or reducing poaching damage and no advantage in terms of herbage or milk production compared with the heavier HF cow.

Manipulation of parental effort affects plumage bacterial load in a wild passerine.

PubMed

Alt, Grete; Saag, Pauli; Mägi, Marko; Kisand, Veljo; Mänd, Raivo

2015-06-01

It has been suggested that plumage microorganisms play an important role in shaping the life histories of wild birds. Some bacteria may act as pathogens or cause damage to feathers, and thereby reduce individual fitness. Intense parental care in birds can result in a reduction of self-maintenance and preening behavior in parents and therefore might affect the dynamics of microbiota living on their feathers. However, experimental evidence of this relationship is virtually absent. We manipulated the parental effort of wild breeding pied flycatcher (Ficedula hypoleuca) females by modifying their brood size or temporarily removing male partners. We expected that experimentally decreasing or increasing parental effort would affect feather sanitation in females and therefore also bacterial density on their plumage. In accordance with this hypothesis, manipulation affected the density of free-living bacteria: females with reduced broods had the lowest number of free-living bacteria on their feathers, while females left without male partners had the highest. However, manipulation did not have a significant effect on the densities of attached bacteria. Our results provide experimental evidence that a trade-off between self-maintenance and parental effort affects plumage bacterial densities in birds.

Determination of dissociation constant of the NFκB p50/p65 heterodimer using fluorescence cross-correlation spectroscopy in the living cell

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

Tiwari, Manisha; Mikuni, Shintaro; Muto, Hideki

Highlights: •We used two-laser-beam FCCS to determine the dissociation constant (K{sub d}) of IPT domain of p50/p65 heterodimer in living cell. •Interaction of p50 and p65 was analyzed in the cytoplasm and nucleus of single living cell. •Binding affinity of p50/p65 heterodimer is higher in cytoplasm than that of nucleus. -- Abstract: Two-laser-beam fluorescence cross-correlation spectroscopy (FCCS) is promising technique that provides quantitative information about the interactions of biomolecules. The p50/p65 heterodimer is the most abundant and well understood of the NFκB dimers in most cells. However, the quantitative value of affinity, namely the K{sub d}, for the heterodimer inmore » living cells is not known yet. To quantify the heterodimerization of the IPT domain of p50/p65 in the living cell, we used two-laser-beam FCCS. The K{sub d} values of mCherry{sub 2}- and EGFP-fused p50 and p65 were determined to be 0.46 μM in the cytoplasm and 1.06 μM in the nucleus of the living cell. These results suggest the different binding affinities of the p50/p65 heterodimer in the cytoplasm and nucleus of the living cell and different complex formation in each region.« less

Microfluidics microFACS for Life Detection

NASA Technical Reports Server (NTRS)

Platt, Donald W.; Hoover, Richard B.

2010-01-01

A prototype micro-scale Fluorescent Activated Cell Sorter (microFACS) for life detection has been built and is undergoing testing. A functional miniature microfluidics instrument with the ability to remotely distinguish live or dead bacterial cells from abiotic particulates in ice or permafrost of icy bodies of the solar system would be of fundamental value to NASA. The use of molecular probes to obtain the bio-signature of living or dead cells could answer the most fundamental question of Astrobiology: Does life exist beyond Earth? The live-dead fluorescent stains to be used in the microFACS instrument function only with biological cell walls. The detection of the cell membranes of living or dead bacteria (unlike PAH's and many other Biomarkers) would provide convincing evidence of present or past life. This miniature device rapidly examine large numbers of particulates from a polar ice or permafrost sample and distinguish living from dead bacteria cells and biological cells from mineral grains and abiotic particulates and sort the cells and particulates based on a staining system. Any sample found to exhibit fluorescence consistent with living cells could then be used in conjunction with a chiral labeled release experiment or video microscopy system to seek addition evidence for cellular metabolism or motility. Results of preliminary testing and calibration of the microFACS prototype instrument system with pure cultures and enrichment assemblages of microbial extremophiles will be reported.

Lipopolysaccharide effects on the proliferation of NRK52E cells via alternations in gap-junction function.

PubMed

Hei, Ziqing; Zhang, Ailan; Wei, Jing; Gan, Xiaoliang; Wang, Yanling; Luo, Gangjian; Li, Xiaoyun

2012-07-01

Gap junctions regulate proper kidney function by facilitating intercellular communication, vascular conduction, and tubular purinergic signaling. However, no clear relationship has been described between gap-junction function and acute kidney injury induced by the endotoxin lipopolysaccharide (LPS). Normal rat kidney epithelial cells (NRK52E cells) were seeded at high and low densities to promote or impede gap-junction formation, respectively, and establish distinctive levels of intercellular communication in culture. Cells were then challenged with LPS at various concentrations (10-1,000 ng/mL). LPS-induced formation and function of gap junctions were assessed by measuring changes in cell proliferation and colony-forming rates, fluorescent dye transmission to adjacent cells, expression levels of connexin43, and repositioning of confluent cells in response to the gap junction inhibitor oleamide or agonist retinoic acid. The cell proliferation rate and colony-forming rate of high- and low-density NRK52E cells were decreased upon LPS challenge, in a dose-dependent manner. The colony-forming rate of confluent high-density cells was significantly lower than that of low-density cells. Oleamide treatment raised the LPS-induced colony-forming rate of high-density cells, whereas retinoic acid decreased the rate. Neither oleamide nor retinoic acid significantly affected the LPS-induced colony-forming rate of low-density cells. Fluorescence transmission of high-density cells was reduced by LPS challenge, in a dose-dependent manner, but inclusion of retinoic acid increased the LPS-induced transmission of fluorescence. LPS challenge of either high- or low-density NRK52E cells resulted in down-regulated connexin43 expression. Gap-junction function plays an important role in concentration-dependent cytotoxic effect of LPS on normal rat kidney cells in vitro.

Non-rigid multi-frame registration of cell nuclei in live cell fluorescence microscopy image data.

PubMed

Tektonidis, Marco; Kim, Il-Han; Chen, Yi-Chun M; Eils, Roland; Spector, David L; Rohr, Karl

2015-01-01

The analysis of the motion of subcellular particles in live cell microscopy images is essential for understanding biological processes within cells. For accurate quantification of the particle motion, compensation of the motion and deformation of the cell nucleus is required. We introduce a non-rigid multi-frame registration approach for live cell fluorescence microscopy image data. Compared to existing approaches using pairwise registration, our approach exploits information from multiple consecutive images simultaneously to improve the registration accuracy. We present three intensity-based variants of the multi-frame registration approach and we investigate two different temporal weighting schemes. The approach has been successfully applied to synthetic and live cell microscopy image sequences, and an experimental comparison with non-rigid pairwise registration has been carried out. Copyright © 2014 Elsevier B.V. All rights reserved.

Does living density matter for nonfatal unintentional home injury in Asian urban settings? Evidence from Hong Kong.

PubMed

Chan, Emily Y Y; Kim, Jean H; Griffiths, Sian M; Lau, Joseph T F; Yu, Ignatius

2009-11-01

Injury is a major global disease burden for the twenty-first century. There are, however, few studies of unintentional household injury in Asian urban settings where living environments are characterized by extremely compact, high-living-density, multistory apartments. This study investigated the association between nonfatal unintentional household injuries with the resident's sociodemographic attributes and household characteristics in Hong Kong, the city with the world's highest population density. A cross-sectional retrospective recall study was conducted in May 2007 using a random telephone survey with a modified Chinese version of the World Health Organization Injury and Violence instrument. The study sample included 1,001 noninstitutionalized Cantonese-speaking Hong Kong residents of all ages, including foreign live-in domestic helpers. Multivariate regression was conducted to identify risk factors for nonfatal unintentional injuries in Hong Kong. Among a predominantly adult sample, household size and time spent at home were not associated with nonfatal unintentional household injuries in the general population in Hong Kong. The multivariate analyses indicated that female gender, owners of private homes, lower square footage of living space per person, and those with slip prevention devices in the bathroom were significantly associated with household injuries. Injured and noninjured groups were found to have adopted different injury prevention strategies toward household injuries. The results identified potential target groups for household injury prevention programs.

Integration of Metabolic and Quorum Sensing Signals Governing the Decision to Cooperate in a Bacterial Social Trait

PubMed Central

Boyle, Kerry E.; Monaco, Hilary; van Ditmarsch, Dave; Deforet, Maxime; Xavier, Joao B.

2015-01-01

Many unicellular organisms live in multicellular communities that rely on cooperation between cells. However, cooperative traits are vulnerable to exploitation by non-cooperators (cheaters). We expand our understanding of the molecular mechanisms that allow multicellular systems to remain robust in the face of cheating by dissecting the dynamic regulation of cooperative rhamnolipids required for swarming in Pseudomonas aeruginosa. We combine mathematical modeling and experiments to quantitatively characterize the integration of metabolic and population density signals (quorum sensing) governing expression of the rhamnolipid synthesis operon rhlAB. The combined computational/experimental analysis reveals that when nutrients are abundant, rhlAB promoter activity increases gradually in a density dependent way. When growth slows down due to nutrient limitation, rhlAB promoter activity can stop abruptly, decrease gradually or even increase depending on whether the growth-limiting nutrient is the carbon source, nitrogen source or iron. Starvation by specific nutrients drives growth on intracellular nutrient pools as well as the qualitative rhlAB promoter response, which itself is modulated by quorum sensing. Our quantitative analysis suggests a supply-driven activation that integrates metabolic prudence with quorum sensing in a non-digital manner and allows P. aeruginosa cells to invest in cooperation only when the population size is large enough (quorum sensing) and individual cells have enough metabolic resources to do so (metabolic prudence). Thus, the quantitative description of rhlAB regulatory dynamics brings a greater understating to the regulation required to make swarming cooperation stable. PMID:26102206

Influence of electric current on bacterial viability in wastewater treatment.

PubMed

Wei, V; Elektorowicz, M; Oleszkiewicz, J A

2011-10-15

Minimizing the influence of electric current on bacterial viability in the electro-technologies such as electrophoresis and electrocoagulation is crucial in designing and operating the electric hybrid wastewater treatment system. In this study the biomass from a membrane bioreactor (MBR) was subjected to constant direct current and the bacterial viability was monitored against electrical intensity, duration as well as the spatial vicinity related to the electrodes. It was found that the bacterial viability was not significantly affected (less than 10% of death percentage) when the applied electric current density (CD) was less than 6.2 A/m2 after 4 h. The percentage of live cell dropped by 15% and 29% at CD of 12.3 A/m2 and 24.7 A/m2, respectively. The pH of electrolytic biomass fluid has shifted to alkaline (from nearly neutral to around pH 10) at CD above 12.3 A/m2, which could have been the contributing factor for the bacterial inactivation. The temperature change in the electrolytic media at all current densities during 4 h of experiment was less than 2 °C, thus temperature effects were negligible. Bacteria experienced different micro-environments in the electrochemical reactor. Bacterial cells on the cathode surface exhibited highest death rate, whereas bacteria outside the space between electrodes were the least affected. It was concluded that in an electro-technology integrated wastewater treatment process, sufficient mixing should be used to avoid localized inactivation of bacterial cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

The Storage Cell for the Tri-Experiment at COSY-JÜLICH

NASA Astrophysics Data System (ADS)

Felden, O.; Gebel, R.; Glende, M.; Lehrach, A.; Maier, R.; Prasuhn, D.; von Rossen, P.; Bisplinghoff, J.; Eversheim, P. D.; Hinterberger, F.

2002-04-01

At the EDDA experiment in the cooler synchrotron COSY in Jülich an atomic beam target is used which provides the designed polarization and density distribution. To increase the target density significantly a storage cell has been developed and implemented. This will contribute to a higher accuracy for the test of Time Reversal Invariance (TRI) which will be performed at the EDDA target place. To obtain the higher luminosity the target density and the transmission of the COSY beam through the cell were determined in their dependence on the cell aperture. Low storage cell apertures increase the target density in the cell but reduce the transmission of the circulating proton beam. To find the optimal cell design the transmission of the COSY beam was measured with movable scrapers and tested with an aperture at EDDA simulating the storage cell. The target density was calculated by Monte Carlo simulations for several cell geometries. An additional gain in target density is achieved by cooling the cell. A Teflon coating of the cell reduces depolarization of the target gas. First measurements with the EDDA detector have shown that the target density as well as the polarization are within the range of the expected values.

Importance of Sox2 in maintenance of cell proliferation and multipotency of mesenchymal stem cells in low-density culture.

PubMed

Yoon, D S; Kim, Y H; Jung, H S; Paik, S; Lee, J W

2011-10-01

This study has aimed to repopulate 'primitive' cells from late-passage mesenchymal stem cells (MSCs) of poor multipotentiality and low cell proliferation rate, by simply altering plating density. Effects of low density culture compared t high density culture on late-passage bone marrow (BM)-derived MSCs and pluripotency markers of multipotentiality were investigated. Cell proliferation, gene expression, RNA interference and differentiation potential were assayed. We repopulated 'primitive' cells by replating late-passage MSCs at low density (17 cells/cm(2) ) regardless of donor age. Repopulated MSCs from low-density culture were smaller cells with spindle shaped morphology compared to MSCs from high-density culture. The latter had enhanced colony-forming ability, proliferation rate, and adipogenic and chondrogenic potential. Strong expression of osteogenic-related genes (Cbfa1, Dlx5, alkaline phosphatase and type Ι collagen) in late-passage MSCs was reduced by replating at low density, whereas expression of three pluripotency markers (Sox2, Nanog and Oct-4), Osterix and Msx2 reverted to levels of early-passage MSCs. Knockdown of Sox2 and Msx2 but not Nanog, using RNA interference, showed significant decrease in colony-forming ability. Specifically, knockdown of Sox2 significantly inhibited multipotentiality and cell proliferation. Our data suggest that plating density should be considered to be a critical factor for enrichment of 'primitive' cells from heterogeneous BM and that replicative senescence and multipotentiality of MSCs during in vitro expansion may be predominantly regulated through Sox2. © 2011 Blackwell Publishing Ltd.

Understanding dynamic changes in live cell adhesion with neutron reflectometry

PubMed Central

JUNGHANS, ANN; WALTMAN, MARY JO; SMITH, HILLARY L.; POCIVAVSEK, LUKA; ZEBDA, NOUREDDINE; BIRUKOV, KONSTANTIN; VIAPIANO, MARIANO; MAJEWSKI, JAROSLAW

2015-01-01

Neutron reflectometry (NR) was used to examine various live cells adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutron reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell – surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies. PMID:25705067

Mediating Effects of Social Support and Internalized Homonegativity on the Association Between Population Density and Mental Health Among Gay and Bisexual Men.

PubMed

Cain, Demetria N; Mirzayi, Chloe; Rendina, H Jonathon; Ventuneac, Ana; Grov, Christian; Parsons, Jeffrey T

2017-10-01

Depression negatively impacts the health and well-being of gay and bisexual men (GBM). However, little is known about the contexts in which rural GBM live relative to those living in urban areas and their overall mental health. The aim of this study was to examine associations between population density and depressive symptoms and the role of internalized homonegativity and social support as potential mediators. A nationally representative sample of 1071 GBM (mean age = 40.24) was enrolled. Participants provided their zip codes, which were categorized according to population density and rank-normalized. In a path analysis model adjusted for race/ethnicity, college education, age, and relationship status, higher population density was significantly associated with increased social support (B = 0.11, P = 0.002) and decreased internalized homonegativity (B = -0.06, P < 0.001). In turn, lower social support (B = -2.93, P < 0.001) and greater internalized homonegativity (B = 4.93, P < 0.001) were significantly associated with greater depressive symptoms. The indirect effects of population density on depression through social support (B = -0.33, P < 0.001) and internalized homonegativity (B = -0.31, P < 0.001) were statistically significant, suggesting evidence for mediation of the effects. These results indicate that living in less inhabited areas acts on depressive symptoms through mechanisms of lower social support and higher internalized homonegativity. These findings suggest that social contexts in which GBM live can affect mental health outcomes and indicate the need for further support and inclusion of GBM, especially in less inhabited areas.

Mediating Effects of Social Support and Internalized Homonegativity on the Association Between Population Density and Mental Health Among Gay and Bisexual Men

PubMed Central

Cain, Demetria N.; Mirzayi, Chloe; Rendina, H. Jonathon; Ventuneac, Ana; Grov, Christian

2017-01-01

Abstract Purpose: Depression negatively impacts the health and well-being of gay and bisexual men (GBM). However, little is known about the contexts in which rural GBM live relative to those living in urban areas and their overall mental health. The aim of this study was to examine associations between population density and depressive symptoms and the role of internalized homonegativity and social support as potential mediators. Methods: A nationally representative sample of 1071 GBM (mean age = 40.24) was enrolled. Participants provided their zip codes, which were categorized according to population density and rank-normalized. Results: In a path analysis model adjusted for race/ethnicity, college education, age, and relationship status, higher population density was significantly associated with increased social support (B = 0.11, P = 0.002) and decreased internalized homonegativity (B = −0.06, P < 0.001). In turn, lower social support (B = −2.93, P < 0.001) and greater internalized homonegativity (B = 4.93, P < 0.001) were significantly associated with greater depressive symptoms. The indirect effects of population density on depression through social support (B = −0.33, P < 0.001) and internalized homonegativity (B = −0.31, P < 0.001) were statistically significant, suggesting evidence for mediation of the effects. Conclusions: These results indicate that living in less inhabited areas acts on depressive symptoms through mechanisms of lower social support and higher internalized homonegativity. These findings suggest that social contexts in which GBM live can affect mental health outcomes and indicate the need for further support and inclusion of GBM, especially in less inhabited areas. PMID:28792886

Cell tracking using iron oxide fails to distinguish dead from living transplanted cells in the infarcted heart.

PubMed

Winter, E M; Hogers, B; van der Graaf, L M; Gittenberger-de Groot, A C; Poelmann, R E; van der Weerd, L

2010-03-01

Recently, debate has arisen about the usefulness of cell tracking using iron oxide-labeled cells. Two important issues in determining the usefulness of cell tracking with MRI are generally overlooked; first, the effect of graft rejection in immunocompetent models, and second, the necessity for careful histological confirmation of the fate of the labeled cells in the presence of iron oxide. Therefore, both iron oxide-labeled living as well as dead epicardium-derived cells (EPDCs) were investigated in ischemic myocardium of immunodeficient non-obese diabetic (NOD)/acid: non-obese diabetic severe combined immunodeficient (NOD/scid) mice with 9.4T MRI until 6 weeks after surgery, at which time immunohistochemical analysis was performed. In both groups, voids on MRI scans were observed that did not change in number, size, or localization over time. Based on MRI, no distinction could be made between living and dead injected cells. Prussian blue staining confirmed that the hypointense spots on MRI corresponded to iron-loaded cells. However, in the dead-EPDC recipients, all iron-positive cells appeared to be macrophages, while the living-EPDC recipients also contained engrafted iron-loaded EPDCs. Iron labeling is inadequate for determining the fate of transplanted cells in the immunodeficient host, since dead cells produce an MRI signal indistinguishable from incorporated living cells. (c) 2010 Wiley-Liss, Inc.

Nanoscale live cell imaging using hopping probe ion conductance microscopy

PubMed Central

Novak, Pavel; Li, Chao; Shevchuk, Andrew I.; Stepanyan, Ruben; Caldwell, Matthew; Hughes, Simon; Smart, Trevor G.; Gorelik, Julia; Ostanin, Victor P.; Lab, Max J.; Moss, Guy W. J.; Frolenkov, Gregory I.; Klenerman, David; Korchev, Yuri E.

2009-01-01

We describe a major advance in scanning ion conductance microscopy: a new hopping mode that allows non-contact imaging of the complex surfaces of live cells with resolution better than 20 nm. The effectiveness of this novel technique was demonstrated by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allows studying nanoscale phenomena on the surface of live cells under physiological conditions. PMID:19252505

Phage-Bacterial Dynamics with Spatial Structure: Self Organization around Phage Sinks Can Promote Increased Cell Densities

PubMed Central

Bull, James J.; Christensen, Kelly A.; Scott, Carly; Crandall, Cameron J.; Krone, Stephen M.

2018-01-01

Bacteria growing on surfaces appear to be profoundly more resistant to control by lytic bacteriophages than do the same cells grown in liquid. Here, we use simulation models to investigate whether spatial structure per se can account for this increased cell density in the presence of phages. A measure is derived for comparing cell densities between growth in spatially structured environments versus well mixed environments (known as mass action). Maintenance of sensitive cells requires some form of phage death; we invoke death mechanisms that are spatially fixed, as if produced by cells. Spatially structured phage death provides cells with a means of protection that can boost cell densities an order of magnitude above that attained under mass action, although the effect is sometimes in the opposite direction. Phage and bacteria self organize into separate refuges, and spatial structure operates so that the phage progeny from a single burst do not have independent fates (as they do with mass action). Phage incur a high loss when invading protected areas that have high cell densities, resulting in greater protection for the cells. By the same metric, mass action dynamics either show no sustained bacterial elevation or oscillate between states of low and high cell densities and an elevated average. The elevated cell densities observed in models with spatial structure do not approach the empirically observed increased density of cells in structured environments with phages (which can be many orders of magnitude), so the empirical phenomenon likely requires additional mechanisms than those analyzed here. PMID:29382134

Theoretical tuning of the firefly bioluminescence spectra by the modification of oxyluciferin

NASA Astrophysics Data System (ADS)

Cheng, Yuan-Yuan; Zhu, Jia; Liu, Ya-Jun

2014-01-01

Extending the firefly bioluminescence is of practical significance for the improved visualization of living cells and the development of a multicolor reporter. Tuning the color of bioluminescence in fireflies mainly involves the modification of luciferase and luciferin. In this Letter, we theoretically studied the emission spectra of 9 firefly oxyluciferin analogs in the gas phase and in solutions. Three density functionals, including B3LYP, CAM-B3LYP and M06-2X, were employed to theoretically predict the efficiently luminescent analogs. The reliable functionals for calculating the targeted systems were suggested. The luminescence efficiency, solvent effects, and substituent effects are discussed based on the calculated results.

Heterotypic interactions regulate cell shape and density during color pattern formation in zebrafish.

PubMed

Mahalwar, Prateek; Singh, Ajeet Pratap; Fadeev, Andrey; Nüsslein-Volhard, Christiane; Irion, Uwe

2016-11-15

The conspicuous striped coloration of zebrafish is produced by cell-cell interactions among three different types of chromatophores: black melanophores, orange/yellow xanthophores and silvery/blue iridophores. During color pattern formation xanthophores undergo dramatic cell shape transitions and acquire different densities, leading to compact and orange xanthophores at high density in the light stripes, and stellate, faintly pigmented xanthophores at low density in the dark stripes. Here, we investigate the mechanistic basis of these cell behaviors in vivo, and show that local, heterotypic interactions with dense iridophores regulate xanthophore cell shape transition and density. Genetic analysis reveals a cell-autonomous requirement of gap junctions composed of Cx41.8 and Cx39.4 in xanthophores for their iridophore-dependent cell shape transition and increase in density in light-stripe regions. Initial melanophore-xanthophore interactions are independent of these gap junctions; however, subsequently they are also required to induce the acquisition of stellate shapes in xanthophores of the dark stripes. In summary, we conclude that, whereas homotypic interactions regulate xanthophore coverage in the skin, their cell shape transitions and density is regulated by gap junction-mediated, heterotypic interactions with iridophores and melanophores. © 2016. Published by The Company of Biologists Ltd.

Predicted energy densitites for nickel-hydrogen and silver-hydrogen cells embodying metallic hydrides for hydrogen storage

NASA Technical Reports Server (NTRS)

Easter, R. W.

1974-01-01

Simplified design concepts were used to estimate gravimetric and volumetric energy densities for metal hydrogen battery cells for assessing the characteristics of cells containing metal hydrides as compared to gaseous storage cells, and for comparing nickel cathode and silver cathode systems. The silver cathode was found to yield superior energy densities in all cases considered. The inclusion of hydride forming materials yields cells with very high volumetric energy densities that also retain gravimetric energy densities nearly as high as those of gaseous storage cells.

Exploring the Leishmania Hydrophilic Acylated Surface Protein B (HASPB) Export Pathway by Live Cell Imaging Methods.

PubMed

MacLean, Lorna; Price, Helen; O'Toole, Peter

2016-01-01

Leishmania major is a human-infective protozoan parasite transmitted by the bite of the female phlebotomine sand fly. The L. major hydrophilic acylated surface protein B (HASPB) is only expressed in infective parasite stages suggesting a role in parasite virulence. HASPB is a "nonclassically" secreted protein that lacks a conventional signal peptide, reaching the cell surface by an alternative route to the classical ER-Golgi pathway. Instead HASPB trafficking to and exposure on the parasite plasma membrane requires dual N-terminal acylation. Here, we use live cell imaging methods to further explore this pathway allowing visualization of key events in real time at the individual cell level. These methods include live cell imaging using fluorescent reporters to determine the subcellular localization of wild type and acylation site mutation HASPB18-GFP fusion proteins, fluorescence recovery after photobleaching (FRAP) to analyze the dynamics of HASPB in live cells, and live antibody staining to detect surface exposure of HASPB by confocal microscopy.

Using In Vitro Live-cell Imaging to Explore Chemotherapeutics Delivered by Lipid-based Nanoparticles.

PubMed

Seynhaeve, Ann L B; Ten Hagen, Timo L M

2017-11-01

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied.

Synthetic-Molecule/Protein Hybrid Probe with Fluorogenic Switch for Live-Cell Imaging of DNA Methylation.

PubMed

Hori, Yuichiro; Otomura, Norimichi; Nishida, Ayuko; Nishiura, Miyako; Umeno, Maho; Suetake, Isao; Kikuchi, Kazuya

2018-02-07

Hybrid probes consisting of synthetic molecules and proteins are powerful tools for detecting biological molecules and signals in living cells. To date, most targets of the hybrid probes have been limited to pH and small analytes. Although biomacromolecules are essential to the physiological function of cells, the hybrid-probe-based approach has been scarcely employed for live-cell detection of biomacromolecules. Here, we developed a hybrid probe with a chemical switch for live-cell imaging of methylated DNA, an important macromolecule in the repression of gene expression. Using a protein labeling technique, we created a hybrid probe containing a DNA-binding fluorogen and a methylated-DNA-binding domain. The hybrid probe enhanced fluorescence intensity upon binding to methylated DNA and successfully monitored methylated DNA during mitosis. The hybrid probe offers notable advantages absent from probes based on small molecules or fluorescent proteins and is useful for live-cell analyses of epigenetic phenomena and diseases related to DNA methylation.

Monoxenic liquid culture with Escherichia coli of the free-living nematode Panagrolaimus sp. (strain NFS 24-5), a potential live food candidate for marine fish and shrimp larvae.

PubMed

Ayub, Farhana; Seychelles, Laurent; Strauch, Olaf; Wittke, Martina; Ehlers, Ralf-Udo

2013-09-01

The free-living, bacterial-feeding nematode Panagrolaimus sp. (strain NFS 24-5) has potential for use as live food for marine shrimp and fish larvae. Mass production in liquid culture is a prerequisite for its commercial exploitation. Panagrolaimus sp. was propagated in monoxenic liquid culture on Escherichia coli and parameters, like nematode density, population dynamics and biomass were recorded and compared with life history table data. A mean maximum nematode density of 174,278 mL(-1) and a maximum of 251,000 mL(-1) were recorded on day 17 after inoculation. Highest average biomass was 40 g L(-1) at day 13. The comparison with life history table data indicated that the hypothetical potential of liquid culture is much higher than documented during this investigation. Nematode development is delayed in liquid culture and egg production per female is more than five times lower than reported from life history trait analysis. The latter assessed a nematode generation time of 7.1 days, whereas the process time at maximum nematode density in liquid culture was 16 days indicating that a reduction of the process time can be achieved by further investigating the influence of nematode inoculum density on population development. The results challenge future research to reduce process time and variability and improve population dynamics also during scale-up of the liquid culture process.

[Effect evaluation of water conservancy project with ditches managed against Oncomelania hupensis in Yunnan Province].

PubMed

Ning-Bo, Huang; Peng, Huang; Zong-Ti, Shao; Xi-Guang, Feng; Yi, Dong; Guang-Huai, Yang; Jin-Song, Li; Yan-Hong, Zhang; Shao-Yun, Chen; Shou-Ju, Nie; Wen, Li

2016-03-11

To evaluate the effect of hydraulic schistosomiasis control project with ditches managed on Oncomelania hupensis snail control. From 2009 to 2011, the snail investigations and schistosomiasis surveillance were carried out in Dali City and Yongsheng County, two sites of national schistosomiasis surveillance. The history data of schistosomiasis control were collected and analyzed. At the harden sections of the water conservancy project with ditches managed in Shajing Village of Dali City, only one snail was found in 2010 with the density of living snails of 0.004 snails/0.1 m 2 , while the densities of living snails were respectively 0.080, 0.002 snails/0.1 m 2 and 0.007 snails/0.1 m2 in unhardened sections of the project from 2009 to 2011. No snails were found in the harden sections of the water conservancy project with ditches managed in Gaojiacun Village of Yongsheng County, while the densities of living snails were respectively 0.040, 0.030 snails/0.1 m2 and 0.040 snails/0.1 m2 in unhardened sections of the project from 2009 to 2011. After the ditches were hardened, no infected snails were found from 2009 to 2011, and the appearance rate of frames with snails and density of living snails were both decreased, while they were both higher in unhardened ditches. The hydraulic schistosomiasis control project has obvious effect on control snails, but the maintain work should be strengthened after the project is completed.

Fourier-transform infrared spectroscopy for rapid screening and live-cell monitoring: application to nanotoxicology.

PubMed

Sundaram, S K; Sacksteder, Colette A; Weber, Thomas J; Riley, Brian J; Addleman, R Shane; Harrer, Bruce J; Peterman, John W

2013-01-01

A significant challenge to realize the full potential of nanotechnology for therapeutic and diagnostic applications is to understand and evaluate how live cells interact with an external stimulus, such as a nanosized particle, and the toxicity and broad risk associated with these stimuli. It is difficult to capture the complexity and dynamics of these interactions by following omics-based approaches exclusively, which can be expensive and time-consuming. Attenuated total reflectance-Fourier transform infrared spectroscopy is well suited to provide noninvasive live-cell monitoring of cellular responses to potentially toxic nanosized particles or other stimuli. This alternative approach provides the ability to carry out rapid toxicity screenings and nondisruptive monitoring of live-cell cultures. We review the technical basis of the approach, the instrument configuration and interface with the biological media, the various effects that impact the data, subsequent data analysis and toxicity, and present some preliminary results on live-cell monitoring.

In-vivo measurement of relationship between applied current amplitude and current density magnitude from 10 mA to 110 mA.

PubMed

DeMonte, Tim P; Wang, Dinghui; Ma, Weijing; Gao, Jia-Hong; Joy, Michael L G

2009-01-01

Current density imaging (CDI) is a magnetic resonance imaging (MRI) technique used to quantitatively measure current density vectors throughout the volume of an object/subject placed in the MRI system. Electrical current pulses are applied externally to the object/subject and are synchronized with the MRI sequence. In this work, CDI is used to measure average current density magnitude in the torso region of an in-vivo piglet for applied current pulse amplitudes ranging from 10 mA to 110 mA. The relationship between applied current amplitude and current density magnitude is linear in simple electronic elements such as wires and resistors; however, this relationship may not be linear in living tissue. An understanding of this relationship is useful for research in defibrillation, human electro-muscular incapacitation (e.g. TASER(R)) and other bioelectric stimulation devices. This work will show that the current amplitude to current density magnitude relationship is slightly nonlinear in living tissue in the range of 10 mA to 110 mA.

Traffic density in area of residence is associated with health-related quality of life in women, the community-based Hordaland Health Study.

PubMed

Gundersen, Hilde; Magerøy, Nils; Moen, Bente E; Bråtveit, Magne

2013-01-01

Vehicle traffic is increasing worldwide, and this is a major concern because traffic-related air pollution and noise may influence health. The aim of this study was to evaluate whether health-related quality of life (HRQoL) is associated with vehicle traffic density in area of residence. A total of 16,410 individuals, 40 to 45 years old, were asked to participate in this study (response rate: 55% for men, 66% for women). Using the 12-Item Short Form Health Survey (SF-12) questionnaire, both physical and mental HRQoL were investigated. Multiple linear regression analyses showed that women living in areas with high traffic density had significantly poorer physical HRQoL than women living in areas with moderate or low vehicle traffic density. There were no similar findings among men. Mental HRQoL was not associated with vehicle traffic density in the area of residence, neither for women nor for men. There is an association between high vehicle traffic density in residential area and reduced HRQoL in women.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

PubMed Central

Almassalha, Luay M.; Bauer, Greta M.; Chandler, John E.; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K.; Subramanian, Hariharan; Chandel, Navdeep S.; Szleifer, Igal; Backman, Vadim

2016-01-01

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells. PMID:27702891

Apoptotic death in cerebral hemisphere cells is density dependent and modulated by transient oxygen and glucose deprivation.

PubMed

Yavin, E; Billia, D M

1997-03-01

Flow cytometry, light and fluorescence microscopy, and designated biochemical techniques were used to examine the type of death which occurs in cerebral cortex cells when grown under crowded vs. sparse conditions or after brief anoxia/hypoglycemia. A 4 hr episode of anoxia combined with glucose deprivation enhanced apoptotic cell death as assessed by 4',6-diamidino-2-phenylindole (DAPI) staining and reduced neutral red eye uptake. An additional form of cell death involving exclusion of the nucleus was recorded by time lapse cinematography and DAPI stain. The presence of the endonuclease inhibitor aurintricarboxylic acid (0.1 mM) reduced cell death by 56.6%, while the protein and RNA synthesis inhibitors actinomycin D and cycloheximide (each at 5 micrograms/ml) effectively decreased cell death by 83.3% and 90.6%, respectively. In contrast, 5 mM glutamate had no effect on cell death in accord with the immature state of the cells. Growth of cells under crowded conditions improved cell survival; after 2 h or 4 days in culture, cells seeded at high density (34 microgram cellular DNA/cm2) showed a nearly 3-fold decline in the amount of cell death in comparison to cells seeded at low density (5 micrograms cellular DNA/cm2). At high cell density, anoxic episodes enhanced cell death most likely by preventing a cell density-mediated rescue. Neutral red dye uptake, an index for cell viability, was enhanced with increasing cell density and in vitro maturation, but was reduced in dense cultures exposed to anoxic/hypoglycemic conditions. The data suggest that cell density may play a critical role in brain organogenesis and that anoxic stress is more deleterious in dense than sparse cell assemblies.

Single-molecule microscopy reveals membrane microdomain organization of cells in a living vertebrate.

PubMed

Schaaf, Marcel J M; Koopmans, Wiepke J A; Meckel, Tobias; van Noort, John; Snaar-Jagalska, B Ewa; Schmidt, Thomas S; Spaink, Herman P

2009-08-19

It has been possible for several years to study the dynamics of fluorescently labeled proteins by single-molecule microscopy, but until now this technology has been applied only to individual cells in culture. In this study, it was extended to stem cells and living vertebrate organisms. As a molecule of interest we used yellow fluorescent protein fused to the human H-Ras membrane anchor, which has been shown to serve as a model for proteins anchored in the plasma membrane. We used a wide-field fluorescence microscopy setup to visualize individual molecules in a zebrafish cell line (ZF4) and in primary embryonic stem cells. A total-internal-reflection microscopy setup was used for imaging in living organisms, in particular in epidermal cells in the skin of 2-day-old zebrafish embryos. Our results demonstrate the occurrence of membrane microdomains in which the diffusion of membrane proteins in a living organism is confined. This membrane organization differed significantly from that observed in cultured cells, illustrating the relevance of performing single-molecule microscopy in living organisms.

Synthetic high-density lipoprotein nanoconjugate targets neuroblastoma stem cells, blocking migration and self-renewal.

PubMed

Subramanian, Chitra; White, Peter T; Kuai, Rui; Kalidindi, Avinaash; Castle, Valerie P; Moon, James J; Timmermann, Barbara N; Schwendeman, Anna; Cohen, Mark S

2018-05-09

Pathways critical for neuroblastoma cancer stem cell function are targeted by 4,19,27-triacetyl withalongolide A (WGA-TA). Because neuroblastoma cells and their cancer stem cells highly overexpress the scavenger receptor class B type 1 receptor that binds to synthetic high-density lipoprotein, we hypothesized that a novel mimetic synthetic high-density lipoprotein nanoparticle would be an ideal carrier for the delivery of 4,19,27-triacetyl withalongolide to neuroblastoma and neuroblastoma cancer stem cells. Expression of scavenger receptor class B type 1 in validated human neuroblastoma cells was evaluated by quantitative polymerase chain reaction (qPCR) and Western blot. In vitro cellular uptake of synthetic high-density lipoprotein nanoparticles was observed with a fluorescence microscope. In vivo biodistribution of synthetic high-density lipoprotein nanoparticles was investigated with IVIS imaging. Self-renewal and migration/invasion were assessed by sphere formation and Boyden chamber assays, respectively. Viability was analyzed by CellTiter-Glo assay. Cancer stem cell markers were evaluated by flow cytometry. qPCR and Western blot analysis revealed a higher level of scavenger receptor class B type 1 expression and drug uptake in N-myc amplified neuroblastoma cells. In vitro uptake of synthetic high-density lipoprotein was almost completely blocked by excess synthetic high-density lipoprotein. The synthetic high-density lipoprotein nanoparticles mainly accumulated in the tumor and liver, but not in other organs. Synthetic HDL-4,19,27-triacetyl withalongolide showed a 1,000-fold higher potency than the carrier (synthetic high-density lipoprotein) alone (P < .01) to kill neuroblastoma cells. Additionally, a dose-dependent decrease in sphere formation, invasion, migration, and cancer stem cell markers was observed after treatment of neuroblastoma cells with synthetic high-density lipoprotein-4,19,27-triacetyl withalongolide A. Synthetic high-density lipoprotein is a promising platform to improve the delivery of anticancer drug 4,19,27-triacetyl withalongolide A to neuroblastomas and neuroblastoma cancer stem cells through SR-B1 targeting in vitro and in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Long term imaging of living brain cancer cells

NASA Astrophysics Data System (ADS)

Farias, Patricia M. A.; Galembeck, André; Milani, Raquel; Andrade, Arnaldo C. D. S.; Stingl, Andreas

2018-02-01

QDs synthesized in aqueous medium and functionalized with polyethylene glycol were used as fluorescent probes. They label and monitor living healthy and cancer brain glial cells in culture. Physical-chemical characterization was performed. Toxicological studies were performed by in vivo short and long-term inhalation in animal models. Healthy and cancer glial living cells were incubated in culture media with highly controlled QDs. Specific features of glial cancer cells were enhanced by QD labelling. Cytoplasmic labelling pattern was clearly distinct for healthy and cancer cells. Labelled cells kept their normal activity for same period as non-labelled control samples.

Decorating an individual living cell with a shell of controllable thickness by cytocompatible surface initiated graft polymerization.

PubMed

Wang, Guan; Zhang, Kai; Wang, Yindian; Zhao, Changwen; He, Bin; Ma, Yuhong; Yang, Wantai

2018-05-03

Surface engineering of individual living cells is a promising field for cell-based applications. However, engineering individual cells with controllable thickness by chemical methods has been rarely studied. This article describes the development of a new cytocompatible chemical strategy to decorate individual living cells. The thicknesses of the crosslinked shells could be conveniently controlled by the irradiation time, visible light intensity, or monomer concentration. Moreover, the lag phase of the yeast cell division was extended and their stability against lysis was improved, which could also be tuned by controlling the shell thickness.

Acid base activity of live bacteria: Implications for quantifying cell wall charge

NASA Astrophysics Data System (ADS)

Claessens, Jacqueline; van Lith, Yvonne; Laverman, Anniet M.; Van Cappellen, Philippe

2006-01-01

To distinguish the buffering capacity associated with functional groups in the cell wall from that resulting from metabolic processes, base or acid consumption by live and dead cells of the Gram-negative bacterium Shewanella putrefaciens was measured in a pH stat system. Live cells exhibited fast consumption of acid (pH 4) or base (pH 7, 8, 9, and 10) during the first few minutes of the experiments. At pH 5.5, no acid or base was required to maintain the initial pH constant. The initial amounts of acid or base consumed by the live cells at pH 4, 8, and 10 were of comparable magnitudes as those neutralized at the same pHs by intact cells killed by exposure to gamma radiation or ethanol. Cells disrupted in a French press required higher amounts of acid or base, due to additional buffering by intracellular constituents. At pH 4, acid neutralization by suspensions of live cells stopped after 50 min, because of loss of viability. In contrast, under neutral and alkaline conditions, base consumption continued for the entire duration of the experiments (5 h). This long-term base neutralization was, at least partly, due to active respiration by the cells, as indicated by the build-up of succinate in solution. Qualitatively, the acid-base activity of live cells of the Gram-positive bacterium Bacillus subtilis resembled that of S. putrefaciens. The pH-dependent charging of ionizable functional groups in the cell walls of the live bacteria was estimated from the initial amounts of acid or base consumed in the pH stat experiments. From pH 4 to 10, the cell wall charge increased from near-zero values to about -4 × 10 -16 mol cell -1 and -6.5 × 10 -16 mol cell -1 for S. putrefaciens and B. subtilis, respectively. The similar cell wall charging of the two bacterial strains is consistent with the inferred low contribution of lipopolysaccharides to the buffering capacity of the Gram-negative cell wall (of the order of 10%).

Is Echinometra viridis facilitating a phase shift on an Acropora cervicornis patch reef in Belize?

NASA Astrophysics Data System (ADS)

Stefanic, C. M.; Greer, L.; Norvell, D.; Benson, W.; Curran, H.

2012-12-01

Coral reef health is in rapid decline across the Caribbean due to a number of anthropogenic and natural disturbances. A phase shift from coral- to macroalgae-dominant reefs is pervasive and has been well documented. Acropora cervicornis (Staghorn Coral) has been particularly affected by this shift due to mass mortality of this species since the 1980s. In recent years few Caribbean A. cervicornis refugia have been documented. This study characterizes the relationship between coral and grazing urchins on a rare patch reef system dominated by A. cervicornis off the coast of Belize. To assess relative abundance of live A. cervicornis and the urchin Echinometra viridis, photographs and urchin abundance data were collected from 132 meter square quadrats along five transects across the reef. Photographs were digitized and manually segmented using Adobe Illustrator, and percent live coral cover and branch tip densities were calculated using Matlab. Mean percent live coral cover across all transects was 24.4 % with a high of 65% live coral per meter square. Average urchin density was 18.5 per quadrat, with an average density per transect ranging from 22.1 to 0.5 per quadrat. Up to over 400 live A. cervicornis branch tips per quadrat were observed. Data show a positive correlation between E. viridis abundance and live A. cervicornis, suggesting that these urchins are facilitating recovery or persistence of this endangered coral species. These results suggest the relationship between E. viridis and A. cervicornis could be a key element in a future reversal of the coral to macroalgae phase shift on some Caribbean coral reefs.

THE SEPARATION OF DIFFERENT CELL CLASSES FROM LYMPHOID ORGANS

PubMed Central

Shortman, Ken; Seligman, Kathrin

1969-01-01

1. Mammalian erythrocytes swell as the pH of the isotonic suspending medium is lowered, as a direct consequence of the specialized permeability properties of the erythrocyte membrane. Lymphocytes and granulocytes from a variety of sources did not exhibit this property. 2. The behaviour of mouse bone marrow erythroid cells at various stages of differentiation was studied by using a change in buoyant density with pH as an index of swelling. The ability to swell with a pH drop was acquired while the cell was still nucleated. All non-nucleated cells showed swelling. Most small erythroblasts shared this property, whereas most large erythroblasts did not. 3. The density shift with pH was used to provide a purification scheme specific for erythroid cells. The bone marrow cells were first centrifuged to equilibrium in an isotonic albumin density gradient at neutral pH. Regions of the gradient containing the erythroid cells were collected, and the cells were recovered and redistributed in an albumin gradient at acid pH. The erythroid cells showed a specific density shift which removed them from contaminants. Preparations containing 90–97% erythroblasts were obtained by this technique. 4. Differentiation within the erythroid series was accompanied by a general increase in cell buoyant density at neutral pH. This density increase may have been a discontinuous process, since erythroid cells appeared to form a number of density peaks. 5. The pH shift technique, in association with established density distribution and sedimentation velocity procedures, provides a range of cell separation techniques for biological or biochemical studies of erythroid cell differentiation in the complex cell mixtures in bone marrow or spleen. PMID:5801428

FRET and BRET-based biosensors in live cell compound screens.

PubMed

Robinson, Katie Herbst; Yang, Jessica R; Zhang, Jin

2014-01-01

Live cell compound screening with genetically encoded fluorescence or bioluminescence-based biosensors offers a potentially powerful approach to identify novel regulators of a signaling event of interest. In particular, compound screening in living cells has the added benefit that the entire signaling network remains intact, and thus the screen is not just against a single molecule of interest but against any molecule within the signaling network that may modulate the distinct signaling event reported by the biosensor in use. Furthermore, only molecules that are cell permeable or act at cell surface receptors will be identified as "hits," thus reducing further optimization of the compound in terms of cell penetration. Here we discuss a detailed protocol for using genetically encoded biosensors in living cells in a 96-well format for the execution of high throughput compound screens and the identification of small molecules which modulate a signaling event of interest.

Live-cell imaging of cyanobacteria.

PubMed

Yokoo, Rayka; Hood, Rachel D; Savage, David F

2015-10-01

Cyanobacteria are a diverse bacterial phylum whose members possess a high degree of ultrastructural organization and unique gene regulatory mechanisms. Unraveling this complexity will require the use of live-cell fluorescence microscopy, but is impeded by the inherent fluorescent background associated with light-harvesting pigments and the need to feed photosynthetic cells light. Here, we outline a roadmap for overcoming these challenges. Specifically, we show that although basic cyanobacterial biology creates challenging experimental constraints, these restrictions can be mitigated by the careful choice of fluorophores and microscope instrumentation. Many of these choices are motivated by recent successful live-cell studies. We therefore also highlight how live-cell imaging has advanced our understanding of bacterial microcompartments, circadian rhythm, and the organization and segregation of the bacterial nucleoid.

A study of the diffusion dynamics and concentration distribution of gold nanospheres (GNSs) without fluorescent labeling inside live cells using fluorescence single particle spectroscopy.

PubMed

Liu, Fangchao; Dong, Chaoqing; Ren, Jicun

2018-03-15

Colloidal gold nanospheres (GNSs) have become important nanomaterials in biomedical applications due to their special optical properties, good chemical stability, and biocompatibility. However, measuring the diffusion coefficients or concentration distribution of GNSs within live cells accurately without any extra fluorescent labeling in situ has still not been resolved. In this work, a single particle method is developed to study the concentration distribution of folic acid-modified GNSs (FA-GNSs) internalized via folate receptors, and investigates their diffusion dynamics within live cells using single particle fluorescence correlation spectroscopy (FCS). We optimized the experimental conditions and verified the feasibility of 30 nm GNSs without extra fluorescence labeling being used for single particle detection inside live cells. Then, the FCS characterization strategy was used to measure the concentration and diffusion coefficient distributions of GNSs inside live cells and the obtained results were basically in agreement with those obtained by TEM. The results demonstrate that our strategy is characterized as an in situ, nondestructive, rapid and dynamic method for the assay of live cells, and it may be widely used in the further design of GNP-based drug delivery and therapeutics.

The density and spatial arrangement of the invasive oyster Crassostrea gigas determines its impact on settlement of native oyster larvae

PubMed Central

Wilkie, Emma M; Bishop, Melanie J; O'Connor, Wayne A

2013-01-01

Understanding how the density and spatial arrangement of invaders is critical to developing management strategies of pest species. The Pacific oyster, Crassostrea gigas, has been translocated around the world for aquaculture and in many instances has established wild populations. Relative to other species of bivalve, it displays rapid suspension feeding, which may cause mortality of pelagic invertebrate larvae. We compared the effect on settlement of Sydney rock oyster, Saccostrea glomerata, larvae of manipulating the spatial arrangement and density of native S. glomerata, and non-native C. gigas. We hypothesized that while manipulations of dead oysters would reveal the same positive relationship between attachment surface area and S. glomerata settlement between the two species, manipulations of live oysters would reveal differing density-dependent effects between the native and non-native oyster. In the field, whether oysters were live or dead, more larvae settled on C. gigas than S. glomerata when substrate was arranged in monospecific clumps. When, however, the two species were interspersed, there were no differences in larval settlement between them. By contrast, in aquaria simulating a higher effective oyster density, more larvae settled on live S. glomerata than C. gigas. When C. gigas was prevented from suspension feeding, settlement of larvae on C. gigas was enhanced. By contrast, settlement was similar between the two species when dead. While the presently low densities of the invasive oyster C. gigas may enhance S. glomerata larval settlement in east Australian estuaries, future increases in densities could produce negative impacts on native oyster settlement. Synthesis and applications: Our study has shown that both the spatial arrangement and density of invaders can influence their impact. Hence, management strategies aimed at preventing invasive populations reaching damaging sizes should not only consider the threshold density at which impacts exceed some acceptable limit, but also how patch formation modifies this. PMID:24455120

Treatment of osteoporosis with TheraCyte-encapsulated parathyroid cells: a study in a rat model.

PubMed

Chou, F-F; Huang, S-C; Chen, S-S; Wang, P-W; Huang, P-H; Lu, K-Y

2006-01-01

The purpose of this study was to evaluate parathyroid function at monthly intervals following the implantation of TheraCyte-encapsulated live human parathyroid cells into ovariectomized rats and to determine the effect on bone mineral density (BMD) 4 months after ovariectomy ( 3 months after implantation). Parathyroid tissues were obtained from patients undergoing surgery for secondary hyperparathyroidism. In total, 21 Sprague-Dawley rats divided randomly into three groups were subjected to one of three treatments: (1) implanted with TheraCyte A-encapsulated 4x10(6) live parathyroid cells; (2) implanted with TheraCyte B-encapsulated 4x10(5) live parathyroid cells; (3) a sham operation; the control group. Rats were ovariectomized 1 month prior to the implantation of the TheraCyte. Blood was drawn at the time of implantation and at monthly intervals thereafter for 3 months to check the levels of calcium, phosphorus and intact parathyroid hormone (iPTH). The BMD of the lumbar spine (L1-L5) and of the left femoral bone was measured with dual-energy-X-ray absorptiometry (DEXA) 1 month after ovariectomy and 3 months after implantation of the TheraCyte (4 months after ovariectomy). We found that the viability ratio of cryopreserved tissues was between 55 and 79% after thawing. In the control group, the BMD of the lumbar spine (L1-L5) had not decreased significantly (p=0.237) nor had the BMD of the left femoral bone increased significantly (p=0.063) 3 months after implantation. In the TheraCyte A group, the BMD of both the lumbar spine (p=0.018) and left femoral bone (p=0.018) had increased significantly 3 months after implantation. In the TheraCyte B group, the BMD of both the lumbar spine (p=0.017) and the left femoral bone (p=0.025) had also increased significantly 3 months after implantation. Serum iPTH levels were higher in the TheraCyte A group than in the TheraCyte B group (p=0.006), and higher in the TheraCyte B group than in the control group (p=0.040). Serum calcium levels were not significantly higher in the TheraCyte group A than in the TheraCyte B group or in the control group. Serum phosphorus levels were not significantly different between the TheraCyte A and TheraCyte B groups. Implantation of TheraCyte A-encapsulated 4x10(5) live parathyroid cells and TheraCyte B-encapsulated 4x10(6) cells can increase the BMD of ovariectomized rats within 3 months of implantation. Neither cause high serum calcium and low phosphorus concentrations.

Orai1 as New Therapeutic Target for Inhibiting Breast Tumor Metastasis

DTIC Science & Technology

2009-09-01

includes focal adhesion assembly (formation of focal complex) and focal adhesion disassembly, we used live - cell imaging to quantify the rates of assembly...A and B) Live cell imaging of paxillin-GFP transfected MEF cells in the absence (A) or presence (B) of SKF96365. Scale bar: 10 µm. (C and D...includes focal adhesion assembly (formation of focal complexes) and focal adhesion disassembly, we used live - cell imaging to quantify the rates of focal

Live-cell imaging of cell signaling using genetically encoded fluorescent reporters.

PubMed

Ni, Qiang; Mehta, Sohum; Zhang, Jin

2018-01-01

Synergistic advances in fluorescent protein engineering and live-cell imaging techniques in recent years have fueled the concurrent development and application of genetically encoded fluorescent reporters that are tailored for tracking signaling dynamics in living systems over multiple length and time scales. These biosensors are uniquely suited for this challenging task, owing to their specificity, sensitivity, and versatility, as well as to the noninvasive and nondestructive nature of fluorescence and the power of genetic encoding. Over the past 10 years, a growing number of fluorescent reporters have been developed for tracking a wide range of biological signals in living cells and animals, including second messenger and metabolite dynamics, enzyme activation and activity, and cell cycle progression and neuronal activity. Many of these biosensors are gaining wide use and are proving to be indispensable for unraveling the complex biological functions of individual signaling molecules in their native environment, the living cell, shedding new light on the structural and molecular underpinnings of cell signaling. In this review, we highlight recent advances in protein engineering that are likely to help expand and improve the design and application of these valuable tools. We then turn our focus to specific examples of live-cell imaging using genetically encoded fluorescent reporters as an important platform for advancing our understanding of G protein-coupled receptor signaling and neuronal activity. © 2017 Federation of European Biochemical Societies.

Modeling T-cell proliferation: an investigation of the consequences of the Hayflick limit.

PubMed

Pilyugin, S; Mittler, J; Antia, R

1997-05-07

Somatic cells, including immune cells such as T-cells have a limited capacity for proliferation and can only replicate for a finite number of generations (known as the Hayflick limit) before dying. In this paper we use mathematical models to investigate the consequences of introducing a Hayflick limit on the dynamics of T-cells stimulated with specific antigen. We show that while the Hayflick limit does not alter the dynamics of T-cell response to antigen over the short term, it may have a profound effect on the long-term immune response. In particular we show that over the long term the Hayflick limit may be important in determining whether an immune response can be maintained to a persistent antigen (or parasite). The eventual outcome is determined by the magnitude of the Hayflick limit, the extent to which antigen reduces the input of T-cells from the thymus, and the rate of antigen-induced proliferation of T-cells. Counter to what might be expected we show that the persistence of an immune response (immune memory) requires the density of persistent antigen to be less than a defined threshold value. If the amount of persistent antigen (or parasite) is greater than this threshold value then immune memory will be relatively short lived. The consequences of this threshold for persistent mycobacterial and HIV infections and for the generation of vaccines are discussed.

Parasite Specific Antibody Increase Induced by an Episode of Acute P. falciparum Uncomplicated Malaria.

PubMed

Kaddumukasa, Mark; Lwanira, Catherine; Lugaajju, Allan; Katabira, Elly; Persson, Kristina E M; Wahlgren, Mats; Kironde, Fred

2015-01-01

There is no approved vaccine for malaria, and precisely how human antibody responses to malaria parasite components and potential vaccine molecules are developed and maintained remains poorly defined. In this study, antibody anamnestic or memory response elicited by a single episode of P. falciparum infection was investigated. This study involved 362 malaria patients aged between 6 months to 60 years, of whom 19% were early-diagnosed people living with HIV/AIDS (PLWHA). On the day malaria was diagnosed and 42 days later, blood specimens were collected. Parasite density, CD4+ cells, and antibodies specific to synthetic peptides representing antigenic regions of the P. falciparum proteins GLURP, MSP3 and HRPII were measured. On the day of malaria diagnosis, Immunoglobulin (IgG) antibodies against GLURP, MSP3 and HRP II peptides were present in the blood of 75%, 41% and 60% of patients, respectively. 42 days later, the majority of patients had boosted their serum IgG antibody more than 1.2 fold. The increase in level of IgG antibody against the peptides was not affected by parasite density at diagnosis. The median CD4+ cell counts of PLWHAs and HIV negative individuals were not statistically different, and median post-infection increases in anti-peptide IgG were similar in both groups of patients. In the majority (70%) of individuals, an infection of P. falciparum elicits at least 20% increase in level of anti-parasite IgG. This boost in anti-P. falciparum IgG is not affected by parasite density on the day of malaria diagnosis, or by HIV status.

Origin and Function of Circulating Plasmablasts during Acute Viral Infections.

PubMed

Fink, Katja

2012-01-01

Activated B cells proliferate and differentiate into antibody-producing cells, long-lived plasma cells, and memory B cells after immunization or infection. Repeated encounter of the same antigen triggers the rapid re-activation of pre-existing specific memory B cells, which then potentially enter new germinal center reactions and differentiate into short-lived plasmablasts or remain in the system as memory B cells. Short-lived class-switched IgG and IgA plasmablasts appear in the circulation transiently and the frequency of these cells can be remarkably high. The specificities and affinities of single plasmablasts in humans have been reported for several viral infections, so far most extensively for influenza and HIV. In general, the immunoglobulin variable regions of plasmablasts are highly mutated and diverse, suggesting that plasmablasts are derived from memory B cells, yet it is unclear which memory B cell subsets are activated and whether activated memory B cells adapt or mature before differentiation. This review summarizes what is known about the phenotype and the origin of human plasmablasts in the context of viral infections and whether these cells can be predictors of long-lived immunity.

Mechanical behavior in living cells consistent with the tensegrity model

NASA Technical Reports Server (NTRS)

Wang, N.; Naruse, K.; Stamenovic, D.; Fredberg, J. J.; Mijailovich, S. M.; Tolic-Norrelykke, I. M.; Polte, T.; Mannix, R.; Ingber, D. E.

2001-01-01

Alternative models of cell mechanics depict the living cell as a simple mechanical continuum, porous filament gel, tensed cortical membrane, or tensegrity network that maintains a stabilizing prestress through incorporation of discrete structural elements that bear compression. Real-time microscopic analysis of cells containing GFP-labeled microtubules and associated mitochondria revealed that living cells behave like discrete structures composed of an interconnected network of actin microfilaments and microtubules when mechanical stresses are applied to cell surface integrin receptors. Quantitation of cell tractional forces and cellular prestress by using traction force microscopy confirmed that microtubules bear compression and are responsible for a significant portion of the cytoskeletal prestress that determines cell shape stability under conditions in which myosin light chain phosphorylation and intracellular calcium remained unchanged. Quantitative measurements of both static and dynamic mechanical behaviors in cells also were consistent with specific a priori predictions of the tensegrity model. These findings suggest that tensegrity represents a unified model of cell mechanics that may help to explain how mechanical behaviors emerge through collective interactions among different cytoskeletal filaments and extracellular adhesions in living cells.

Live-cell Imaging of Pol II Promoter Activity to Monitor Gene expression with RNA IMAGEtag reporters

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

Shin, Ilchung; Ray, Judhajeet; Gupta, Vinayak

2014-04-20

We describe a ribonucleic acid (RNA) reporter system for live-cell imaging of gene expression to detect changes in polymerase II activity on individual promoters in individual cells. The reporters use strings of RNA aptamers that constitute IMAGEtags (Intracellular MultiAptamer GEnetic tags) that can be expressed from a promoter of choice. For imaging, the cells are incubated with their ligands that are separately conjugated with one of the FRET pair, Cy3 and Cy5. The IMAGEtags were expressed in yeast from the GAL1, ADH1 or ACT1 promoters. Transcription from all three promoters was imaged in live cells and transcriptional increases from themore » GAL1 promoter were observed with time after adding galactose. Expression of the IMAGEtags did not affect cell proliferation or endogenous gene expression. Advantages of this method are that no foreign proteins are produced in the cells that could be toxic or otherwise influence the cellular response as they accumulate, the IMAGEtags are short lived and oxygen is not required to generate their signals. The IMAGEtag RNA reporter system provides a means of tracking changes in transcriptional activity in live cells and in real time.« less

Corneal endothelial cell density after femtosecond thin-flap LASIK and PRK for myopia: a contralateral eye study.

PubMed

Smith, Ryan T; Waring, George O; Durrie, Daniel S; Stahl, Jason E; Thomas, Priscilla

2009-12-01

To compare the effect of femtosecond thinflap LASIK and photorefractive keratectomy (PRK) on postoperative endothelial cell density. In a prospective, randomized, contralateral, single-center clinical trial, 25 patients (mean age: 30+/-5 years [range: 21 to 38 years]) underwent PRK in one eye and thin-flap LASIK in the fellow eye for the correction of myopia using a wavefront-guided platform. The central corneal endothelial cell density was measured using the NIDEK Confoscan 4 preoperatively, and at 1 and 3 months postoperatively. Changes in endothelial cell density were analyzed over time between the two refractive techniques. In PRK, the average preoperative endothelial cell density was 3011+/-329 cells/mm(2), which decreased to 2951+/-327 cells/mm(2) at 1 month (P=.5736) and 2982+/-365 cells/mm(2) at 3 months (P=.6513). In thinflap LASIK, the average preoperative endothelial cell density was 2995+/-325 cells/mm(2), which decreased to 2977+/-358 cells/mm(2) at 1 month (P=.5756) and 2931+/-369 cells/mm(2) at 3 months (P=.4106). No statistically significant difference was found between the two groups at 1 (P=.7404) or 3 (P=.3208) months postoperatively. No statistically significant change was noted in endothelial cell density following either PRK or thin-flap LASIK for the treatment of myopia. Furthermore, no statistically significant difference was found between the two groups out to 3 months postoperatively, indicating that thin-flap LASIK is as safe as PRK with regards to endothelial health.

Associations between proximity and density of local alcohol outlets and alcohol use among Scottish adolescents.

PubMed

Young, Robert; Macdonald, Laura; Ellaway, Anne

2013-01-01

Associations between different alcohol outcomes and outlet density measures vary between studies and may not be generalisable to adolescents. In a cross-sectional study of 979 15-year old Glaswegians, we investigated the association between alcohol outlet availability (outlet density and proximity), outlet type (on-premise vs. off-premise) and frequent (weekly) alcohol consumption. We adjusted for social background (gender, social class, family structure). Proximity and density of on-premise outlets were not associated with weekly drinking. However, adolescents living close (within 200 m) to an off-sales outlet were more likely to drink frequently (OR 1.97, p=0.004), as were adolescents living in areas with many nearby off-premises outlets (OR 1.60, p=0.016). Our findings suggest that certain alcohol behaviours (e.g. binge drinking) may be linked to the characteristics of alcohol outlets in the vicinity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Associations between proximity and density of local alcohol outlets and alcohol use among Scottish adolescents

PubMed Central

Young, Robert; Macdonald, Laura; Ellaway, Anne

2013-01-01

Associations between different alcohol outcomes and outlet density measures vary between studies and may not be generalisable to adolescents. In a cross-sectional study of 979 15-year old Glaswegians, we investigated the association between alcohol outlet availability (outlet density and proximity), outlet type (on-premise vs. off-premise) and frequent (weekly) alcohol consumption. We adjusted for social background (gender, social class, family structure). Proximity and density of on-premise outlets were not associated with weekly drinking. However, adolescents living close (within 200 m) to an off-sales outlet were more likely to drink frequently (OR 1.97, p=0.004), as were adolescents living in areas with many nearby off-premises outlets (OR 1.60, p=0.016). Our findings suggest that certain alcohol behaviours (e.g. binge drinking) may be linked to the characteristics of alcohol outlets in the vicinity. PMID:23220375

NUTRITION OF CELLULAR SLIME MOLDS I.

PubMed Central

Hohl, Hans-Rudolf; Raper, Kenneth B.

1963-01-01

Hohl, Hans-Rudolf (University of Wisconsin, Madison) and Kenneth B. Raper. Nutrition of cullular slime molds. I. Growth on living and dead bacteria. J. Bacteriol. 85:191–198. 1963.—Methods for growing selected species of cellular slime molds in liquid culture on living and dead bacteria are described. Species investigated included Polysphondylium pallidum, P. violaceum, Dictyostelium discoideum, and D. purpureum. Maximal growth of myxamoebae occurred in suspensions of 1010 living bacteria (Escherichia coli B/r)/ml in Sörensen's phosphate buffer (pH 6.0), reaching a density of 107 to 2 × 107 cells/ml in 48 hr. The generation time for the different slime molds ranged from 2.4 hr for P. violaceum to 2.9 hr for D. discoideum (strain V-12). Good growth of P. pallidum occurred between pH 3.6 and 7.8. The slime molds grew less well on dead (autoclaved) than on living bacteria and, except for P. pallidum, the amount and rate of growth decreased markedly as the time of autoclaving was increased from 2.5 to 80 min. Bacteria killed with propylene oxide supported growth equal to those autoclaved for a few minutes. The myxamoebae were very sensitive to the osmotic pressure of the culture medium, especially in the presence of living bacteria, and addition of as little as 0.01 m NaCl caused a measurable decrease in slime mold growth. The culture techniques employed afford useful methods for investigating the nutritional requirements of the cellular slime molds, and the experiments described provide the bases for subsequent studies relating to the axenic cultivation of these singular microorganisms. Images PMID:13961228

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy

PubMed Central

Vélez-Ortega, A. Catalina; Frolenkov, Gregory I.

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3 to 4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette –which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier– is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface. Here we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations. PMID:27259929

Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.

PubMed

Vélez-Ortega, A Catalina; Frolenkov, Gregory I

2016-01-01

The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

Non-protein thiol imaging and quantification in live cells with a novel benzofurazan sulfide triphenylphosphonium fluorogenic compound.

PubMed

Yang, Yang; Guan, Xiangming

2017-05-01

Thiols (-SH) play various roles in biological systems. They are divided into protein thiols (PSH) and non-protein thiols (NPSH). Due to the significant roles thiols play in various physiological/pathological functions, numerous analytical methods have been developed for thiol assays. Most of these methods are developed for glutathione, the major form of NPSH. Majority of these methods require tissue/cell homogenization before analysis. Due to a lack of effective thiol-specific fluorescent/fluorogenic reagents, methods for imaging and quantifying thiols in live cells are limited. Determination of an analyte in live cells can reveal information that cannot be revealed by analysis of cell homogenates. Previously, we reported a thiol-specific thiol-sulfide exchange reaction. Based on this reaction, a benzofurazan sulfide thiol-specific fluorogenic reagent was developed. The reagent was able to effectively image and quantify total thiols (PSH+NPSH) in live cells through fluorescence microscopy. The reagent was later named as GUALY's reagent. Here we would like to report an extension of the work by synthesizing a novel benzofurazan sulfide triphenylphosphonium derivative [(((7,7'-thiobis(benzo[c][1,2,5]oxadiazole-4,4'-sulfonyl))bis(methylazanediyl))bis(butane-4,1-diyl))bis(triphenylphosphonium) (TBOP)]. Like GUALY's reagent, TBOP is a thiol-specific fluorogenic agent that is non-fluorescent but forms fluorescent thiol adducts in a thiol-specific fashion. Different than GUALY's reagent, TBOP reacts only with NPSH but not with PSH. TBOP was effectively used to image and quantify NPSH in live cells using fluorescence microscopy. TBOP is a complementary reagent to GUALY's reagent in determining the roles of PSH, NPSH, and total thiols in thiol-related physiological/pathological functions in live cells through fluorescence microscopy. Graphical Abstract Live cell imaging and quantification of non-protein thiols by TBOP.

Non-interferometric quantitative phase imaging of yeast cells

NASA Astrophysics Data System (ADS)

Poola, Praveen K.; Pandiyan, Vimal Prabhu; John, Renu

2015-12-01

Real-time imaging of live cells is quite difficult without the addition of external contrast agents. Various methods for quantitative phase imaging of living cells have been proposed like digital holographic microscopy and diffraction phase microscopy. In this paper, we report theoretical and experimental results of quantitative phase imaging of live yeast cells with nanometric precision using transport of intensity equations (TIE). We demonstrate nanometric depth sensitivity in imaging live yeast cells using this technique. This technique being noninterferometric, does not need any coherent light sources and images can be captured through a regular bright-field microscope. This real-time imaging technique would deliver the depth or 3-D volume information of cells and is highly promising in real-time digital pathology applications, screening of pathogens and staging of diseases like malaria as it does not need any preprocessing of samples.

In situ cannulation, microgrid follow-up and low-density plating provide first passage endothelial cell masscultures for in vitro lining.

PubMed

Zilla, P; Fasol, R; Dudeck, U; Siedler, S; Preiss, P; Fischlein, T; Müller-Glauser, W; Baitella, G; Sanan, D; Odell, J

1990-08-01

A rapid and reliable harvest and culture technique was developed to provide a sufficient number of autologous endothelial cells for the confluent in vitro lining of cardiovascular prostheses. Enzymatic endothelial cell detachment was achieved by the in situ application of collagenase to short vessel segments. This harvest technique resulted in a complete lack of contaminating smooth muscle cells in all of 124 cultures from nonhuman primates and 13 cultures from human adults. The use of a microgrid technique enabled the daily in situ quantification of available endothelial cells. To assess ideal plating densities after passage the population doubling time was continuously related to the cell density. Surprisingly, a low plating density of 1.5 X 10(3) endothelial cells/cm2 achieved 43% shorter cell cycles than the usual plating density of 1.0 X 10(4) endothelial cells/cm2. Moreover, low density plating enabled mass cultures after one single cell passage, thereby reducing the cell damaging effect of trypsin. When the growth characteristics of endothelial cells from five anatomically different vessel sites were compared, the external jugular vein--which would be easily accessible and dispensable in each patient--proved to be an excellent source for endothelial cell cultures. By applying in situ administration of collagenase, low density plating and microgrid follow-up to adult human saphenous vein endothelial cells, 14,000,000 first passage endothelial cells--sufficient for the in vitro lining of long vascular prostheses--were obtained 26.2 days after harvest. (95% confidence interval:22.3 to 32.2 days).

Long-lived light mediator to dark matter and primordial small scale spectrum

DOE PAGES

Zhang, Yue

2015-05-01

We calculate the early universe evolution of perturbations in the dark matter energy density in the context of simple dark sector models containing a GeV scale light mediator. We consider the case that the mediator is long-lived, with lifetime up to a second, and before decaying it temporarily dominates the energy density of the universe. We show that for primordial perturbations that enter the horizon around this period, the interplay between linear growth during matter domination and collisional damping can generically lead to a sharp peak in the spectrum of dark matter density perturbation. Finally, as a result, the populationmore » of the smallest DM halos gets enhanced. Possible implications of this scenario are discussed.« less

Characterization and optimization of cell seeding in scaffolds by factorial design: quality by design approach for skeletal tissue engineering.

PubMed

Chen, Yantian; Bloemen, Veerle; Impens, Saartje; Moesen, Maarten; Luyten, Frank P; Schrooten, Jan

2011-12-01

Cell seeding into scaffolds plays a crucial role in the development of efficient bone tissue engineering constructs. Hence, it becomes imperative to identify the key factors that quantitatively predict reproducible and efficient seeding protocols. In this study, the optimization of a cell seeding process was investigated using design of experiments (DOE) statistical methods. Five seeding factors (cell type, scaffold type, seeding volume, seeding density, and seeding time) were selected and investigated by means of two response parameters, critically related to the cell seeding process: cell seeding efficiency (CSE) and cell-specific viability (CSV). In addition, cell spatial distribution (CSD) was analyzed by Live/Dead staining assays. Analysis identified a number of statistically significant main factor effects and interactions. Among the five seeding factors, only seeding volume and seeding time significantly affected CSE and CSV. Also, cell and scaffold type were involved in the interactions with other seeding factors. Within the investigated ranges, optimal conditions in terms of CSV and CSD were obtained when seeding cells in a regular scaffold with an excess of medium. The results of this case study contribute to a better understanding and definition of optimal process parameters for cell seeding. A DOE strategy can identify and optimize critical process variables to reduce the variability and assists in determining which variables should be carefully controlled during good manufacturing practice production to enable a clinically relevant implant.

Effect of three months of soft contact lens wear on conjunctival cytology.

PubMed

Sapkota, Kishor; Franco, Sandra; Sampaio, Paula; Lira, Madalena

2016-07-01

The purpose of this study was to investigate the effect of three months of soft contact lens wear on conjunctival goblet cell density and epithelial cell morphology. This was a longitudinal clinical trial. Conjunctival impression cytology was performed on the superior palpebral conjunctiva in fifty-four eyes of twenty-seven neophyte contact lens wearers before and after three months of contact lens wear. Goblet cell density was determined by optical microscopy and epithelial cell morphology was classified according to the Tseng classification. Changes in goblet cell density as well as epithelial cell grading were determined. The effects of lens material and wearing modality on cytological changes were also investigated. Goblet cell density reduced significantly by 85 ± 151 cells/mm(2) (p < 0.001) after three months of contact lens wear. Reduction in goblet cell density was associated with lens materials; it was higher in conventional hydrogel lenses in comparison to silicone-hydrogel lenses (p = 0.008). The highest reduction in goblet cell density was found with Nelfilcon A lens wear (p = 0.002) and the lowest with Comfilcon A lens wear (p = 0.414). There was no statistically significant difference in grading of epithelial metaplasia before and after three months of contact lens wear (p = 0.075). Age was not correlated with the reduction in goblet cell density (r = -0.196, p = 0.160) but it was associated with the change in epithelial cell morphology (p = 0.036). Three months of soft contact lens wear statistically significantly reduced goblet cell density; however, no significant changes were found in the grading of epithelial metaplasia. Contact lenses with lower oxygen permeability, higher Young modulus and higher thickness highly affected the conjunctival cytology. © 2016 Optometry Australia.

Live cell imaging of actin dynamics in dexamethasone-treated porcine trabecular meshwork cells.

PubMed

Fujimoto, Tomokazu; Inoue, Toshihiro; Inoue-Mochita, Miyuki; Tanihara, Hidenobu

2016-04-01

The regulation of the actin cytoskeleton in trabecular meshwork (TM) cells is important for controlling outflow of the aqueous humor. In some reports, dexamethasone (DEX) increased the aqueous humor outflow resistance and induced unusual actin structures, such as cross-linked actin networks (CLAN), in TM cells. However, the functions and dynamics of CLAN in TM cells are not completely known, partly because actin stress fibers have been observed only in fixed cells. We conducted live-cell imaging of the actin dynamics in TM cells with or without DEX treatment. An actin-green fluorescent protein (GFP) fusion construct with a modified insect virus was transfected into porcine TM cells. Time-lapse imaging of live TM cells treated with 25 μM Y-27632 and 100 nM DEX was performed using an inverted fluorescence microscope. Fluorescent images were recorded every 15 s for 30 min after Y-27632 treatment or every 30 min for 72 h after DEX treatment. The GFP-actin was expressed in 22.7 ± 10.9% of the transfected TM cells. In live TM cells, many actin stress fibers were observed before the Y-27632 treatment. Y-27632 changed the cell shape and decreased stress fibers in a time-dependent manner. In fixed cells, CLAN-like structures were seen in 26.5 ± 1.7% of the actin-GFP expressed PTM cells treated with DEX for 72 h. In live imaging, there was 28% CLAN-like structure formation at 72 h after DEX treatment, and the lifetime of CLAN-like structures increased after DEX treatment. The DEX-treated cells with CLAN-like structures showed less migration than DEX-treated cells without CLAN-like structures. Furthermore, the control cells (without DEX treatment) with CLAN-like structures also showed less migration than the control cells without CLAN-like structures. These results suggested that CLAN-like structure formation was correlated with cell migration in TM cells. Live cell imaging of the actin cytoskeleton provides valuable information on the actin dynamics in TM cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

Simple and Effective Methods of Freezing Capercaillie (Tetrao urogallus L.) Semen

PubMed Central

Kowalczyk, Artur; Łukaszewicz, Ewa

2015-01-01

A continuous decline in the number and range of capercaillie (Tetrao urogallus L.) in many European countries can be observed, mostly due to habitat destruction by human activity, unecological forestry management, and increased density of natural predators. Ex situ in vitro gene banks provide a unique opportunity to preserve the genetic material for future generations. Simple and effective cryopreservation methods for capercaillie semen are discussed. Semen was collected from seven males kept in the Capercaillie Breeding Centre at Forestry Wisła in Poland. Within five minutes after collection, ejaculates were diluted with EK diluent, then divided into two parts, and subjected to two freezing procedures: in pellets and in straws. In fresh semen, ejaculate clearness, viscosity, color and volume, as well as sperm concentration, motility and morphology, were evaluated, while in frozen-thawed semen only motility and morphology of sperm were determined. Fertilizing ability of thawed semen was examined for samples frozen in straws. Significant (P<0.05) differences between individual males were found in relation to the majority of fresh semen traits: ejaculate volume averaged 102.1 µL (varying from 49.0 to 205.0); average sperm concentration was 632.5 x106 mL-1 (178.8–1257.1); percentage of live normal cells varied from 39.2 to 70.3% (58.7% on an average); percentage of motile cells ranged from 76.0 to 85.7%) and motility parameters were male dependent, as well. Both cryopreservation methods had a negative effect on morphology and motility of frozen-thawed semen; however, the straw method yielded 60.7% and the pellet method 42.5% of live cells in total in thawed semen (P<0.05), while the number of live normal (intact) cells was similar (22.4 and 22.2%, respectively). Egg fertility varied between 77.8 and 91.7% (average 84.4%). Both freezing procedures seem to be effective in obtaining acceptable viability and high fertilizing potency of thawed sperm and can be used to create a gene bank of capercaillie semen. PMID:25615640

Changes in cytokine production associated with acquired immunity to Plasmodium falciparum malaria

PubMed Central

Rhee, M S M; Akanmori, B D; Waterfall, M; Riley, E M

2001-01-01

Individuals living in malaria-endemic areas eventually develop clinical immunity to Plasmodium falciparum. That is, they are able to limit blood parasite densities to extremely low levels and fail to show symptoms of infection. As the clinical symptoms of malaria infection are mediated in part by pro-inflammatory cytokines it is not clear whether the acquisition of clinical immunity is due simply to the development of antiparasitic mechanisms or whether the ability to regulate inflammatory cytokine production is also involved. We hypothesize that there is a correlation between risk of developing clinical malaria and the tendency to produce high levels of proinflammatory cytokines in response to malaria infection. In order to test this hypothesis, we have compared the ability of peripheral blood mononuclear cells from malaria-naive and malaria-exposed adult donors to proliferate and to secrete IFN-γ in response to P. falciparum schizont extract (PfSE). In order to determine how PfSE-induced IFN-γ production is regulated, we have also measured production of IL-12p40 and IL-10 from PfSE-stimulated PBMC and investigated the role of neutralizing antibody to IL-12 in modulating IFN-γ production. We find that cells from naive donors produce moderate amounts of IFN-γ in response to PfSE and that IFN-γ production is strongly IL-12 dependent. Cells from malaria-exposed donors living in an area of low malaria endemicity produce much higher levels of IFN-γ and this response is also at least partially IL-12 dependent. In complete contrast, cells from donors living in an area of very high endemicity produce minimal amounts of IFN-γ. No significant differences were detected between the groups in IL-10 production, suggesting that this cytokine does not play a major role in regulating malaria-induced IFN-γ production. The data from this study thus strongly support the hypothesis that down-regulation of inflammatory cytokine production may be a component of acquired clinical immunity to malaria but the mechanism by which this is achieved remains to be elucidated. PMID:11737069

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

A coupled diffusion-fluid pressure model to predict cell density distribution for cells encapsulated in a porous hydrogel scaffold under mechanical loading.

PubMed

Zhao, Feihu; Vaughan, Ted J; Mc Garrigle, Myles J; McNamara, Laoise M

2017-10-01

Tissue formation within tissue engineering (TE) scaffolds is preceded by growth of the cells throughout the scaffold volume and attachment of cells to the scaffold substrate. It is known that mechanical stimulation, in the form of fluid perfusion or mechanical strain, enhances cell differentiation and overall tissue formation. However, due to the complex multi-physics environment of cells within TE scaffolds, cell transport under mechanical stimulation is not fully understood. Therefore, in this study, we have developed a coupled multiphysics model to predict cell density distribution in a TE scaffold. In this model, cell transport is modelled as a thermal conduction process, which is driven by the pore fluid pressure under applied loading. As a case study, the model is investigated to predict the cell density patterns of pre-osteoblasts MC3T3-e1 cells under a range of different loading regimes, to obtain an understanding of desirable mechanical stimulation that will enhance cell density distribution within TE scaffolds. The results of this study have demonstrated that fluid perfusion can result in a higher cell density in the scaffold region closed to the outlet, while cell density distribution under mechanical compression was similar with static condition. More importantly, the study provides a novel computational approach to predict cell distribution in TE scaffolds under mechanical loading. Copyright © 2017 Elsevier Ltd. All rights reserved.

Dependency of subcellular reactions during PDT on the metabolic state of cell cultures probed by different microscopic techniques

NASA Astrophysics Data System (ADS)

Rueck, Angelika C.; Schneckenburger, Herbert; Strauss, Wolfgang S. L.; Gschwend, Michael H.; Beck, Gerd C.; Kunzi-Rapp, Karin; Steiner, Rudolf W.

1994-02-01

Various microscopic techniques were used to study the dependency of photodynamically induced subcellular reactions on the metabolic state of cell cultures. TPPS4 and AlS2-3Pc were incubated in RR 1022 epithelial cells with varying cell density. To attain almost isolated cells (low cell density) or confluent growing cells (high cell density) 25 cells/mm2 or 500 cells/mm2 were seeded, respectively. Low cell density irradiation with blue light led to a change in the initial cytoplasmatic fluorescence pattern. For both sensitizers, TPPS4 as well as AlS2-3, a fluorescence relocalization and fluorescence intensity increase could be detected, moreover in the case of TPPS4 a fluorescence formation in the nucleus and nucleoli were detected. In contrast, for confluent growing cells no redistribution was observed.

Pathological significance and prognostic roles of densities of CD57+ cells, CD68+ cells, and mast cells, and their ratios in clear cell renal cell carcinoma.

PubMed

Nakanishi, Hiromi; Miyata, Yasuyoshi; Mochizuki, Yasushi; Yasuda, Takuji; Nakamura, Yuichiro; Araki, Kyohei; Sagara, Yuji; Matsuo, Tomohiro; Ohba, Kojiro; Sakai, Hideki

2018-05-19

The immune system is closely associated with malignant behavior in renal cell carcinoma (RCC). Therefore, understanding the pathological roles of immune cells in tumor stroma is essential to discuss the pathological characteristics of RCC. In this study, the clinical significance of densities of CD57+ cells, CD68+ cells, and mast cells, and their ratios were investigated in patients with clear cell RCC. The densities of CD57+, CD68+, and mast cells were evaluated by immunohistochemical techniques in 179 patients. Proliferation index (PI), apoptotic index (AI), and microvessel density (MVD) were evaluated by using anti-Ki-67, anti-cleaved caspase-3, and anti-CD31 antibodies, respectively. The density of CD57+ cell was negatively correlated with grade, pT stage, and metastasis, although densities of CD68+ cell and mast cell were positively correlated. Ratios of CD68+ cell/CD57+ cell and mast cell/CD57+ cell were significantly correlated with grade, pT stage, and metastasis. Survival analyses showed that the CD68+ cell/CD57+ cell ratio was a significant predictor for cause-specific survival by multi-variate analyses (hazard ratio=1.41, 95% confidential interval=1.03-1.93, P=.031), and was significantly correlated with PI, AI, and MVD (r=.47; P <. 001, r=-.31, P<.001, and r=.40, P<.001, respectively). In conclusion, CD57+ cell, CD68+ cell, and mast cell played important roles in malignancy in clear cell RCC. The CD68+ cell/CD57+ cell ratio was strongly correlated with pathological features and prognosis in these patients because this ratio reflected the status of cancer cell proliferation, apoptosis, and angiogenesis. Copyright © 2018. Published by Elsevier Inc.

Understanding dynamic changes in live cell adhesion with neutron reflectometry

DOE PAGES

Junghans, Ann; Waltman, Mary Jo; Smith, Hillary L.; ...

2014-12-10

In this study, neutron reflectometry (NR) was used to examine various live cells' adhesion to quartz substrates under different environmental conditions, including flow stress. To the best of our knowledge, these measurements represent the first successful visualization and quantization of the interface between live cells and a substrate with sub-nanometer resolution. In our first experiments, we examined live mouse fibroblast cells as opposed to past experiments using supported lipids, proteins, or peptide layers with no associated cells. We continued the NR studies of cell adhesion by investigating endothelial monolayers and glioblastoma cells under dynamic flow conditions. We demonstrated that neutronmore » reflectometry is a powerful tool to study the strength of cellular layer adhesion in living tissues, which is a key factor in understanding the physiology of cell interactions and conditions leading to abnormal or disease circumstances. Continuative measurements, such as investigating changes in tumor cell — surface contact of various glioblastomas, could impact advancements in tumor treatments. In principle, this can help us to identify changes that correlate with tumor invasiveness. Pursuit of these studies can have significant medical impact on the understanding of complex biological problems and their effective treatment, e.g. for the development of targeted anti-invasive therapies.« less

Nanofiber Nerve Guide for Peripheral Nerve Repair and Regeneration

DTIC Science & Technology

2014-01-01

observing cell migration using live - cell imaging microscopy, and analyzing cell migration with our MATLAB-based programs. Our studies...are then pipetted into the chamber and their path of migration is observed using a live - cell imaging microscope (Fig. 6d). Utilizing this migration

Potential of Staphylococcus xylosus strain for recovering nickel ions from aqueous solutions

NASA Astrophysics Data System (ADS)

Al-Gheethi, A.; Efaq, A. N.; Mohamed, R. M.; Abdel-Monem, M.; Amir Hashim, M.

2017-10-01

The potential of bacterial biomass for the biosorption of heavy metals has investigated extensively. However, the bacterial species exhibited different affinities toward the heavy metals ions based on their differences in cell wall characteristics, structure and physiological status (living or dead cells). In this study, the potential of living and dead cells of Staphylococcus xylosus 222W for removal nickel ions from aqueous solution as a function for physiological status, nickel and biomass concentrations, time, pH and temperature was investigated. The pre-treatment of bacterial cells was performed by the heating at 100 °C for 15 min. The removal experiments were conducted in the lab scale. The results revealed that the dead cells exhibited more efficiency in removing nickel ions than living cells at all investigated concentrations (2 to 10 mM). The biosorption efficiency (E %) increased with increasing in biomass cells to limit concentrations (0.1 to 1 g dry wt L-1). The maximum removal of nickel was 81.41 vs. 77.10 % by living and dead cells, respectively achieved after 9 and 10 hrs of the incubation period, respectively. The acidic conditions decrease the efficiency of metal removal, while the optimal removal was recorded at pH 8 for both biomass (living and dead cells). The maximum uptake capacity of S. xylosus 222W (living and dead cells) was recorded at 37oC, the percentage removed being 75.90 vs. 84.92 %, respectively. It can be concluded that S. xylosus 222W exhibited high potential and affinity to remove of nickel ions from aqueous solution.

Physiological responses to acid stress by Saccharomyces cerevisiae when applying high initial cell density

PubMed Central

2016-01-01

High initial cell density is used to increase volumetric productivity and shorten production time in lignocellulosic hydrolysate fermentation. Comparison of physiological parameters in high initial cell density cultivation of Saccharomyces cerevisiae in the presence of acetic, formic, levulinic and cinnamic acids demonstrated general and acid-specific responses of cells. All the acids studied impaired growth and inhibited glycolytic flux, and caused oxidative stress and accumulation of trehalose. However, trehalose may play a role other than protecting yeast cells from acid-induced oxidative stress. Unlike the other acids, cinnamic acid did not cause depletion of cellular ATP, but abolished the growth of yeast on ethanol. Compared with low initial cell density, increasing initial cell density reduced the lag phase and improved the bioconversion yield of cinnamic acid during acid adaptation. In addition, yeast cells were able to grow at elevated concentrations of acid, probable due to the increase in phenotypic cell-to-cell heterogeneity in large inoculum size. Furthermore, the specific growth rate and the specific rates of glucose consumption and metabolite production were significantly lower than at low initial cell density, which was a result of the accumulation of a large fraction of cells that persisted in a viable but non-proliferating state. PMID:27620460

NanoFlares for the detection, isolation, and culture of live tumor cells from human blood.

PubMed

Halo, Tiffany L; McMahon, Kaylin M; Angeloni, Nicholas L; Xu, Yilin; Wang, Wei; Chinen, Alyssa B; Malin, Dmitry; Strekalova, Elena; Cryns, Vincent L; Cheng, Chonghui; Mirkin, Chad A; Thaxton, C Shad

2014-12-02

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood—so-called circulating tumor cells (CTCs)—may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy.

NanoFlares for the detection, isolation, and culture of live tumor cells from human blood

PubMed Central

Halo, Tiffany L.; McMahon, Kaylin M.; Angeloni, Nicholas L.; Xu, Yilin; Wang, Wei; Chinen, Alyssa B.; Malin, Dmitry; Strekalova, Elena; Cryns, Vincent L.; Cheng, Chonghui; Mirkin, Chad A.; Thaxton, C. Shad

2014-01-01

Metastasis portends a poor prognosis for cancer patients. Primary tumor cells disseminate through the bloodstream before the appearance of detectable metastatic lesions. The analysis of cancer cells in blood—so-called circulating tumor cells (CTCs)—may provide unprecedented opportunities for metastatic risk assessment and investigation. NanoFlares are nanoconstructs that enable live-cell detection of intracellular mRNA. NanoFlares, when coupled with flow cytometry, can be used to fluorescently detect genetic markers of CTCs in the context of whole blood. They allow one to detect as few as 100 live cancer cells per mL of blood and subsequently culture those cells. This technique can also be used to detect CTCs in a murine model of metastatic breast cancer. As such, NanoFlares provide, to our knowledge, the first genetic-based approach for detecting, isolating, and characterizing live cancer cells from blood and may provide new opportunities for cancer diagnosis, prognosis, and personalized therapy. PMID:25404304

Live cell refractometry using microfluidic devices.

PubMed

Lue, Niyom; Popescu, Gabriel; Ikeda, Takahiro; Dasari, Ramachandra R; Badizadegan, Kamran; Feld, Michael S

2006-09-15

Using Hilbert phase microscopy for extracting quantitative phase images, we measured the average refractive index associated with live cells in culture. To decouple the contributions to the phase signal from the cell refractive index and thickness, we confined the cells in microchannels. The results are confirmed by comparison with measurements of spherical cells in suspension.

CD3+/CD8+ T-cell density and tumoral PD-L1 predict survival irrespective of rituximab treatment in Chinese diffuse large B-cell lymphoma patients.

PubMed

Shi, Yunfei; Deng, Lijuan; Song, Yuqin; Lin, Dongmei; Lai, Yumei; Zhou, LiXin; Yang, Lei; Li, Xianghong

2018-05-10

To investigate the prognostic value of tumor-infiltrating T-cell density and programmed cell death ligand-1 (PD-L1) expression in diffuse large B cell lymphoma (DLBCL). One-hundred-twenty-five Chinese DLBCL patients were enrolled in our study and provided samples; 76 of all cases were treated with rituximab (R). Tumor tissues were immunostained and analyzed for CD3+ and CD8+ tumor-infiltrating T-cell density, tumoral PD-L1, and microenvironmental PD-L1 (mPD-L1). The density of CD3 was rated as high in 33.6% cases, while 64.0% of DLBCLs were classified as high CD8 density. Of all cases, 16.8% were PD-L1+. Of the remaining PD-L1-DLBCLs, 29.8% positively expressed mPD-L1. Both CD3 high density and CD8 high density were associated with mPD-L1 positivity (P = 0.001 and P = 0.0001). In multivariate analysis, independently, high CD3 density predicted better OS (P = 0.023), while CD8 high density and PD-L1 positivity were both associated with prolonged PFS (P = 0.013 and P = 0.036, respectively). Even in the subgroup treated with R, univariate analyses indicated that high CD3 density and PD-L1 positivity were associated with better OS (P = 0.041) and PFS (P = 0.033), respectively. The infiltrating densities of CD3+ T-cells, CD8+ T-cells, and PD-L1 expression are predictive of survival in DLBCLs, irrespective of R usage.

Cell lineage and cell cycling analyses of the 4d micromere using live imaging in the marine annelid Platynereis dumerilii

PubMed Central

Handberg-Thorsager, Mette; Vervoort, Michel

2017-01-01

Cell lineage, cell cycle, and cell fate are tightly associated in developmental processes, but in vivo studies at single-cell resolution showing the intricacies of these associations are rare due to technical limitations. In this study on the marine annelid Platynereis dumerilii, we investigated the lineage of the 4d micromere, using high-resolution long-term live imaging complemented with a live-cell cycle reporter. 4d is the origin of mesodermal lineages and the germline in many spiralians. We traced lineages at single-cell resolution within 4d and demonstrate that embryonic segmental mesoderm forms via teloblastic divisions, as in clitellate annelids. We also identified the precise cellular origins of the larval mesodermal posterior growth zone. We found that differentially-fated progeny of 4d (germline, segmental mesoderm, growth zone) display significantly different cell cycling. This work has evolutionary implications, sets up the foundation for functional studies in annelid stem cells, and presents newly established techniques for live imaging marine embryos. PMID:29231816

Pancreatin-EDTA treatment affects buoyancy of cells in Cohn fraction V protein density gradients without residual effect on cell size.

PubMed

Sheridan, J W; Simmons, R J

1983-12-01

The buoyancy of suspension-grown Mastocytoma P815 X-2 cells in albumin-rich Cohn fraction V protein (CFVP) density gradients was found to be affected by prior incubation of the cells in pancreatin-EDTA salt solution. Whereas in pH 5.2 CFVP, pancreatin-EDTA treated cells behaved as if of reduced density when compared with the control 'undigested' group, in pH 7.3 CFVP they behaved as if of increased density. By contrast, pancreatin-EDTA treatment had no effect on the buoyancy of mastocytoma cells in polyvinylpyrrolidone-coated colloidal silica (PVP-CS, Percoll T.M.) density gradients of either pH 5.2 or pH 7.3. As cell size determinations failed to reveal alterations in cell size either as a direct result of pancreatin-EDTA treatment or as a combined consequence of such treatment and exposure to CFVP either with or without centrifugation, a mechanism involving a change in cell density other than during the centrifugation process itself seems unlikely. Binding studies employing 125I-CFVP, although indicating that CFVP bound to cells at 4 degrees, failed to reveal a pancreatin-EDTA treatment-related difference in the avidity of this binding. Although the mechanism of the pancreatin-EDTA-induced buoyancy shift in CFVP remains obscure, the absence of such an effect in PVP-CS suggests that the latter cell separation solution may more accurately be used to determine cell density.

Implications of seed banking for recruitment of Southern Appalachian woody species

Treesearch

Janneke Hille Ris Lambers; James S. Clark; Michael Lavine

2002-01-01

Seed dormancy is assumed to be unimportant for population dynamics of temperate woody species, because seeds occur at low densities and are short lived in forest soils. However, low soil seed densities may result from low seed production, and even modest seed longevity can buffer against fluctuating seed production, potentially limiting density-dependent mortality and...

A novel "Turn-On" fluorescent probe for F(-) detection in aqueous solution and its application in live-cell imaging.

PubMed

Xu, Jian; Sun, Shaobo; Li, Qian; Yue, Ying; Li, Yingdong; Shao, Shijun

2014-11-07

A novel probe incorporating quaternized 4-pyridinium group into a BODIPY molecule was synthesized and studied for the selective detection of fluoride ions (F(-)) in aqueous solution. The design was based on a fluoride-specific desilylation reaction and the "Turn-On" fluorescent response of probe 1 to F(-) was ascribed to the inhibition of photoinduced electron transfer (PET) process. The probe displayed many desired properties such as high specificity, appreciable solubility, desirable response time and low toxicity to mammalian cells. There was a good linearity between the fluorescence intensity and the concentrations of F(-) in the range of 0.1-1mM with a detection limit of 0.02 mM. The sensing mechanism was confirmed by the NMR, electrospray ionization mass spectrum, optical spectroscopy and the mechanism of "Turn-On" fluorescent response was also determinated by a density functional theory (DFT) calculation using Gaussian 03 program. Moreover, the probe was successfully applied for the fluorescence imaging of F(-) in human epithelial lung cancer (A549) cells and alveolar type II (ATII) cells under physiological conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

Motion mechanics of non-adherent giant liposomes with a combined optical and atomic force microscope

NASA Astrophysics Data System (ADS)

Moreno-Flores, Susana; Ortíz, Rocío

2017-11-01

Herein we present an investigation of the motional dynamics of single mesoscopic bodies of biological relevance with an AFM-based macromanipulation tool and an optical microscope. Giant liposomes are prominent case examples as minimal cell models; studying their mechanics provides a means to address the influence of structural components in the mechanical behaviour of living cells. However, they also pose an experimental challenge due to their lightness, fragility, and high mobility. Their entrapment in wells in a fluid of lower density allows their study under conditions of constrained motion, which enables the synchronous measurement of nanoforces with motion tracking. The procedure enables to estimate sliding friction coefficients and masses of vesicles, and sheds light upon the region between the vesicle and the underlying substrate. The present study paves the way for the investigation of motion and deformation mechanics with one combined technique and a single type of experiment traditionally vetoed to objects that can move as well as deform. Such an approach can be directly applied to cells in suspension, adherent cells or cellular 3D-assemblies so as to assess substrate biocompatibility, monitor adhesion, detachment, motility as well as deformability.

Investigate The Role Of Cells To Cell Adhesion During Wound Healing

NASA Astrophysics Data System (ADS)

Montes, D. R.; Rodriguez, A. J.; Uyoh, E.; Antwi, P.; Olusegun, S.; Murray, P.

2017-12-01

Improvements in wound healing can save living organism and cut misery in health care centers and on the military battlefield. Microscopic examination of how Madin-Darby Canine Kidney (MDCK) epithelial cells respond to wounds under various treatments that block general calcium absorption and e-cadherin mechanics may yield novel wound treatment insights. We used MDCK cells as our tissue model for this experiment.Each tissue was grown on mattek dishes at high, middle, and low densities. Next, we incubated the tissue at 37 degrees celsius overnight. The next day, we wounded the MDCK tissue by scratching them with a 21G1 ½(38.1 mm) syringe needle. Before the scratch we took an image of the tissues. With the tissue wound we made a time-lapse movie of about 3 hours long with a interval of 1 min. Afterward, we compared each MDCK tissue by adding a calcium chelator named EGTA. As so, this will sequester calcium use as well to block e-cadherin mechanics. We then looked at the relationship between a wounded tissue and a healthy tissue. As such, wound dynamics can be observed. We asked ourselves, will EGTA prevent wound closure?

Biocompatibility of Hydrogen-Diluted Amorphous Silicon Carbide Thin Films for Artificial Heart Valve Coating

NASA Astrophysics Data System (ADS)

Rizal, Umesh; Swain, Bhabani S.; Rameshbabu, N.; Swain, Bibhu P.

2018-01-01

Amorphous silicon carbide (a-SiC:H) thin films were synthesized using trichloromethylsilane by a hot wire chemical vapor deposition process. The deposited films were characterized by Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, x-ray diffraction and x-ray photoelectron spectroscopy to confirm its chemical bonding, structural network and composition of the a-SiC:H films. The optical microscopy images reveal that hydrogen dilution increased the surface roughness and pore density of a-SiC:H thin film. The Raman spectroscopy and FTIR spectra reveal chemical network consisting of Si-Si, C-C and Si-C bonds, respectively. The XRD spectroscopy and Raman spectroscopy indicate a-SiC:H still has short-range order. In addition, in vitro cytotoxicity test ensures the behavior of cell-semiconductor hybrid to monitor the proper coordination. The live-dead assays and MTT assay reveal an increase in green nucleus cell, and cell viability is greater than 88%, respectively, showing non-toxic nature of prepared a-SiC:H film. Moreover, the result indicated by direct contact assay, and cell prefers to adhere and proliferate on a-SiC:H thin films having a positive effect as artificial heart valve coating material.

High CD4(+) T-cell surface CXCR4 density as a risk factor for R5 to X4 switch in the course of HIV-1 infection.

PubMed

Fiser, Anne-Laure; Vincent, Thierry; Brieu, Natalie; Lin, Yea-Lih; Portalès, Pierre; Mettling, Clément; Reynes, Jacques; Corbeau, Pierre

2010-12-15

For unclear reasons, about 50% of HIV-infected subjects harbour CXCR4-using (X4) viral strains in addition of CCR5-using (R5) viral strains at late stages of the disease. One hypothesis is that a low CD4(+) T-cell surface CCR5 density could facilitate the emergence of X4 strains. Alternatively, one could argue that a high CD4(+) T-cell surface CXCR4 density that is observed in individuals presenting with X4 strains, could favour R5 to X4 switch. Here, we tested both hypotheses. In vivo, we observed by quantitative flow cytometry no difference in CD4(+) T-cell surface CCR5 densities between patients with or without X4 strains. In the course of an in vitro R5 infection, the delay of emergence of X4 mutants was similar between cells expressing 2 distinct cell surface CCR5 densities, but shorter (12 ± 0 days and 21 ± 0 days, respectively, P = 0.01) in cells expressing a high surface CXCR4 density as compared with cells with a low surface CXCR4 density. These data argue for a role of CXCR4 density, but not of CCR5 density, in the emergence of X4 strains. They are reassuring concerning the risk of inducing an R5 to X4 switch using CCR5 antagonists to treat HIV infection.

Dynamics of the DNA damage response: insights from live-cell imaging

PubMed Central

Karanam, Ketki; Loewer, Alexander

2013-01-01

All organisms have to safeguard the integrity of their genome to prevent malfunctioning and oncogenic transformation. Sophisticated DNA damage response mechanisms have evolved to detect and repair genomic lesions. With the emergence of live-cell microscopy of individual cells, we now begin to appreciate the complex spatiotemporal kinetics of the DNA damage response and can address the causes and consequences of the heterogeneity in the responses of genetically identical cells. Here, we highlight key discoveries where live-cell imaging has provided unprecedented insights into how cells respond to DNA double-strand breaks and discuss the main challenges and promises in using this technique. PMID:23292635

Patients with Dry Eye Disease and Low Subbasal Nerve Density are at High Risk for an Accelerated Corneal Endothelial Cell Loss

PubMed Central

Kheirkhah, Ahmad; Satitpitakul, Vannarut; Hamrah, Pedram; Dana, Reza

2016-01-01

Purpose To evaluate the changes in corneal endothelial cell density (CECD) over time in patients with dry eye disease (DED) and to correlate the endothelial cell loss with corneal subbasal nerve density. Methods This retrospective study included 40 eyes of 20 patients with DED. Laser in vivo confocal microscopy had been performed in the central cornea of both eyes at an initial visit and repeated after a mean follow-up of 33.2 ± 10.2 months. The densities of corneal endothelial cells and subbasal nerves were measured in both visits and compared with 13 eyes of 13 normal age-matched controls. Results At the initial visit, the DED group had lower densities of corneal endothelial cells (2620 ± 386 cells/mm2) and subbasal nerves (17.8 ± 7.5 mm/mm2) compared with the control group (2861 ± 292 cells/mm2 and 22.8 ± 3.0 mm/mm2, with P=0.08 and P=0.01, respectively). At the end of follow-up, although there was no significant change in subbasal nerve density (16.7 ± 7.2 mm/mm2, P=0.43), the mean CECD significantly decreased to 2465 ± 391 cells/mm2 (P=0.01), with a mean corneal endothelial cell loss of 2.1 ± 3.6% per year. The endothelial cell loss showed a statistically significant negative correlation with the initial subbasal nerve density (Rs= −0.55, P=0.02). Conclusion Patients with DED have an accelerated corneal endothelial cell loss which is more than what has been reported in the literature for normal aging. Those with lower subbasal nerve density, in particular, are at a higher risk for endothelial cell loss over time. PMID:28060067

Stretchable living materials and devices with hydrogel–elastomer hybrids hosting programmed cells

PubMed Central

Liu, Xinyue; Tang, Tzu-Chieh; Tham, Eléonore; Yuk, Hyunwoo; Lin, Shaoting; Lu, Timothy K.; Zhao, Xuanhe

2017-01-01

Living systems, such as bacteria, yeasts, and mammalian cells, can be genetically programmed with synthetic circuits that execute sensing, computing, memory, and response functions. Integrating these functional living components into materials and devices will provide powerful tools for scientific research and enable new technological applications. However, it has been a grand challenge to maintain the viability, functionality, and safety of living components in freestanding materials and devices, which frequently undergo deformations during applications. Here, we report the design of a set of living materials and devices based on stretchable, robust, and biocompatible hydrogel–elastomer hybrids that host various types of genetically engineered bacterial cells. The hydrogel provides sustainable supplies of water and nutrients, and the elastomer is air-permeable, maintaining long-term viability and functionality of the encapsulated cells. Communication between different bacterial strains and with the environment is achieved via diffusion of molecules in the hydrogel. The high stretchability and robustness of the hydrogel–elastomer hybrids prevent leakage of cells from the living materials and devices, even under large deformations. We show functions and applications of stretchable living sensors that are responsive to multiple chemicals in a variety of form factors, including skin patches and gloves-based sensors. We further develop a quantitative model that couples transportation of signaling molecules and cellular response to aid the design of future living materials and devices. PMID:28202725

Live-cell imaging of budding yeast telomerase RNA and TERRA.

PubMed

Laprade, Hadrien; Lalonde, Maxime; Guérit, David; Chartrand, Pascal

2017-02-01

In most eukaryotes, the ribonucleoprotein complex telomerase is responsible for maintaining telomere length. In recent years, single-cell microscopy techniques such as fluorescent in situ hybridization and live-cell imaging have been developed to image the RNA subunit of the telomerase holoenzyme. These techniques are now becoming important tools for the study of telomerase biogenesis, its association with telomeres and its regulation. Here, we present detailed protocols for live-cell imaging of the Saccharomyces cerevisiae telomerase RNA subunit, called TLC1, and also of the non-coding telomeric repeat-containing RNA TERRA. We describe the approach used for genomic integration of MS2 stem-loops in these transcripts, and provide information for optimal live-cell imaging of these non-coding RNAs. Copyright © 2016 Elsevier Inc. All rights reserved.

Live-cell imaging of mammalian RNAs with Spinach2.

PubMed

Strack, Rita L; Jaffrey, Samie R

2015-01-01

The ability to monitor RNAs of interest in living cells is crucial to understanding the function, dynamics, and regulation of this important class of molecules. In recent years, numerous strategies have been developed with the goal of imaging individual RNAs of interest in living cells, each with their own advantages and limitations. This chapter provides an overview of current methods of live-cell RNA imaging, including a detailed discussion of genetically encoded strategies for labeling RNAs in mammalian cells. This chapter then focuses on the development and use of "RNA mimics of GFP" or Spinach technology for tagging mammalian RNAs and includes a detailed protocol for imaging 5S and CGG60 RNA with the recently described Spinach2 tag. © 2015 Elsevier Inc. All rights reserved.

Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells.

PubMed

Wilson, Hannah K; Canfield, Scott G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

2015-05-21

Brain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood-brain barrier (BBB) phenotype. A range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay. The initial hPSC seeding density of approximately 30,000 cells/cm(2) served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm(2)) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype. Given the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.