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Sample records for quantitative cellular level

  1. Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

    PubMed

    Streets, Aaron M; Li, Ang; Chen, Tao; Huang, Yanyi

    2014-09-02

    Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

  2. Transcript quantitation in total yeast cellular RNA using kinetic PCR

    PubMed Central

    Kang, John J.; Watson, Robert M.; Fisher, Mary E.; Higuchi, Russell; Gelfand, David H.; Holland, Michael J.

    2000-01-01

    Kinetically monitored, reverse transcriptase-initiated PCR (kinetic RT–PCR, kRT–PCR) is a novel application of kinetic PCR for high throughput transcript quantitation in total cellular RNA. The assay offers the simplicity and flexibility of an enzyme assay with distinct advantages over DNA microarray hybridization and SAGE technologies for certain applications. The reproducibility, sensitivity and accuracy of the kRT–PCR were assessed for yeast transcripts previously quantitated by a variety of methods including SAGE analysis. Changes in transcript levels between different genetic or physiological cell states were reproducibly quantitated with an accuracy of ±20%. The assay was sufficiently sensitive to quantitate yeast transcripts over a range of more than five orders of magnitude, including low abundance transcripts encoding cell cycle and transcriptional regulators. PMID:10606670

  3. Quantitative analysis of cellular metabolic dissipative, self-organized structures.

    PubMed

    de la Fuente, Ildefonso Martínez

    2010-09-27

    One of the most important goals of the postgenomic era is understanding the metabolic dynamic processes and the functional structures generated by them. Extensive studies during the last three decades have shown that the dissipative self-organization of the functional enzymatic associations, the catalytic reactions produced during the metabolite channeling, the microcompartmentalization of these metabolic processes and the emergence of dissipative networks are the fundamental elements of the dynamical organization of cell metabolism. Here we present an overview of how mathematical models can be used to address the properties of dissipative metabolic structures at different organizational levels, both for individual enzymatic associations and for enzymatic networks. Recent analyses performed with dissipative metabolic networks have shown that unicellular organisms display a singular global enzymatic structure common to all living cellular organisms, which seems to be an intrinsic property of the functional metabolism as a whole. Mathematical models firmly based on experiments and their corresponding computational approaches are needed to fully grasp the molecular mechanisms of metabolic dynamical processes. They are necessary to enable the quantitative and qualitative analysis of the cellular catalytic reactions and also to help comprehend the conditions under which the structural dynamical phenomena and biological rhythms arise. Understanding the molecular mechanisms responsible for the metabolic dissipative structures is crucial for unraveling the dynamics of cellular life.

  4. Quantitative Analysis of Cellular Metabolic Dissipative, Self-Organized Structures

    PubMed Central

    de la Fuente, Ildefonso Martínez

    2010-01-01

    One of the most important goals of the postgenomic era is understanding the metabolic dynamic processes and the functional structures generated by them. Extensive studies during the last three decades have shown that the dissipative self-organization of the functional enzymatic associations, the catalytic reactions produced during the metabolite channeling, the microcompartmentalization of these metabolic processes and the emergence of dissipative networks are the fundamental elements of the dynamical organization of cell metabolism. Here we present an overview of how mathematical models can be used to address the properties of dissipative metabolic structures at different organizational levels, both for individual enzymatic associations and for enzymatic networks. Recent analyses performed with dissipative metabolic networks have shown that unicellular organisms display a singular global enzymatic structure common to all living cellular organisms, which seems to be an intrinsic property of the functional metabolism as a whole. Mathematical models firmly based on experiments and their corresponding computational approaches are needed to fully grasp the molecular mechanisms of metabolic dynamical processes. They are necessary to enable the quantitative and qualitative analysis of the cellular catalytic reactions and also to help comprehend the conditions under which the structural dynamical phenomena and biological rhythms arise. Understanding the molecular mechanisms responsible for the metabolic dissipative structures is crucial for unraveling the dynamics of cellular life. PMID:20957111

  5. Quantitative comparison of cellular patterns of stable and unstable mixtures

    NASA Astrophysics Data System (ADS)

    Zhao, H.; Lee, J. H. S.; Lee, J.; Zhang, Y.

    2016-09-01

    This study describes a comparison of smoked foils from five different mixtures ({{C}}2{{H}}2+2.5{{O}}2+85 %{{Ar}}, 2{{H}}2+{{O}}2+50 %{{Ar}}, {{C}}2{{H}}2+2.5{{O}}2+70 %{{Ar}}, {C}2{{H}}2+5{{N}}2{{O}}, and {{CH}}4+2{{O}}2) that produced transverse waves of regular and irregular spacing. Histograms, variance, and the autocorrelation function (ACF) were used to quantify the spacing irregularity. Each smoked foil was first digitized then separated into left-running and right-running waves for subsequent analysis. The five mixtures showed different degrees of irregularity in the analysis of the histograms and the ACF of the spacing of the transverse waves. The dominant mode was readily found from the peak in the histogram and the first peak in the ACF result. The spacing of the main transverse waves provided by the histogram and the first peak of the ACF were much closer to the spacing of the transverse waves measured by eye for stable mixtures than for unstable ones due to their stronger dominant mode. In certain cases, other modes besides the dominant one were observed, such as two peaks in the histogram and other large peaks in the ACF result. Variance was used as a quantitative measurement of the cellular pattern irregularity level.

  6. In situ quantitative imaging of cellular lipids using molecular sensors

    NASA Astrophysics Data System (ADS)

    Yoon, Youngdae; Lee, Park J.; Kurilova, Svetlana; Cho, Wonhwa

    2011-11-01

    Membrane lipids are dynamic molecules that play important roles in cell signalling and regulation, but an in situ imaging method for quantitatively tracking lipids in living cells is lacking at present. Here, we report a new chemical method of quantitative lipid imaging using sensors engineered by labelling proteins with an environmentally sensitive fluorophore. A prototype sensor for phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2)—a key signalling lipid in diverse cellular processes—was generated by covalently attaching a single 2-dimethylamino-6-acyl-naphthalene group to the N-terminal α-helix of the engineered epsin1 ENTH domain, a protein that selectively binds PtdIns(4,5)P2. The sensor allows robust and sensitive in situ quantitative imaging in mammalian cells, providing new insight into the spatiotemporal dynamics and fluctuation of this key signalling lipid. Application of the sensor to immune cells reveals the presence of a local threshold PtdIns(4,5)P2 concentration required for triggering phagocytosis. This sensor strategy is generally applicable to in situ quantification of other cellular lipids.

  7. Quantitative Analysis of Cellular Senescence in Culture and In Vivo.

    PubMed

    Zhao, Jing; Fuhrmann-Stroissnigg, Heike; Gurkar, Aditi U; Flores, Rafael R; Dorronsoro, Akaitz; Stolz, Donna B; St Croix, Claudette M; Niedernhofer, Laura J; Robbins, Paul D

    2017-01-05

    Cellular senescence refers to the irreversible growth arrest of normally dividing cells in response to various types of stress. Cellular senescence is induced by telomere shortening due to repeated cell division, which causes a DNA damage response, as well as genotoxic, oxidative, and inflammatory stress. Strong mitogenic signaling, such as oncogene activation, also drives cells into a senescent state. Senescent cells express a specific subset of genes, termed the senescence-associated secretory phenotype (SASP), including pro-inflammatory factors, growth factors, and matrix metalloproteinases, which together promote non-cell autonomous, secondary senescence. Clearance of senescent cells that accumulate with age improves health span, implicating cellular senescence as a contributing factor to the aging process. Thus, there is a need for methods to identify and quantify cellular senescence, both in cultured cells and in vivo. Here, methods for the most well-characterized and widely used senescent assays are described, from cell morphology and senescence-associated β-galactosidase (SA-βgal) staining to nuclear biomarkers, SASP, and altered levels of tumor suppressors. © 2017 by John Wiley & Sons, Inc.

  8. Gene Essentiality Is a Quantitative Property Linked to Cellular Evolvability.

    PubMed

    Liu, Gaowen; Yong, Mei Yun Jacy; Yurieva, Marina; Srinivasan, Kandhadayar Gopalan; Liu, Jaron; Lim, John Soon Yew; Poidinger, Michael; Wright, Graham Daniel; Zolezzi, Francesca; Choi, Hyungwon; Pavelka, Norman; Rancati, Giulia

    2015-12-03

    Gene essentiality is typically determined by assessing the viability of the corresponding mutant cells, but this definition fails to account for the ability of cells to adaptively evolve to genetic perturbations. Here, we performed a stringent screen to assess the degree to which Saccharomyces cerevisiae cells can survive the deletion of ~1,000 individual "essential" genes and found that ~9% of these genetic perturbations could in fact be overcome by adaptive evolution. Our analyses uncovered a genome-wide gradient of gene essentiality, with certain essential cellular functions being more "evolvable" than others. Ploidy changes were prevalent among the evolved mutant strains, and aneuploidy of a specific chromosome was adaptive for a class of evolvable nucleoporin mutants. These data justify a quantitative redefinition of gene essentiality that incorporates both viability and evolvability of the corresponding mutant cells and will enable selection of therapeutic targets associated with lower risk of emergence of drug resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Membrane wound healing at single cellular level.

    PubMed

    Afrin, Rehana; Saito, Masakazu; Watanabe-Nakayama, Takahiro; Ikai, Atsushi

    2017-10-01

    We report a nano-technological method of creating a micrometer sized hole on the live cell membrane using atomic force microscope (AFM) and its resealing process at the single cellular level as a model of molecular level wound healing. First, the cell membrane was fluorescently labeled with Kusabira Orange (KO) which was tagged to a lipophilic membrane-sorting peptide. Then a glass bead glued on an AFM cantilever and modified with phospholipase A2 was made to contact the cell membrane. A small dark hole (4-14 μm(2) in area) was created on the otherwise fluorescent cell surface often being accompanied by bleb formation. Refilling of holes with KO fluorescence proceeded at an average rate of ~0.014μm(2)s(-1). The fluorescent lumps which initially surrounded the hole were gradually lost. We compared the present result with our previous ones on the repair processes of artificially damaged stress fibers (Graphical Abstract: Figure S2). Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Cellular-level surgery using nano robots.

    PubMed

    Song, Bo; Yang, Ruiguo; Xi, Ning; Patterson, Kevin Charles; Qu, Chengeng; Lai, King Wai Chiu

    2012-12-01

    The atomic force microscope (AFM) is a popular instrument for studying the nano world. AFM is naturally suitable for imaging living samples and measuring mechanical properties. In this article, we propose a new concept of an AFM-based nano robot that can be applied for cellular-level surgery on living samples. The nano robot has multiple functions of imaging, manipulation, characterizing mechanical properties, and tracking. In addition, the technique of tip functionalization allows the nano robot the ability for precisely delivering a drug locally. Therefore, the nano robot can be used for conducting complicated nano surgery on living samples, such as cells and bacteria. Moreover, to provide a user-friendly interface, the software in this nano robot provides a "videolized" visual feedback for monitoring the dynamic changes on the sample surface. Both the operation of nano surgery and observation of the surgery results can be simultaneously achieved. This nano robot can be easily integrated with extra modules that have the potential applications of characterizing other properties of samples such as local conductance and capacitance.

  11. Cellular-Level Surgery Using Nano Robots

    PubMed Central

    Song, Bo; Yang, Ruiguo; Xi, Ning; Patterson, Kevin Charles; Qu, Chengeng; Lai, King Wai Chiu

    2014-01-01

    The atomic force microscope (AFM) is a popular instrument for studying the nano world. AFM is naturally suitable for imaging living samples and measuring mechanical properties. In this article, we propose a new concept of an AFM-based nano robot that can be applied for cellular-level surgery on living samples. The nano robot has multiple functions of imaging, manipulation, characterizing mechanical properties, and tracking. In addition, the technique of tip functionalization allows the nano robot the ability for precisely delivering a drug locally. Therefore, the nano robot can be used for conducting complicated nano surgery on living samples, such as cells and bacteria. Moreover, to provide a user-friendly interface, the software in this nano robot provides a “videolized” visual feedback for monitoring the dynamic changes on the sample surface. Both the operation of nano surgery and observation of the surgery results can be simultaneously achieved. This nano robot can be easily integrated with extra modules that have the potential applications of characterizing other properties of samples such as local conductance and capacitance. PMID:23015517

  12. Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

    NASA Astrophysics Data System (ADS)

    Esposito, Alessandro

    2006-05-01

    This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

  13. Facilitating arrhythmia simulation: the method of quantitative cellular automata modeling and parallel running

    PubMed Central

    Zhu, Hao; Sun, Yan; Rajagopal, Gunaretnam; Mondry, Adrian; Dhar, Pawan

    2004-01-01

    Background Many arrhythmias are triggered by abnormal electrical activity at the ionic channel and cell level, and then evolve spatio-temporally within the heart. To understand arrhythmias better and to diagnose them more precisely by their ECG waveforms, a whole-heart model is required to explore the association between the massively parallel activities at the channel/cell level and the integrative electrophysiological phenomena at organ level. Methods We have developed a method to build large-scale electrophysiological models by using extended cellular automata, and to run such models on a cluster of shared memory machines. We describe here the method, including the extension of a language-based cellular automaton to implement quantitative computing, the building of a whole-heart model with Visible Human Project data, the parallelization of the model on a cluster of shared memory computers with OpenMP and MPI hybrid programming, and a simulation algorithm that links cellular activity with the ECG. Results We demonstrate that electrical activities at channel, cell, and organ levels can be traced and captured conveniently in our extended cellular automaton system. Examples of some ECG waveforms simulated with a 2-D slice are given to support the ECG simulation algorithm. A performance evaluation of the 3-D model on a four-node cluster is also given. Conclusions Quantitative multicellular modeling with extended cellular automata is a highly efficient and widely applicable method to weave experimental data at different levels into computational models. This process can be used to investigate complex and collective biological activities that can be described neither by their governing differentiation equations nor by discrete parallel computation. Transparent cluster computing is a convenient and effective method to make time-consuming simulation feasible. Arrhythmias, as a typical case, can be effectively simulated with the methods described. PMID:15339335

  14. Quantitative analysis of cellular proteome alterations of Pseudomonas putida to naphthalene-induced stress.

    PubMed

    Li, Shan-Shan; Hu, Xiao; Zhao, Hong; Li, Ya-Xiao; Zhang, Li; Gong, Li-Jun; Guo, Jin; Zhao, Hua-Bing

    2015-08-01

    To investigate whether genes other than the operons nahAaAbAcAdBFCED and nahGTHINLOMKJ of Pseudomonas putida are involved in the tolerance of the bacterium to naphthalene. Cellular responses of P. putida ND6 grown with 2 and 4 g naphthalene/l were investigated using a quantitative proteomic-based approach. Comparative analysis of the proteome data identified that the expression levels of 22 proteins involved in heat shock and universal stress response, naphthalene degradation, cell envelope synthesis, and motility were up-regulated; while the expression levels of 26 proteins involved in protein and fatty acid synthesis, carbon compound, nucleotide, and amino acid metabolism, and small molecule transport were down-regulated. Our findings offer insights into the cellular response of P. putida to high naphthalene concentrations at the protein level.

  15. Adenovirus infection elevates levels of cellular topoisomerase I.

    PubMed Central

    Chow, K C; Pearson, G D

    1985-01-01

    We have developed a specific, sensitive, and quantitative assay for topoisomerase I, which is based on the formation of a covalent enzyme-DNA intermediate. Our assay measures the quantitative transfer of 32P radioactivity from 32P-labeled DNA to topoisomerase I. Since 32P-labeled topoisomerase molecules are resolved by NaDodSO4/PAGE, HeLa topoisomerase I (100 kDa) and calf thymus topoisomerase I (82 kDa) can be quantitatively assayed in the same reaction mixture. The assay can detect at least 0.3 ng (3 fmol) of topoisomerase I. We have used our assay to measure the levels of topoisomerase I activity in crude extracts of nuclei prepared from uninfected, adenovirus-infected, and adenovirus-transformed human cells. The evidence suggests that an adenovirus early gene product, presumably a protein encoded in early region 1A (E1A), increases cellular topoisomerase I activity at least 10-fold. Immunoblotting analysis with antiserum against calf thymus topoisomerase I shows that the increase in activity is due to an increase in the amount of enzyme. Images PMID:2986107

  16. Sub-cellular and Multi-cellular Signaling Mechanisms Revealed by Quantitative Laser Microscopies

    NASA Astrophysics Data System (ADS)

    Piston, David

    2005-03-01

    Newly developed instrumentation and optical probes allows us to image quantitatively dynamic processes within ever more complicated biological systems. Using methods such as fluorescence recovery after photobleaching (FRAP) and Förster resonance energy transfer (FRET) of GFPs fused to the glucose sensing enzyme glucokinase (GK), we have discovered that the location and activity of beta cell GK is acutely regulated by insulin. These findings provide a mechanism whereby the glucose sensing ability of the beta cell is tightly coupled to insulin signaling. We have also measured pancreatic β-cell metabolism during glucose stimulation by quantitative two-photon NAD(P)H imaging. We have developed methods to delineate quantitatively the NAD(P)H signals from the cytoplasm and mitochondria, and show that the metabolic response of these two compartments are differentially stimulated by glucose and other metabolites. Absolute levels of NAD(P)H were determined using two-photon excited fluorescence lifetime imaging (FLIM). These findings elucidate the relative contributions of glycolytic and citric acid cycle metabolism in normal and diabetic cells.

  17. Systematic quantitative characterization of cellular responses induced by multiple signals

    PubMed Central

    2011-01-01

    Background Cells constantly sense many internal and environmental signals and respond through their complex signaling network, leading to particular biological outcomes. However, a systematic characterization and optimization of multi-signal responses remains a pressing challenge to traditional experimental approaches due to the arising complexity associated with the increasing number of signals and their intensities. Results We established and validated a data-driven mathematical approach to systematically characterize signal-response relationships. Our results demonstrate how mathematical learning algorithms can enable systematic characterization of multi-signal induced biological activities. The proposed approach enables identification of input combinations that can result in desired biological responses. In retrospect, the results show that, unlike a single drug, a properly chosen combination of drugs can lead to a significant difference in the responses of different cell types, increasing the differential targeting of certain combinations. The successful validation of identified combinations demonstrates the power of this approach. Moreover, the approach enables examining the efficacy of all lower order mixtures of the tested signals. The approach also enables identification of system-level signaling interactions between the applied signals. Many of the signaling interactions identified were consistent with the literature, and other unknown interactions emerged. Conclusions This approach can facilitate development of systems biology and optimal drug combination therapies for cancer and other diseases and for understanding key interactions within the cellular network upon treatment with multiple signals. PMID:21624115

  18. Quantitative analysis of cellular proteome alterations in human influenza A virus-infected mammalian cell lines.

    PubMed

    Vester, Diana; Rapp, Erdmann; Gade, Dörte; Genzel, Yvonne; Reichl, Udo

    2009-06-01

    Over the last years virus-host cell interactions were investigated in numerous studies. Viral strategies for evasion of innate immune response, inhibition of cellular protein synthesis and permission of viral RNA and protein production were disclosed. With quantitative proteome technology, comprehensive studies concerning the impact of viruses on the cellular machinery of their host cells at protein level are possible. Therefore, 2-D DIGE and nanoHPLC-nanoESI-MS/MS analysis were used to qualitatively and quantitatively determine the dynamic cellular proteome responses of two mammalian cell lines to human influenza A virus infection. A cell line used for vaccine production (MDCK) was compared with a human lung carcinoma cell line (A549) as a reference model. Analyzing 2-D gels of the proteomes of uninfected and influenza-infected host cells, 16 quantitatively altered protein spots (at least +/-1.7-fold change in relative abundance, p<0.001) were identified for both cell lines. Most significant changes were found for keratins, major components of the cytoskeleton system, and for Mx proteins, interferon-induced key components of the host cell defense. Time series analysis of infection processes allowed the identification of further proteins that are described to be involved in protein synthesis, signal transduction and apoptosis events. Most likely, these proteins are required for supporting functions during influenza viral life cycle or host cell stress response. Quantitative proteome-wide profiling of virus infection can provide insights into complexity and dynamics of virus-host cell interactions and may accelerate antiviral research and support optimization of vaccine manufacturing processes.

  19. Evidence for the coordinate control of glycogen synthesis, glucose utilization, and glycolysis in Escherichia coli. II. Quantitative correlation of the inhibition of glycogen synthesis and the stimulation of glucose utilization by 2,4-dinitrophenol with the effects on the cellular levels of glucose 6-phosphate, fructose, 1,6-diphosphate, and total adenylates.

    PubMed

    Dietzler, D N; Leckie, M P; Magnani, J L; Sughrue, M J; Bergstein, P E

    1975-09-25

    In cultures of Escherichia coli W4597(K) and G34 under various nutritional conditions the rates of glucose utilization and cellular levels of fructose-1,6-P2 are quantitatively related by the Hill equation where the value of the Hill coefficient is approximately equal to 2. This is the first evidence that fructose-P2, or any metabolite which covaries with fructose-P2, modulates glucose utilization in E. coli. In light of previous observations from our laboratory this new observation and those in the succeeding report provide the first evidence that in E. coli glycolsis, glycogen synthesis and glucose utilization are coordinately regulated, thus providing for the coupling of ATP utilization and production under various metabolic circumstances. Alterations in the level of ATP apparently affect the velocity of phosphofructokinase, the rate-limiting enzyme in glycolsis, altering the cellular levels of glucose-6-P or fructose-P2. Changes in the levels of these hexose phosphates are quantitatively related to alterations in the rates of glucose utilization and glycogen synthesis in the intact E. coli cell.

  20. Simulating Quantitative Cellular Responses Using Asynchronous Threshold Boolean Network Ensembles

    EPA Science Inventory

    With increasing knowledge about the potential mechanisms underlying cellular functions, it is becoming feasible to predict the response of biological systems to genetic and environmental perturbations. Due to the lack of homogeneity in living tissues it is difficult to estimate t...

  1. Simulating Quantitative Cellular Responses Using Asynchronous Threshold Boolean Network Ensembles

    EPA Science Inventory

    With increasing knowledge about the potential mechanisms underlying cellular functions, it is becoming feasible to predict the response of biological systems to genetic and environmental perturbations. Due to the lack of homogeneity in living tissues it is difficult to estimate t...

  2. Development of Cellular Absorptive Tracers (CATs) for a Quantitative Characterization of Microbial Mass in Flow Systems

    SciTech Connect

    Saripalli, Prasad; Brown, Christopher F.; Lindberg, Michael J.

    2005-03-16

    We report on a new Cellular Absorptive Tracers (CATs) method, for a simple, non-destructive characterization of bacterial mass in flow systems. Results show that adsorption of a CAT molecule into the cellular mass results in its retardation during flow, which is a good, quantitative measure of the biomass quantity and distribution. No such methods are currently available for a quantitative characterization of cell mass.

  3. Modeling approaches for qualitative and semi-quantitative analysis of cellular signaling networks

    PubMed Central

    2013-01-01

    A central goal of systems biology is the construction of predictive models of bio-molecular networks. Cellular networks of moderate size have been modeled successfully in a quantitative way based on differential equations. However, in large-scale networks, knowledge of mechanistic details and kinetic parameters is often too limited to allow for the set-up of predictive quantitative models. Here, we review methodologies for qualitative and semi-quantitative modeling of cellular signal transduction networks. In particular, we focus on three different but related formalisms facilitating modeling of signaling processes with different levels of detail: interaction graphs, logical/Boolean networks, and logic-based ordinary differential equations (ODEs). Albeit the simplest models possible, interaction graphs allow the identification of important network properties such as signaling paths, feedback loops, or global interdependencies. Logical or Boolean models can be derived from interaction graphs by constraining the logical combination of edges. Logical models can be used to study the basic input–output behavior of the system under investigation and to analyze its qualitative dynamic properties by discrete simulations. They also provide a suitable framework to identify proper intervention strategies enforcing or repressing certain behaviors. Finally, as a third formalism, Boolean networks can be transformed into logic-based ODEs enabling studies on essential quantitative and dynamic features of a signaling network, where time and states are continuous. We describe and illustrate key methods and applications of the different modeling formalisms and discuss their relationships. In particular, as one important aspect for model reuse, we will show how these three modeling approaches can be combined to a modeling pipeline (or model hierarchy) allowing one to start with the simplest representation of a signaling network (interaction graph), which can later be refined to

  4. Quantitative study of cellular heterogeneity in doxorubicin uptake and its pharmacological effect on cancer cells.

    PubMed

    Deng, Bin; Wang, Zhi-Ming; Zhou, Zi-Hao; Liu, Yi-Meng; Yang, Xi-Liang; Song, Jian; Xiao, Yu-Xiu

    2014-10-01

    Cellular heterogeneity in doxorubicin (DOX) uptake and its relationship with pharmacological effect on cancer cells were quantitatively investigated for the first time. An in vitro experimental model was established by treating human leukemia K562 and breast cancer MCF-7 cells with different schedules of DOX with or without surface P-glycoprotein (P-gp) inhibitor verapamil (VER). The cellular heterogeneity in DOX uptake was quantitatively examined by single-cell analysis using capillary electrophoresis coupled with laser-induced fluorescence detection. The corresponding cytotoxic effect was tested by cellular morphology, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium and flow cytometry assays. The expression of cellular membrane surface P-gp was determined by flow cytometry. Results showed that the cellular heterogeneity exists in DOX uptake. The single-high DOX schedule leads to lower uptake heterogeneity and higher mean drug uptake. The cellular heterogeneity in DOX uptake was found to be negatively correlated with drug cytotoxicity and surface P-gp expression, with r = -0.7680 to ~ -0.9587. VER reduces the cellular variation in DOX uptake, suggesting that surface P-gp may be one of the causes of the cellular heterogeneity in DOX uptake. This research demonstrates the importance of quantitative study of cellular heterogeneity in drug uptake and its potential application in drug schedule design, response prediction and therapy modulation.

  5. Ultrasensitive proteomic quantitation of cellular signaling by digitized nanoparticle-protein counting

    PubMed Central

    Jacob, Thomas; Agarwal, Anupriya; Ramunno-Johnson, Damien; O’Hare, Thomas; Gönen, Mehmet; Tyner, Jeffrey W.; Druker, Brian J.; Vu, Tania Q.

    2016-01-01

    Many important signaling and regulatory proteins are expressed at low abundance and are difficult to measure in single cells. We report a molecular imaging approach to quantitate protein levels by digitized, discrete counting of nanoparticle-tagged proteins. Digitized protein counting provides ultrasensitive molecular detection of proteins in single cells that surpasses conventional methods of quantitating total diffuse fluorescence, and offers a substantial improvement in protein quantitation. We implement this digitized proteomic approach in an integrated imaging platform, the single cell-quantum dot platform (SC-QDP), to execute sensitive single cell phosphoquantitation in response to multiple drug treatment conditions and using limited primary patient material. The SC-QDP: 1) identified pAKT and pERK phospho-heterogeneity and insensitivity in individual leukemia cells treated with a multi-drug panel of FDA-approved kinase inhibitors, and 2) revealed subpopulations of drug-insensitive CD34+ stem cells with high pCRKL and pSTAT5 signaling in chronic myeloid leukemia patient blood samples. This ultrasensitive digitized protein detection approach is valuable for uncovering subtle but important differences in signaling, drug insensitivity, and other key cellular processes amongst single cells. PMID:27320899

  6. Modeling cellular deformations using the level set formalism

    PubMed Central

    Yang, Liu; Effler, Janet C; Kutscher, Brett L; Sullivan, Sarah E; Robinson, Douglas N; Iglesias, Pablo A

    2008-01-01

    Background Many cellular processes involve substantial shape changes. Traditional simulations of these cell shape changes require that grids and boundaries be moved as the cell's shape evolves. Here we demonstrate that accurate cell shape changes can be recreated using level set methods (LSM), in which the cellular shape is defined implicitly, thereby eschewing the need for updating boundaries. Results We obtain a viscoelastic model of Dictyostelium cells using micropipette aspiration and show how this viscoelastic model can be incorporated into LSM simulations to recreate the observed protrusion of cells into the micropipette faithfully. We also demonstrate the use of our techniques by simulating the cell shape changes elicited by the chemotactic response to an external chemoattractant gradient. Conclusion Our results provide a simple but effective means of incorporating cellular deformations into mathematical simulations of cell signaling. Such methods will be useful for simulating important cellular events such as chemotaxis and cytokinesis. PMID:18652669

  7. Building quantitative, three dimensional atlases of gene expression and morphology at cellular resolution

    PubMed Central

    Knowles, David W.; Biggin, Mark D.

    2013-01-01

    Animals comprise dynamic three-dimensional arrays of cells that express gene products in intricate spatial and temporal patterns that determine cellular differentiation and morphogenesis. A rigorous understanding of these developmental processes requires automated methods that quantitatively record and analyze complex morphologies and their associated patterns of gene expression at cellular resolution. Here we summarize light microscopy based approaches to establish permanent, quantitative datasets—atlases—that record this information. We focus on experiments that capture data for whole embryos or large areas of tissue in three dimensions, often at multiple time points. We compare and contrast the advantages and limitations of different methods and highlight some of the discoveries made. We emphasize the need for interdisciplinary collaborations and integrated experimental pipelines that link sample preparation, image acquisition, image analysis, database design, visualization and quantitative analysis. PMID:24123936

  8. Protein Quantitative Trait Loci Identify Novel Candidates Modulating Cellular Response to Chemotherapy

    PubMed Central

    Gorsic, Lidija K.; Antao, Nirav N.; Wong, Shan S.; Chung, Sophie H.; Gill, Daniel F.; Im, Hae K.; Myers, Jamie L.; White, Kevin P.; Jones, Richard Baker; Dolan, M. Eileen

    2014-01-01

    Annotating and interpreting the results of genome-wide association studies (GWAS) remains challenging. Assigning function to genetic variants as expression quantitative trait loci is an expanding and useful approach, but focuses exclusively on mRNA rather than protein levels. Many variants remain without annotation. To address this problem, we measured the steady state abundance of 441 human signaling and transcription factor proteins from 68 Yoruba HapMap lymphoblastoid cell lines to identify novel relationships between inter-individual protein levels, genetic variants, and sensitivity to chemotherapeutic agents. Proteins were measured using micro-western and reverse phase protein arrays from three independent cell line thaws to permit mixed effect modeling of protein biological replicates. We observed enrichment of protein quantitative trait loci (pQTLs) for cellular sensitivity to two commonly used chemotherapeutics: cisplatin and paclitaxel. We functionally validated the target protein of a genome-wide significant trans-pQTL for its relevance in paclitaxel-induced apoptosis. GWAS overlap results of drug-induced apoptosis and cytotoxicity for paclitaxel and cisplatin revealed unique SNPs associated with the pharmacologic traits (at p<0.001). Interestingly, GWAS SNPs from various regions of the genome implicated the same target protein (p<0.0001) that correlated with drug induced cytotoxicity or apoptosis (p≤0.05). Two genes were functionally validated for association with drug response using siRNA: SMC1A with cisplatin response and ZNF569 with paclitaxel response. This work allows pharmacogenomic discovery to progress from the transcriptome to the proteome and offers potential for identification of new therapeutic targets. This approach, linking targeted proteomic data to variation in pharmacologic response, can be generalized to other studies evaluating genotype-phenotype relationships and provide insight into chemotherapeutic mechanisms. PMID:24699359

  9. Self-organisation in Cellular Automata with Coalescent Particles: Qualitative and Quantitative Approaches

    NASA Astrophysics Data System (ADS)

    Hellouin de Menibus, Benjamin; Sablik, Mathieu

    2017-06-01

    This article introduces new tools to study self-organisation in a family of simple cellular automata which contain some particle-like objects with good collision properties (coalescence) in their time evolution. We draw an initial configuration at random according to some initial shift-ergodic measure, and use the limit measure to describe the asymptotic behaviour of the automata. We first take a qualitative approach, i.e. we obtain information on the limit measure(s). We prove that only particles moving in one particular direction can persist asymptotically. This provides some previously unknown information on the limit measures of various deterministic and probabilistic cellular automata: 3 and 4-cyclic cellular automata [introduced by Fisch (J Theor Probab 3(2):311-338, 1990; Phys D 45(1-3):19-25, 1990)], one-sided captive cellular automata [introduced by Theyssier (Captive Cellular Automata, 2004)], the majority-traffic cellular automaton, a self stabilisation process towards a discrete line [introduced by Regnault and Rémila (in: Mathematical Foundations of Computer Science 2015—40th International Symposium, MFCS 2015, Milan, Italy, Proceedings, Part I, 2015)]. In a second time we restrict our study to a subclass, the gliders cellular automata. For this class we show quantitative results, consisting in the asymptotic law of some parameters: the entry times [generalising K ůrka et al. (in: Proceedings of AUTOMATA, 2011)], the density of particles and the rate of convergence to the limit measure.

  10. Game level layout generation using evolved cellular automata

    NASA Astrophysics Data System (ADS)

    Pech, Andrew; Masek, Martin; Lam, Chiou-Peng; Hingston, Philip

    2016-01-01

    Design of level layouts typically involves the production of a set of levels which are different, yet display a consistent style based on the purpose of a particular level. In this paper, a new approach to the generation of unique level layouts, based on a target set of attributes, is presented. These attributes, which are learned automatically from an example layout, are used for the off-line evolution of a set of cellular automata rules. These rules can then be used for the real-time generation of level layouts that meet the target parameters. The approach is demonstrated on a set of maze-like level layouts. Results are presented to show the effect of various CA parameters and rule representation.

  11. Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide.

    PubMed

    Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

    2017-02-10

    Paralytic peptide (PP) participates in diverse physiological processes as an insect cytokine, such as immunity control, paralysis induction, regulation of cell morphology and proliferation. To investigate the molecular mechanism underlying those physiological activities, we systematically investigated the global phosphorylation events in fat body of silkworm larvae induced by PP through label-free quantitative phosphoproteomics. 2534 phosphosites were finally identified, of which the phosphorylation level of 620 phosphosites on 244 proteins was significantly up-regulated and 67 phosphosites on 43 proteins was down-regulated. Among those proteins, 13 were protein kinases (PKs), 13 were transcription factors (TFs) across 10 families and 17 were metabolism related enzymes. Meanwhile, Motif-X analysis of the phosphorylation sites showed that 16 motifs are significantly enriched, including 8 novel phosphorylation motifs. In addition, KEGG and functional interacting network analysis revealed that phosphorylation cascades play the crucial regulation roles in PP-dependent signaling pathways, and highlighted the potential central position of the mitogen-activated protein kinases (MAPKs) in them. These analyses provide direct insights into the molecule mechanisms of cellular response induced by PP.

  12. Quantitative Proteomic Analysis Revealed Lovastatin-induced Perturbation of Cellular Pathways in HL-60 Cells

    PubMed Central

    Dong, Xiaoli; Xiao, Yongsheng; Jiang, Xinning; Wang, Yinsheng

    2011-01-01

    Lovastatin, a member of the statin family of drugs, is widely prescribed for treating hypercholesterolemia. Statin family of drugs, however, also show promise for cancer treatment and prevention. Although lovastatin is known to be an inhibitor for HMG-CoA reductase, the precise mechanisms underlying the drug’s antiproliferative activity remain unclearly defined. Here we utilized mass spectrometry, in conjunction with stable isotope labeling by amino acids in cell culture (SILAC), to analyze the perturbation of protein expression in HL-60 cells treated with lovastatin. We were able to quantify ~3200 proteins with both forward and reverse SILAC labeling experiments, among which ~120 exhibited significant alterations in expression levels upon lovastatin treatment. Apart from confirming the expected inhibition of cholesterol biosynthesis pathway, our quantitative proteomic results revealed that lovastatin perturbed estrogen receptor signaling pathway, which was manifested by the diminished expression of estrogen receptor α, steroid receptor RNA activator 1 and other related proteins. Lovastatin also altered glutamate metabolism through down-regulation of glutamine synthetase and γ-glutamylcysteine synthetase. Moreover, lovastatin treatment led to a marked down-regulation of carbonate dehydratase II (a.k.a. carbonic anhydrase II) and perturbed the protein ubiquitination pathway. Together, the results from the present study underscored several new cellular pathways perturbed by lovastatin. PMID:21967149

  13. In situ cellular level Raman spectroscopy of the thyroid

    PubMed Central

    Law, Alan Wing Lun; Ahmed, Rafay; Cheung, Tsz Wing; Mak, Chun Yu; Lau, Condon

    2017-01-01

    We report a novel Raman spectroscopy method for in situ cellular level analysis of the thyroid. Thyroids are harvested from control and lithium treated mice. Lithium is used to treat bipolar disorder, but affects thyroid function. Raman spectra are acquired with a confocal setup (514 nm laser, 20 µm spot) focused on a follicular lumen. Raman peaks are observed at 1440, 1656, and 1746 cm−1, corresponding to tyrosine, an important amino acid for protein synthesis. Peaks are also observed at 563, 1087, 1265 and 1301 cm−1. With lithium, the tyrosine peaks increase, indicating tyrosine buildup. Raman spectroscopy can study the impact of many exogenous treatments on thyroid biochemistry. PMID:28270975

  14. In situ cellular level Raman spectroscopy of the thyroid.

    PubMed

    Law, Alan Wing Lun; Ahmed, Rafay; Cheung, Tsz Wing; Mak, Chun Yu; Lau, Condon

    2017-02-01

    We report a novel Raman spectroscopy method for in situ cellular level analysis of the thyroid. Thyroids are harvested from control and lithium treated mice. Lithium is used to treat bipolar disorder, but affects thyroid function. Raman spectra are acquired with a confocal setup (514 nm laser, 20 µm spot) focused on a follicular lumen. Raman peaks are observed at 1440, 1656, and 1746 cm(-1), corresponding to tyrosine, an important amino acid for protein synthesis. Peaks are also observed at 563, 1087, 1265 and 1301 cm(-1). With lithium, the tyrosine peaks increase, indicating tyrosine buildup. Raman spectroscopy can study the impact of many exogenous treatments on thyroid biochemistry.

  15. Effects of static magnetic fields at the cellular level.

    PubMed

    Miyakoshi, Junji

    2005-01-01

    There have been few studies on the effects of static magnetic fields at the cellular level, compared to those of extremely low frequency magnetic fields. Past studies have shown that a static magnetic field alone does not have a lethal effect on the basic properties of cell growth and survival under normal culture conditions, regardless of the magnetic density. Most but not all studies have also suggested that a static magnetic field has no effect on changes in cell growth rate. It has also been shown that cell cycle distribution is not influenced by extremely strong static magnetic fields (up to a maximum of 10 T). A further area of interest is whether static magnetic fields cause DNA damage, which can be evaluated by determination of the frequency of micronucleus formation. The presence or absence of such micronuclei can confirm whether a particular treatment damages cellular DNA. This method has been used to confirm that a static magnetic field alone has no such effect. However, the frequency of micronucleus formation increases significantly when certain treatments (e.g., X-irradiation) are given prior to exposure to a 10 T static magnetic field. It has also been reported that treatment with trace amounts of ferrous ions in the cell culture medium and exposure to a static magnetic field increases DNA damage, which is detected using the comet assay. In addition, many studies have found a strong magnetic field that can induce orientation phenomena in cell culture.

  16. Ataxia telangiectasia: more variation at clinical and cellular levels.

    PubMed

    Taylor, A M R; Lam, Z; Last, J I; Byrd, P J

    2015-03-01

    Ataxia telangiectasia (A-T) is a rare recessively inherited disorder resulting in a progressive neurological decline. It is caused by biallelic mutation of the ATM gene that encodes a 370 kDa serine/threonine protein kinase responsible for phosphorylating many target proteins. ATM is activated by auto(trans)phosphorylation in response to DNA double strand breaks and leads to the activation of cell cycle checkpoints and either DNA repair or apoptosis as part of the cellular response to DNA damage. The allelic heterogeneity in A-T is striking. While the majority of mutations are truncating, leading to instability and loss of the ATM protein from the allele, a significant proportion of patients carry one of a small number of mutations that are either missense or leaky splice site mutations resulting in retention of some ATM with activity. The allelic heterogeneity in ATM, therefore, results in an equally striking clinical heterogeneity. There is also locus heterogeneity because mutation of the MRE11 gene can cause an obvious A-T like disorder both clinically and also at the cellular level and mutation of the RNF168 gene results in a much milder clinical phenotype, neurologically, with the major clinical feature being an immunological defect. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  17. Cellular chromophores and signaling in low level light therapy

    NASA Astrophysics Data System (ADS)

    Hamblin, Michael R.; Demidova-Rice, Tatiana N.

    2007-02-01

    The use of low levels of visible or near infrared light (LLLT) for reducing pain, inflammation and edema, promoting healing of wounds, deeper tissues and nerves, and preventing tissue damage by reducing cellular apoptosis has been known for almost forty years since the invention of lasers. Originally thought to be a peculiar property of laser light (soft or cold lasers), the subject has now broadened to include photobiomodulation and photobiostimulation using non-coherent light. Despite many reports of positive findings from experiments conducted in vitro, in animal models and in randomized controlled clinical trials, LLLT remains controversial. This likely is due to two main reasons; firstly the biochemical mechanisms underlying the positive effects are incompletely understood, and secondly the complexity of rationally choosing amongst a large number of illumination parameters such as wavelength, fluence, power density, pulse structure and treatment timing has led to the publication of a number of negative studies as well as many positive ones. In recent years major advances have been made in understanding the mechanisms that operate at the cellular and tissue levels during LLLT. Mitochondria are thought to be the main site for the initial effects of light and specifically cytochrome c oxidase that has absorption peaks in the red and near infrared regions of the electromagnetic spectrum matches the action spectra of LLLT effects. The discovery that cells employ nitric oxide (NO) synthesized in the mitochondria by neuronal nitric oxide synthase, to regulate respiration by competitive binding to the oxygen binding of cytochrome c oxidase, now suggests how LLLT can affect cell metabolism. If LLLT photodissociates inhibitory NO from cytochrome c oxidase, this would explain increased ATP production, modulation of reactive oxygen species, reduction and prevention of apoptosis, stimulation of angiogenesis, increase of blood flow and induction of transcription factors. In

  18. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    NASA Astrophysics Data System (ADS)

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-06-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.

  19. Quantitatively Mapping Cellular Viscosity with Detailed Organelle Information via a Designed PET Fluorescent Probe

    PubMed Central

    Liu, Tianyu; Liu, Xiaogang; Spring, David R.; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-01-01

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions. PMID:24957323

  20. Quantitatively mapping cellular viscosity with detailed organelle information via a designed PET fluorescent probe.

    PubMed

    Liu, Tianyu; Liu, Xiaogang; Spring, David R; Qian, Xuhong; Cui, Jingnan; Xu, Zhaochao

    2014-06-24

    Viscosity is a fundamental physical parameter that influences diffusion in biological processes. The distribution of intracellular viscosity is highly heterogeneous, and it is challenging to obtain a full map of cellular viscosity with detailed organelle information. In this work, we report 1 as the first fluorescent viscosity probe which is able to quantitatively map cellular viscosity with detailed organelle information based on the PET mechanism. This probe exhibited a significant ratiometric fluorescence intensity enhancement as solvent viscosity increases. The emission intensity increase was attributed to combined effects of the inhibition of PET due to restricted conformational access (favorable for FRET, but not for PET), and the decreased PET efficiency caused by viscosity-dependent twisted intramolecular charge transfer (TICT). A full map of subcellular viscosity was successfully constructed via fluorescent ratiometric detection and fluorescence lifetime imaging; it was found that lysosomal regions in a cell possess the highest viscosity, followed by mitochondrial regions.

  1. Quantitative rules for lymphocyte regulation: the cellular calculus and decisions between tolerance and activation.

    PubMed

    Hodgkin, P D

    2005-10-01

    The innovation of fluorescent division-tracking techniques has elevated our understanding of lymphocytes to a new level. These techniques applied in vitro have identified quantitative rules for lymphocyte differentiation, proliferation and survival that had previously been hidden. The many patterns of quantitative response revealed by these analyses provide a sharp contrast to the traditional idea that T cells must make a binary choice between tolerance and activation. Here, evidence for the classic dogma of two-signal theory and T-T help is re-examined in the light of the new quantitative view to show how logical difficulties can be resolved.

  2. Purification, Cellular Levels, and Functional Domains of LMF1

    PubMed Central

    Babilonia-Rosa, Melissa; Neher, Saskia B.

    2014-01-01

    Over a third of the US adult population has hypertriglyceridemia, resulting in an increased risk of atherosclerosis, pancreatitis, and metabolic syndrome. Lipoprotein lipase (LPL)1, a dimeric enzyme, is the main lipase responsible for TG clearance from the blood after food intake. LPL requires an endoplasmic reticulum (ER)-resident, transmembrane protein known as lipase maturation factor 1 (LMF1) for secretion and enzymatic activity. LMF1 is believed to act as a client specific chaperone for dimeric lipases, but the precise mechanism by which LMF1 functions is not understood. Here, we examine which domains of LMF1 contribute to dimeric lipase maturation by assessing the function of truncation variants. N-terminal truncations of LMF1 show that all the domains are necessary for LPL maturation. Fluorescence microscopy and protease protection assays confirmed that these variants were properly oriented in the ER. We measured cellular levels of LMF1 and found that it is expressed at low levels and each molecule of LMF1 promotes the maturation of 50 or more molecules of LPL. Thus we provide evidence for the critical role of the N-terminus of LMF1 for the maturation of LPL and relevant ratio of chaperone to substrate. PMID:24909692

  3. A positive feedback at the cellular level promotes robustness and modulation at the circuit level.

    PubMed

    Dethier, Julie; Drion, Guillaume; Franci, Alessio; Sepulchre, Rodolphe

    2015-10-01

    This article highlights the role of a positive feedback gating mechanism at the cellular level in the robustness and modulation properties of rhythmic activities at the circuit level. The results are presented in the context of half-center oscillators, which are simple rhythmic circuits composed of two reciprocally connected inhibitory neuronal populations. Specifically, we focus on rhythms that rely on a particular excitability property, the postinhibitory rebound, an intrinsic cellular property that elicits transient membrane depolarization when released from hyperpolarization. Two distinct ionic currents can evoke this transient depolarization: a hyperpolarization-activated cation current and a low-threshold T-type calcium current. The presence of a slow activation is specific to the T-type calcium current and provides a slow positive feedback at the cellular level that is absent in the cation current. We show that this slow positive feedback is required to endow the network rhythm with physiological modulation and robustness properties. This study thereby identifies an essential cellular property to be retained at the network level in modeling network robustness and modulation. Copyright © 2015 the American Physiological Society.

  4. Study of the cellular sociology through quantitative microscopy and topographical analysis

    NASA Astrophysics Data System (ADS)

    Dussert, Christophe; Palmari, Jacqueline; Rasigni, Monique; Kopp, Francis; Berthois, Yolande; Dong, Xue-Fen; Isnardon, Daniel; Rasigni, Georges; Martin, Pierre-Marie

    1992-06-01

    We have developed a methodology to quantitatively study tumor cell heterogeneity from a topographical point of view through the concept of a minimal spanning tree graph. This concept is applied to the quantitation of the degree of order that may exist in a cell population, and by combining biological and mathematical approaches, to the analysis of dynamic and metabolic interactions responsible for this topographical organization. The method is used to analyze the cell cycle phases in tumor cell lines: the cells are detected from an optical microscopy image of the preparation by using algorithms that preserve the cell topography. The cells appear to be differently, and non-randomly, spatially distributed depending on the cycle phase in which they fall. Those topographical behaviors allow us to deduce some unexpected proliferating characteristics of the cells and to compare them to a numerical model of the cell cycle in an interactive population, developed from the cellular automata theory. The method may as well be applied to the topographical analysis of the cells expressing hormone receptors (namely, oestrogenic ones). More generally it may be used to analyze and quantify the cellular sociology both in its normal (morphogenesis) and pathological (cancer, therapeutic responses, ...) aspects.

  5. Quantitative optical microscopy: measurement of cellular biophysical features with a standard optical microscope.

    PubMed

    Phillips, Kevin G; Baker-Groberg, Sandra M; McCarty, Owen J T

    2014-04-07

    We describe the use of a standard optical microscope to perform quantitative measurements of mass, volume, and density on cellular specimens through a combination of bright field and differential interference contrast imagery. Two primary approaches are presented: noninterferometric quantitative phase microscopy (NIQPM), to perform measurements of total cell mass and subcellular density distribution, and Hilbert transform differential interference contrast microscopy (HTDIC) to determine volume. NIQPM is based on a simplified model of wave propagation, termed the paraxial approximation, with three underlying assumptions: low numerical aperture (NA) illumination, weak scattering, and weak absorption of light by the specimen. Fortunately, unstained cellular specimens satisfy these assumptions and low NA illumination is easily achieved on commercial microscopes. HTDIC is used to obtain volumetric information from through-focus DIC imagery under high NA illumination conditions. High NA illumination enables enhanced sectioning of the specimen along the optical axis. Hilbert transform processing on the DIC image stacks greatly enhances edge detection algorithms for localization of the specimen borders in three dimensions by separating the gray values of the specimen intensity from those of the background. The primary advantages of NIQPM and HTDIC lay in their technological accessibility using "off-the-shelf" microscopes. There are two basic limitations of these methods: slow z-stack acquisition time on commercial scopes currently abrogates the investigation of phenomena faster than 1 frame/minute, and secondly, diffraction effects restrict the utility of NIQPM and HTDIC to objects from 0.2 up to 10 (NIQPM) and 20 (HTDIC) μm in diameter, respectively. Hence, the specimen and its associated time dynamics of interest must meet certain size and temporal constraints to enable the use of these methods. Excitingly, most fixed cellular specimens are readily investigated with

  6. Quantification of Pseudouridine Levels in Cellular RNA Pools with a Modified HPLC-UV Assay.

    PubMed

    Xu, Jialin; Gu, Alice Y; Thumati, Naresh R; Wong, Judy M Y

    2017-09-05

    Pseudouridine (Ψ) is the most abundant post-transcriptionally modified ribonucleoside. Different Ψ modifications correlate with stress responses and are postulated to coordinate the distinct biological responses to a diverse panel of cellular stresses. With the help of different guide RNAs, the dyskerin complex pseudouridylates ribosomal RNA, small nuclear RNA and selective messenger RNAs. To monitor Ψ levels quantitatively, a previously reported high performance liquid chromatography method coupled with ultraviolet detection (HPLC-UV) was modified to determine total Ψ levels in different cellular RNA fractions. Our method was validated to be accurate and precise within the linear range of 0.06-15.36 pmol/μL and to have absolute Ψ quantification levels as low as 3.07 pmol. Using our optimized HPLC assay, we found that 1.20% and 1.94% of all ribonucleosides in nuclear-enriched RNA and small non-coding RNA pools from the HEK293 cell line, and 1.77% and 0.98% of ribonucleosides in 18S and 28S rRNA isolated from the HeLa cell line, were pseudouridylated. Upon knockdown of dyskerin expression, a consistent and significant reduction in total Ψ levels in nuclear-enriched RNA pools was observed. Our assay provides a fast and accurate quantification method to measure changes in Ψ levels of different RNA pools without sample derivatization.

  7. Image-based quantitative analysis of gold immunochromatographic strip via cellular neural network approach.

    PubMed

    Zeng, Nianyin; Wang, Zidong; Zineddin, Bachar; Li, Yurong; Du, Min; Xiao, Liang; Liu, Xiaohui; Young, Terry

    2014-05-01

    Gold immunochromatographic strip assay provides a rapid, simple, single-copy and on-site way to detect the presence or absence of the target analyte. This paper aims to develop a method for accurately segmenting the test line and control line of the gold immunochromatographic strip (GICS) image for quantitatively determining the trace concentrations in the specimen, which can lead to more functional information than the traditional qualitative or semi-quantitative strip assay. The canny operator as well as the mathematical morphology method is used to detect and extract the GICS reading-window. Then, the test line and control line of the GICS reading-window are segmented by the cellular neural network (CNN) algorithm, where the template parameters of the CNN are designed by the switching particle swarm optimization (SPSO) algorithm for improving the performance of the CNN. It is shown that the SPSO-based CNN offers a robust method for accurately segmenting the test and control lines, and therefore serves as a novel image methodology for the interpretation of GICS. Furthermore, quantitative comparison is carried out among four algorithms in terms of the peak signal-to-noise ratio. It is concluded that the proposed CNN algorithm gives higher accuracy and the CNN is capable of parallelism and analog very-large-scale integration implementation within a remarkably efficient time.

  8. Piracy on the molecular level: human herpesviruses manipulate cellular chemotaxis.

    PubMed

    Cornaby, Caleb; Tanner, Anne; Stutz, Eric W; Poole, Brian D; Berges, Bradford K

    2016-03-01

    Cellular chemotaxis is important to tissue homeostasis and proper development. Human herpesvirus species influence cellular chemotaxis by regulating cellular chemokines and chemokine receptors. Herpesviruses also express various viral chemokines and chemokine receptors during infection. These changes to chemokine concentrations and receptor availability assist in the pathogenesis of herpesviruses and contribute to a variety of diseases and malignancies. By interfering with the positioning of host cells during herpesvirus infection, viral spread is assisted, latency can be established and the immune system is prevented from eradicating viral infection.

  9. Confocal reflectance quantitative phase microscope system for cellular membranes dynamics study (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Singh, Vijay Raj; Yaqoob, Zahid; So, Peter T. C.

    2017-02-01

    Quantitative phase microscopy (QPM) techniques developed so far primarily belongs to high speed transmitted light based systems that has enough sensitivity to resolve membrane fluctuations and dynamics, but has no depth resolution. Therefore, most biomechanics studies using QPM today is confined to simple cells, such as RBCs, without internal organelles. An important instrument that will greatly extend the biomedical applications of QPM is to develop next generation microscope with 3D capability and sufficient temporal resolution to study biomechanics of complex eukaryotic cells including the mechanics of their internal compartments. For eukaryotic cells, the depth sectioning capability is critical and should be sufficient to distinguish nucleic membrane fluctuations from plasma membrane fluctuations. Further, this microscope must provide high temporal resolution since typical eukaryotes membranes are substantially stiffer than RBCs. A confocal reflectance quantitative phase microscope is presented based on multi-pinhole scanning, with the capabilities of higher temporal resolution and sensitivity for nucleic and plasma membranes of eukaryotic cells. System hardware is developed based on an array of confocal pinhole generated by using the `ON' state of subset of micro-mirrors of digital micro-mirror device (DMD, from Texas Instruments) and high-speed raster scanning provides 14ms imaging speed in wide-field mode. A common path interferometer is integrated at the imaging arm for detection of specimens' quantitative phase information. Theoretical investigation of quantitative phase reconstructed from system is investigated and application of system is presented for dimensional fluctuations measurements of both cellular plasma and nucleic membranes of embryonic stem cells.

  10. Teratogenic potency of valproate analogues evaluated by quantitative estimation of cellular morphology in vitro.

    PubMed

    Berezin, V; Kawa, A; Bojic, U; Foley, A; Nau, H; Regan, C; Edvardsen, K; Bock, E

    1996-10-01

    To develop a simple prescreening system for teratogenicity testing, a novel in vitro assay was established using computer assisted microscopy allowing automatic delineation of contours of stained cells and thereby quantitative determination of cellular morphology. The effects of valproic acid (VPA) and analogues with high as well as low teratogenic activities-(as previously determined in vivo)-were used as probes for study of the discrimination power of the in vitro model. VPA, a teratogenic analogue (+/-)-4-en-VPA, and a non-teratogenic analogue (E)-2-en-VPA, as well as the purified (S)- and (R)-enantiomers of 4-yn-VPA (teratogenic and non-teratogenic, respectively), were tested for their effects on cellular morphology of cloned mouse fibroblastoid L-cell lines, neuroblastoma N2a cells, and rat glioma BT4Cn cells, and were found to induce varying increases in cellular area: Furthermore, it was demonstrated that under the chosen conditions the increase in area correlated statistically significantly with the teratogenic potency of the employed compounds. Setting the cellular area of mouse L-cells to 100% under control conditions, the most pronounced effect was observed for (S)-4-yn-VPA (211%, P = < 0.001) followed by VPA (186%, P < 0.001), 4-en-VPA (169%, P < 0.001) and non-teratogenic 2-en-VPA (137%, P < 0.005) and (R)-4-yn-VPA (105%). This effect was independent of the choice of substrata, since it was observed on L-cells grown on plastic, fibronectin, laminin and Matrigel. However, when VPA-treated cells were exposed to an arginyl-glycyl-aspartate (RGD)-containing peptide to test whether VPA treatment was able to modulate RGD-dependent integrin interactions with components of the extracellular matrix, hardly any effect could be observed, whereas control cells readily detached from the substratum, indicating a changed substrate adhesion of the VPA-treated cells. The data thus indicate that measurement of cellular area may serve as a simple in vitro test in the

  11. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles.

    PubMed

    Julien, Olivier; Zhuang, Min; Wiita, Arun P; O'Donoghue, Anthony J; Knudsen, Giselle M; Craik, Charles S; Wells, James A

    2016-04-05

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events.

  12. Quantitative MS-based enzymology of caspases reveals distinct protein substrate specificities, hierarchies, and cellular roles

    PubMed Central

    Zhuang, Min; Wiita, Arun P.; O’Donoghue, Anthony J.; Knudsen, Giselle M.; Craik, Charles S.; Wells, James A.

    2016-01-01

    Proteases constitute the largest enzyme family, yet their biological roles are obscured by our rudimentary understanding of their cellular substrates. There are 12 human caspases that play crucial roles in inflammation and cell differentiation and drive the terminal stages of cell death. Recent N-terminomics technologies have begun to enumerate the diverse substrates individual caspases can cleave in complex cell lysates. It is clear that many caspases have shared substrates; however, few data exist about the catalytic efficiencies (kcat/KM) of these substrates, which is critical to understanding their true substrate preferences. In this study, we use quantitative MS to determine the catalytic efficiencies for hundreds of natural protease substrates in cellular lysate for two understudied members: caspase-2 and caspase-6. Most substrates are new, and the cleavage rates vary up to 500-fold. We compare the cleavage rates for common substrates with those found for caspase-3, caspase-7, and caspase-8, involved in apoptosis. There is little correlation in catalytic efficiencies among the five caspases, suggesting each has a unique set of preferred substrates, and thus more specialized roles than previously understood. We synthesized peptide substrates on the basis of protein cleavage sites and found similar catalytic efficiencies between the protein and peptide substrates. These data suggest the rates of proteolysis are dominated more by local primary sequence, and less by the tertiary protein fold. Our studies highlight that global quantitative rate analysis for posttranslational modification enzymes in complex milieus for native substrates is critical to better define their functions and relative sequence of events. PMID:27006500

  13. Cellular identity at the single-cell level.

    PubMed

    Coskun, Ahmet F; Eser, Umut; Islam, Saiful

    2016-10-20

    A single cell creates surprising heterogeneity in a multicellular organism. While every organismal cell shares almost an identical genome, molecular interactions in cells alter the use of DNA sequences to modulate the gene of interest for specialization of cellular functions. Each cell gains a unique identity through molecular coding across the DNA, RNA, and protein conversions. On the other hand, loss of cellular identity leads to critical diseases such as cancer. Most cell identity dissection studies are based on bulk molecular assays that mask differences in individual cells. To probe cell-to-cell variability in a population, we discuss single cell approaches to decode the genetic, epigenetic, transcriptional, and translational mechanisms for cell identity formation. In combination with molecular instructions, the physical principles behind cell identity determination are examined. Deciphering and reprogramming cellular types impact biology and medicine.

  14. Quantitation of the Noncovalent Cellular Retinol-Binding Protein, Type 1 Complex Through Native Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Li, Wenjing; Yu, Jianshi; Kane, Maureen A.

    2017-01-01

    Native mass spectrometry (MS) has become a valuable tool in probing noncovalent protein-ligand interactions in a sample-efficient way, yet the quantitative application potential of native MS has not been fully explored. Cellular retinol binding protein, type I (CrbpI) chaperones retinol and retinal in the cell, protecting them from nonspecific oxidation and delivering them to biosynthesis enzymes where the bound (holo-) and unbound (apo-) forms of CrbpI exert distinct biological functions. Using nanoelectrospray, we developed a native MS assay for probing apo- and holo-CrbpI abundance to facilitate exploring their biological functions in retinoid metabolism and signaling. The methods were developed on two platforms, an Orbitrap-based Thermo Exactive and a Q-IMS-TOF-based Waters Synapt G2S, where similar ion behaviors under optimized conditions were observed. Overall, our results suggested that within the working range ( 1-10 μM), gas-phase ions in the native state linearly correspond to solution concentration and relative ion intensities of the apo- and holo-protein ions can linearly respond to the solution ratios, suggesting native MS is a viable tool for relative quantitation in this system.

  15. Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels

    PubMed Central

    Sugimoto, Shinya; Arita-Morioka, Ken-ichi; Mizunoe, Yoshimitsu; Yamanaka, Kunitoshi; Ogura, Teru

    2015-01-01

    The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively, these results suggest that probing mRNA dynamics with ThT is a broadly applicable approach ranging from the molecular level to the single-cell level. PMID:25883145

  16. Quantitative phase-digital holographic microscopy: a new imaging modality to identify original cellular biomarkers of diseases

    NASA Astrophysics Data System (ADS)

    Marquet, P.; Rothenfusser, K.; Rappaz, B.; Depeursinge, C.; Jourdain, P.; Magistretti, P. J.

    2016-03-01

    Quantitative phase microscopy (QPM) has recently emerged as a powerful label-free technique in the field of living cell imaging allowing to non-invasively measure with a nanometric axial sensitivity cell structure and dynamics. Since the phase retardation of a light wave when transmitted through the observed cells, namely the quantitative phase signal (QPS), is sensitive to both cellular thickness and intracellular refractive index related to the cellular content, its accurate analysis allows to derive various cell parameters and monitor specific cell processes, which are very likely to identify new cell biomarkers. Specifically, quantitative phase-digital holographic microscopy (QP-DHM), thanks to its numerical flexibility facilitating parallelization and automation processes, represents an appealing imaging modality to both identify original cellular biomarkers of diseases as well to explore the underlying pathophysiological processes.

  17. Quantitative high content imaging of cellular adaptive stress response pathways in toxicity for chemical safety assessment.

    PubMed

    Wink, Steven; Hiemstra, Steven; Huppelschoten, Suzanna; Danen, Erik; Niemeijer, Marije; Hendriks, Giel; Vrieling, Harry; Herpers, Bram; van de Water, Bob

    2014-03-17

    Over the past decade, major leaps forward have been made on the mechanistic understanding and identification of adaptive stress response landscapes underlying toxic insult using transcriptomics approaches. However, for predictive purposes of adverse outcome several major limitations in these approaches exist. First, the limited number of samples that can be analyzed reduces the in depth analysis of concentration-time course relationships for toxic stress responses. Second these transcriptomics analysis have been based on the whole cell population, thereby inevitably preventing single cell analysis. Third, transcriptomics is based on the transcript level, totally ignoring (post)translational regulation. We believe these limitations are circumvented with the application of high content analysis of relevant toxicant-induced adaptive stress signaling pathways using bacterial artificial chromosome (BAC) green fluorescent protein (GFP) reporter cell-based assays. The goal is to establish a platform that incorporates all adaptive stress pathways that are relevant for toxicity, with a focus on drug-induced liver injury. In addition, cellular stress responses typically follow cell perturbations at the subcellular organelle level. Therefore, we complement our reporter line panel with reporters for specific organelle morphometry and function. Here, we review the approaches of high content imaging of cellular adaptive stress responses to chemicals and the application in the mechanistic understanding and prediction of chemical toxicity at a systems toxicology level.

  18. Cellular Organization of Normal Mouse Liver: A Histological, Quantitative Immunocytochemical, and Fine Structural Analysis

    PubMed Central

    Baratta, Janie L.; Ngo, Anthony; Lopez, Bryan; Kasabwalla, Natasha; Longmuir, Kenneth J.; Robertson, Richard T.

    2009-01-01

    The cellular organization of normal mouse liver was studied using light and electron microscopy and quantitative immunocytochemical techniques. The general histological organization of the mouse liver is similar to livers of other mammalian species, with a lobular organization based on the distributions of portal areas and central venules. The parenchymal hepatocytes were detected with immunocytochemical techniques to recognize albumin or biotin containing cells. The macrophage Kupffer cells were identified with F4-80 immunocytochemistry, Ito stellate cells were identified with GFAP immunocytochemistry, and endothelial cells were labeled with the CD-34 antibody. Kupffer cells were labeled with intravascularly administered fluorescently labeled latex microspheres of both large (0.5 μm) and small (0.03 μm) diameters, while endothelial cells were labeled only with small diameter microspheres. Neither hepatocytes nor Ito stellate cells were labeled by intravascularly administered latex microspheres. The principal fine structural features of hepatocytes and non-parenchymal cells of mouse liver are similar to those reported for rat. Counts of immunocytochemically labeled cells with stained nuclei indicated that hepatocytes constituted approximately 52% of all labeled cells, Kupffer cells about 18%, Ito cells about 8%, and endothelial cells about 22% of all labeled cells. Approximately 35% of the hepatocytes contained two nuclei; none of the Kupffer or Ito cells were double nucleated. The presence of canaliculi and a bile duct system appear similar to that reported for other mammalian species. The cellular organization of the mouse liver is quite similar to that of other mammalian species, confirming that the mouse presents a useful animal model for studies of liver structure and function. PMID:19255771

  19. Qualitative and quantitative determination of heavy metals in waste cellular phones.

    PubMed

    Maragkos, Konstantinos G; Hahladakis, John N; Gidarakos, Evangelos

    2013-09-01

    Twenty four waste cellular phones, manufactured between 2002 and 2011, were selected in order to determine the total heavy metal content in each of their parts (printed circuit boards (PCBs), plastic housing (PH) and liquid crystal display monitors (LCDs)) and compare the results with the permissible limits set by the 2003 Directive on Restriction of Hazardous Substances (RoHS). All the selected samples were pulverized and digested with strong acids. Inductively coupled plasma-mass spectrometry was used to measure the heavy metal content in each sample. The results revealed that concentration levels of the examined heavy metals were higher in PCBs, followed by PH and LCD in that particular order (PCB>PH>LCD). With the exception of Pb and Cr present in PCBs of mobile phones released before the year 2006, all the other metal concentrations were according to the Directive. Concentration levels of Cd, Hg were lower than the permissible limits set by the EU, either before or after the validity of the 2003 RoHS Directive. Considering their significant heavy metal content, coupled with their large quantities produced worldwide in an annual rate, waste cellular phones need to be treated under an environmentally sound management scheme, prioritizing recycling and at the same time eliminating the possibility of any harm. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. AntiJen: a quantitative immunology database integrating functional, thermodynamic, kinetic, biophysical, and cellular data

    PubMed Central

    Toseland, Christopher P; Clayton, Debra J; McSparron, Helen; Hemsley, Shelley L; Blythe, Martin J; Paine, Kelly; Doytchinova, Irini A; Guan, Pingping; Hattotuwagama, Channa K; Flower, Darren R

    2005-01-01

    AntiJen is a database system focused on the integration of kinetic, thermodynamic, functional, and cellular data within the context of immunology and vaccinology. Compared to its progenitor JenPep, the interface has been completely rewritten and redesigned and now offers a wider variety of search methods, including a nucleotide and a peptide BLAST search. In terms of data archived, AntiJen has a richer and more complete breadth, depth, and scope, and this has seen the database increase to over 31,000 entries. AntiJen provides the most complete and up-to-date dataset of its kind. While AntiJen v2.0 retains a focus on both T cell and B cell epitopes, its greatest novelty is the archiving of continuous quantitative data on a variety of immunological molecular interactions. This includes thermodynamic and kinetic measures of peptide binding to TAP and the Major Histocompatibility Complex (MHC), peptide-MHC complexes binding to T cell receptors, antibodies binding to protein antigens and general immunological protein-protein interactions. The database also contains quantitative specificity data from position-specific peptide libraries and biophysical data, in the form of diffusion co-efficients and cell surface copy numbers, on MHCs and other immunological molecules. The uses of AntiJen include the design of vaccines and diagnostics, such as tetramers, and other laboratory reagents, as well as helping parameterize the bioinformatic or mathematical in silico modeling of the immune system. The database is accessible from the URL: . PMID:16305757

  1. Time- and polarization-resolved cellular autofluorescence towards quantitative biochemistry on living cells

    NASA Astrophysics Data System (ADS)

    Alfveby, John; TImerman, Randi; Soto Velasquez, Monica P.; Wickramasinghe, Dhanushka W. P. M.; Bartusek, Jillian; Heikal, Ahmed A.

    2014-09-01

    Native coenzymes such as the reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavin adenine dinucleotide play pivotal roles in energy metabolism and a myriad of biochemical reactions in living cells/tissues. These coenzymes are naturally fluorescent and, therefore, have the potential to serve as intrinsic biomarkers for mitochondrial activities, programmed cell death (apoptosis), oxidative stress, aging, and neurodegenerative disease. In this contribution, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) and time-resolved anisotropy imaging of intracellular NADH for quantitative, non-invasive biochemistry on living cells in response to hydrogenperoxide- induced oxidative stress. In contrast with steady-state one-photon, UV-excited autofluorescence, two-photon FLIM is sensitive to both molecular conformation and stimuli-induced changes in the local environment in living cells with minimum photodamage and inherently enhanced spatial resolution. On the other hand, time-resolved, two-photon anisotropy imaging of cellular autofluorescence allows for quantitative assessment of binding state and environmental restrictions on the tumbling mobility of intrinsic NADH. Our measurements reveal that free and enzyme-bound NADH exist at equilibrium, with a dominant autofluorescence contribution of the bound fraction in living cells. Parallel studies on NADH-enzyme binding in controlled environments serve as a point of reference in analyzing autofluorescence in living cells. These autofluorescence-based approaches complement the conventional analytical biochemistry methods that require the destruction of cells/tissues, while serving as an important step towards establishing intracellular NADH as a natural biomarker for monitoring changes in energy metabolism and redox state of living cells in response to environmental hazards.

  2. Quantitative analysis of receptor tyrosine kinase-effector coupling at functionally relevant stimulus levels.

    PubMed

    Li, Simin; Bhave, Devayani; Chow, Jennifer M; Riera, Thomas V; Schlee, Sandra; Rauch, Simone; Atanasova, Mariya; Cate, Richard L; Whitty, Adrian

    2015-04-17

    A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pM) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pM ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5-10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25-50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome.

  3. Quantitative Analysis of Receptor Tyrosine Kinase-Effector Coupling at Functionally Relevant Stimulus Levels*♦

    PubMed Central

    Li, Simin; Bhave, Devayani; Chow, Jennifer M.; Riera, Thomas V.; Schlee, Sandra; Rauch, Simone; Atanasova, Mariya; Cate, Richard L.; Whitty, Adrian

    2015-01-01

    A major goal of current signaling research is to develop a quantitative understanding of how receptor activation is coupled to downstream signaling events and to functional cellular responses. Here, we measure how activation of the RET receptor tyrosine kinase on mouse neuroblastoma cells by the neurotrophin artemin (ART) is quantitatively coupled to key downstream effectors. We show that the efficiency of RET coupling to ERK and Akt depends strongly on ART concentration, and it is highest at the low (∼100 pm) ART levels required for neurite outgrowth. Quantitative discrimination between ERK and Akt pathway signaling similarly is highest at this low ART concentration. Stimulation of the cells with 100 pm ART activated RET at the rate of ∼10 molecules/cell/min, leading at 5–10 min to a transient peak of ∼150 phospho-ERK (pERK) molecules and ∼50 pAkt molecules per pRET, after which time the levels of these two signaling effectors fell by 25–50% while the pRET levels continued to slowly rise. Kinetic experiments showed that signaling effectors in different pathways respond to RET activation with different lag times, such that the balance of signal flux among the different pathways evolves over time. Our results illustrate that measurements using high, super-physiological growth factor levels can be misleading about quantitative features of receptor signaling. We propose a quantitative model describing how receptor-effector coupling efficiency links signal amplification to signal sensitization between receptor and effector, thereby providing insight into design principles underlying how receptors and their associated signaling machinery decode an extracellular signal to trigger a functional cellular outcome. PMID:25635057

  4. Integrated multiplatform method for in vitro quantitative assessment of cellular uptake for fluorescent polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Ferrari, Raffaele; Lupi, Monica; Falcetta, Francesca; Bigini, Paolo; Paolella, Katia; Fiordaliso, Fabio; Bisighini, Cinzia; Salmona, Mario; D'Incalci, Maurizio; Morbidelli, Massimo; Moscatelli, Davide; Ubezio, Paolo

    2014-01-01

    Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 103-104 polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.

  5. A quantitative chaperone interaction network reveals the architecture of cellular protein homeostasis pathways

    PubMed Central

    Taipale, Mikko; Tucker, George; Peng, Jian; Krykbaeva, Irina; Lin, Zhen-Yuan; Larsen, Brett; Choi, Hyungwon; Berger, Bonnie; Gingras, Anne-Claude; Lindquist, Susan

    2014-01-01

    Chaperones are abundant cellular proteins that promote the folding and function of their substrate proteins (clients). In vivo, chaperones also associate with a large and diverse set of co-factors (co-chaperones) that regulate their specificity and function. However, how these co-chaperones regulate protein folding and whether they have chaperone-independent biological functions is largely unknown. We have combined mass spectrometry and quantitative high-throughput LUMIER assays to systematically characterize the chaperone/co-chaperone/client interaction network in human cells. We uncover hundreds of novel chaperone clients, delineate their participation in specific co-chaperone complexes, and establish a surprisingly distinct network of protein/protein interactions for co-chaperones. As a salient example of the power of such analysis, we establish that NUDC family co-chaperones specifically associate with structurally related but evolutionarily distinct β-propeller folds. We provide a framework for deciphering the proteostasis network, its regulation in development and disease, and expand the use of chaperones as sensors for drug/target engagement. PMID:25036637

  6. Ethanol teratogenesis in Japanese medaka: effects at the cellular level.

    PubMed

    Wu, Minghui; Chaudhary, Amit; Khan, Ikhlas A; Dasmahapatra, Asok K

    2008-01-01

    The adverse effects of alcohol on the developing humans represent a spectrum of structural and neurobehavioral abnormalities, most appropriately termed as fetal alcohol spectrum disorder (FASD). The mechanism by which ethanol induces FASD is unknown. Human studies of FASD are very limited due to ethical constraints; however, several animal models from nematodes to mammals are utilized to understand the molecular mechanism of this disorder. We have used Japanese medaka (Oryzias latipes) embryo-larval development as a unique non-mammalian model to study the molecular mechanism of FASD. Fertilized medaka eggs were exposed to ethanol (0-400 mM) for 48 h post fertilization (hpf) and then maintained in regular embryo rearing medium without ethanol. Viable embryos were harvested on 0, 2, 4 and 6 day post fertilization (dpf) and analyzed for DNA, RNA and protein contents of the embryos. By applying semi-quantitative RT-PCR (rRT-PCR) and quantitative real-time RT-PCR (qRT-PCR), RNA samples were further analyzed for seven transcription factors, emx2, en2, iro3, otx2, shh, wnt1 and zic5 which are expressed in the neural tube of medaka embryo during early phase of development. RNA and protein contents of the embryos were significantly reduced by ethanol at 400 mM dose on 4 and 6 dpf compared to the control (no ethanol), and 100 mM ethanol treated embryos. However, significant reduction of DNA was observed only in 4 dpf embryos. Total protein contents of yolk remained unaltered after ethanol treatment. Expression pattern of emx2, en2, iro3, otx2, shh, wnt1, and zic5 mRNAs were found to be developmentally regulated, however, remained unaltered after ethanol treatment. It is therefore concluded that alteration of nucleic acid and protein contents of medaka embryo by ethanol could be used as an indicator of embryonic growth retardation which might be the result of disruption of specific gene function during development.

  7. Monitoring the physiological status in bioprocesses on the cellular level.

    PubMed

    Schuster, K C

    2000-01-01

    The trend in bioprocess monitoring and control is towards strategies which are based on the physiological status of the organism in the bioprocess. This requires that the measured process variables should be biologically meaningful in order to apply them in physiologically based control strategies. The on-line monitoring equipment available today mostly derives information on the physiological status indirectly, from external variables outside the cells. The complementary approach reviewed here is to analyse the microbial cells directly, in order to obtain information on the internal variables inside the cells. This overview covers methods for analysis of whole cells (as a population or as a single cell), for groups of cellular components, and for specific compounds which serve as markers for a certain physiological status. Physico-chemical separation methods (chromatography, electrophoresis) and reactive analysis can be used to analyse elemental and macromolecular composition of cells. Spectroscopic methods (mass, dielectric, nuclear magnetic, infrared, and Raman) have only recently been applied to such complex multicomponent mixtures such as microbial cells. Spectroscopy and chemical separation methods produce large amounts of data, which can often be used in the best way by applying chemometrics. Some of the methods can yield information not just on the average of the microbial cell population, but also on the distribution of sub-populations. The suitability of the methods for on-line coupling to the bioprocess is discussed. Others not suitable for on-line coupling can be established in routine off-line analysis procedures. The information gained by the methods discussed can mainly be used to establish better knowledge of the basis for monitoring and control strategies. Some are also applicable in real-time monitoring and control.

  8. A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment

    NASA Astrophysics Data System (ADS)

    Khademolhosseini, F.; Liu, C.-C.; Lim, C. J.; Chiao, M.

    2016-08-01

    Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

  9. Quantitative levels of immunoglobulin E in advanced tuberculosis.

    PubMed

    Casterline, C L; Evans, R; Ward, G W

    1976-07-01

    Quantitative levels of immunoglobulin E (IgE) were determined in samples of sera obtained from 29 patients with proven moderate to far-advanced tuberculosis. The sensitive radioimmunoassay test for IgE was used. Statistical analysis of the results revealed no difference in IgE values as compared to a control group of normal sera. In contrast to other chronic pulmonary infections, such as bronchopulmonary aspergillosis, the IgE level in pulmonary tuberculous infection is of no diagnostic significance. Simultaneous determination of levels of immunoglobulins G, A, M, and D (IgG, IgA, IgM, IgD) in these same sera by radial immunodiffusion showed elevated IgG and lowered IgM levels in the tuberculous patients, confirming previous studies. The significance of these alterations in immunoglobulin levels is unclear and may represent a secondary phenomenon rather than a primary host response.

  10. Pyrazinoic acid decreases the proton motive force, respiratory ATP synthesis activity, and cellular ATP levels.

    PubMed

    Lu, Ping; Haagsma, Anna C; Pham, Hoang; Maaskant, Janneke J; Mol, Selena; Lill, Holger; Bald, Dirk

    2011-11-01

    Pyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels in Mycobacterium bovis BCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

  11. Using a Virtual Tissue Culture System to Assist Students in Understanding Life at the Cellular Level

    ERIC Educational Resources Information Center

    McLauglin, Jacqueline S.; Seaquist, Stephen B.

    2008-01-01

    In every biology course ever taught in the nation's classrooms, and in every biology book ever published, students are taught about the "cell." The cell is as fundamental to biology as the atom is to chemistry. Truly, everything an organism does occurs fundamentally at the cellular level. Beyond memorizing the cellular definition, students are not…

  12. Using a Virtual Tissue Culture System to Assist Students in Understanding Life at the Cellular Level

    ERIC Educational Resources Information Center

    McLauglin, Jacqueline S.; Seaquist, Stephen B.

    2008-01-01

    In every biology course ever taught in the nation's classrooms, and in every biology book ever published, students are taught about the "cell." The cell is as fundamental to biology as the atom is to chemistry. Truly, everything an organism does occurs fundamentally at the cellular level. Beyond memorizing the cellular definition, students are not…

  13. Highly Dynamic Cellular-Level Response of Symbiotic Coral to a Sudden Increase in Environmental Nitrogen

    PubMed Central

    Kopp, C.; Pernice, M.; Domart-Coulon, I.; Djediat, C.; Spangenberg, J. E.; Alexander, D. T. L.; Hignette, M.; Meziane, T.; Meibom, A.

    2013-01-01

    ABSTRACT Metabolic interactions with endosymbiotic photosynthetic dinoflagellate Symbiodinium spp. are fundamental to reef-building corals (Scleractinia) thriving in nutrient-poor tropical seas. Yet, detailed understanding at the single-cell level of nutrient assimilation, translocation, and utilization within this fundamental symbiosis is lacking. Using pulse-chase 15N labeling and quantitative ion microprobe isotopic imaging (NanoSIMS; nanoscale secondary-ion mass spectrometry), we visualized these dynamic processes in tissues of the symbiotic coral Pocillopora damicornis at the subcellular level. Assimilation of ammonium, nitrate, and aspartic acid resulted in rapid incorporation of nitrogen into uric acid crystals (after ~45 min), forming temporary N storage sites within the dinoflagellate endosymbionts. Subsequent intracellular remobilization of this metabolite was accompanied by translocation of nitrogenous compounds to the coral host, starting at ~6 h. Within the coral tissue, nitrogen is utilized in specific cellular compartments in all four epithelia, including mucus chambers, Golgi bodies, and vesicles in calicoblastic cells. Our study shows how nitrogen-limited symbiotic corals take advantage of sudden changes in nitrogen availability; this opens new perspectives for functional studies of nutrient storage and remobilization in microbial symbioses in changing reef environments. PMID:23674611

  14. Networking with noise at the molecular, cellular, and population level

    NASA Astrophysics Data System (ADS)

    Vilar, Jose

    2002-03-01

    The intrinsic stochastic nature of biochemical reactions affects enzymatic and transcriptional networks at different levels. Yet, cells are able to function effectively and consistently amidst such random fluctuations. I will discuss some molecular mechanisms that are able to reduce the intrinsic noise of chemical reactions, how suitable designs can make networks resistant to noise, and what strategies can be used by populations to achieve precise functions.

  15. Modeling psychiatric disorders at the cellular and network levels.

    PubMed

    Brennand, K J; Simone, A; Tran, N; Gage, F H

    2012-12-01

    Although psychiatric disorders such as autism spectrum disorders, schizophrenia and bipolar disorder affect a number of brain regions and produce a complex array of clinical symptoms, basic phenotypes likely exist at the level of single neurons and simple networks. Being highly heritable, it is hypothesized that these disorders are amenable to cell-based studies in vitro. Using induced pluripotent stem cell-derived neurons and/or induced neurons from fibroblasts, limitless numbers of live human neurons can now be generated from patients with a genetic background permissive to the disease state. We predict that cell-based studies will ultimately contribute to our understanding of the initiation, progression and treatment of these psychiatric disorders.

  16. A preparative suspension culture system permitting quantitation of anchorage-independent growth by direct radiolabeling of cellular DNA.

    PubMed

    Assoian, R K; Boardman, L A; Drosinos, S

    1989-02-15

    We have developed a hybrid methylcellulose/agar suspension culture system which permits long-term colony formation of transformed mesenchymal cells. In contrast to traditional agar suspensions, our system allows for recovery of cells and direct biochemical analysis of anchorage-independent growth. The ability to readily radiolabel cellular macromolecules in these preparative cultures permits a quantitative and objective analysis of colony formation by incorporation of [3H]thymidine into newly synthesized DNA.

  17. Evaluation of polymeric nanoparticle formulations by effective imaging and quantitation of cellular uptake for controlled delivery of doxorubicin.

    PubMed

    Win, Khin Yin; Teng, Choon Peng; Ye, Enyi; Low, Michelle; Han, Ming-Yong

    2015-03-01

    Various polymeric nanoparticles have been extensively engineered for applications in controlled drug release delivery in the last decades. Currently, there is a great demand to develop a strategy to qualitatively and quantitatively evaluate these polymeric nanoparticle formulations for producing innovative delivery systems. In this work, a screening platform is developed using luminescent quantum dots as drug model and imaging label to evaluate nanoparticle formulations incorporating either hydrophilic or hydrophobic drugs and imaging agents. It is validated that there is no influence of the incorporated entities on the cellular uptake profile. The use of quantum dots enables efficient detection and precise quantitation of cellular uptake of particles which occupy 25% of the cell volume. The correlation of quantum dot- and doxorubicin-incorporated nanoparticles is useful to develop an evaluation platform for nanoparticle formulations through imaging and quantitation. This platform is also used to observe the surface properties effect of other polymers such as chitosan and poly(ethylene) glycol on the cellular interaction and uptake. Moreover, quantum dots can be used to study microparticle theranostic delivery formulations by deliberately incorporating as visible ring surrounding the microparticles for their easy identifying and tracing in diagnostic and chemotherapeutic applications.

  18. QUANTITATIVE IN VITRO MEASUREMENT OF CELLULAR PROCESSES CRITICAL TO THE DEVELOPMENT OF NEURAL CONNECTIVITY USING HCA.

    EPA Science Inventory

    New methods are needed to screen thousands of environmental chemicals for toxicity, including developmental neurotoxicity. In vitro, cell-based assays that model key cellular events have been proposed for high throughput screening of chemicals for developmental neurotoxicity. Whi...

  19. QUANTITATIVE IN VITRO MEASUREMENT OF CELLULAR PROCESSES CRITICAL TO THE DEVELOPMENT OF NEURAL CONNECTIVITY USING HCA.

    EPA Science Inventory

    New methods are needed to screen thousands of environmental chemicals for toxicity, including developmental neurotoxicity. In vitro, cell-based assays that model key cellular events have been proposed for high throughput screening of chemicals for developmental neurotoxicity. Whi...

  20. Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy.

    PubMed

    Sena, Giovanni; Frentz, Zak; Birnbaum, Kenneth D; Leibler, Stanislas

    2011-01-01

    To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.

  1. Quantitative, Phenotypical, and Functional Characterization of Cellular Immunity in Children and Adolescents With Down Syndrome.

    PubMed

    Schoch, Justine; Rohrer, Tilman R; Kaestner, Michael; Abdul-Khaliq, Hashim; Gortner, Ludwig; Sester, Urban; Sester, Martina; Schmidt, Tina

    2017-05-15

    Infections and autoimmune disorders are more frequent in Down syndrome, suggesting abnormality of adaptive immunity. Although the role of B cells and antibodies is well characterized, knowledge regarding T cells is limited. Lymphocyte subpopulations of 40 children and adolescents with Down syndrome and 51 controls were quantified, and phenotype and functionality of antigen-specific effector T cells were analyzed with flow cytometry after polyclonal and pathogen-specific stimulation (with varicella-zoster virus [VZV] and cytomegalovirus [CMV]). Results were correlated with immunoglobulin (Ig) G responses. Apart from general alterations in the percentage of lymphocytes, regulatory T cells, and T-helper 1 and 17 cells, all major T-cell subpopulations showed higher expression of the inhibitory receptor PD-1. Polyclonally stimulated effector CD4+ T-cell frequencies were significantly higher in subjects with Down syndrome, whereas their inhibitory receptor expression (programmed cell death 1 [PD-1] and cytotoxic T-lymphocyte antigen 4 [CTLA-4]) was similar to that of controls and cytokine expression profiles were only marginally altered. Pathogen-specific immunity showed age-appropriate levels of endemic infection, with correlation of CMV-specific cellular and humoral immunity in all subjects. Among VZV IgG-positive individuals, a higher percentage of VZV-specific T-cell-positive subjects was seen in those with Down syndrome. Despite alterations in lymphocyte subpopulations, individuals with Down syndrome can mount effector T-cell responses with similar phenotype and functionality as controls but may require higher effector T-cell frequencies to ensure pathogen control.

  2. Development of Cellular Absorptive Tracers (CATs) for a Quantitative Characterization of the Complexity of Nanoscale Biological Systems

    NASA Astrophysics Data System (ADS)

    Choi, J.; Saripalli, P.; Meldrum, D.; Lee, S.

    2005-12-01

    A new method is proposed in this study, which is entitled Cellular Absorptive Tracers (CATs; PNNL Invention Disclosure; Saripalli, P. 2001), to characterize the extent, location, and morphology of cell mass in microelectromechanical systems (MEMS), by preferentially absorbing into the living or lysed cells or adsorbing at the cell surfaces. The CATs were used to demonstrate their utility to quantitatively characterize the biomass, its location, and morphology in MEMS. A series of experiments had conducted, using different number/types of cells and morphologic distributions (e.g., concentrated vs. disperse) within the microenvironmental chamber. The experiments were conducted such that, simultaneously with CATs characterization, a number of metabolic parameters (such as oxygen, CO2, glucose, pH, methanol, formate and small molecule metabolites, etc.) also were measured by fluorescence microscopy (Lidstrom and Meldrum, Nature, 2003). Results of partition and transport experiments show that adsorption of a CAT molecule into the cellular mass results in its retardation during flow, which is a good measure of the biomass quantity and distribution. The molecules chosen were used to demonstrate their utility to quantitatively characterize the biomass, its location and morphology in MEMS. The results yield first-of-their-kind data sets relating metabolic parameters to location, heterogeneity, and morphology of cells. The data were used to obtain quantitative information needed for the characterization of biomass in MEMS, and to develop the theory relating the cellular phenomena of interest to cellular heterogeneity and morphology. The proposed research contributes a new set of tools for a rapid, noninvasive characterization of nano-scale biological systems.

  3. In situ sensing and modeling of molecular events at the cellular level

    NASA Astrophysics Data System (ADS)

    Yang, Ruiguo

    We developed the Atomic Force Microscopy (AFM) based nanorobot in combination with other nanomechanical sensors for the investigation of cell signaling pathways. The AFM nanorobotics hinge on the superior spatial resolution of AFM in imaging and extends it into the measurement of biological processes and manipulation of biological matters. A multiple input single output control system was designed and implemented to solve the issues of nanomanipulation of biological materials, feedback, response frequency and nonlinearity. The AFM nanorobotic system therefore provide the human-directed position, velocity and force control with high frequency feedback, and more importantly it can feed the operator with the real-time imaging of manipulation result from the fast-imaging based local scanning. The use of the system has taken the study of cellular process at the molecular scale into a new level. The cellular response to the physiological conditions can be significantly manifested in cellular mechanics. Dynamic mechanical property has been regarded as biomarkers, sometimes even regulators of the signaling and physiological processes, thus the name mechanobiology. We sought to characterize the relationship between the structural dynamics and the molecular dynamics and the role of them in the regulation of cell behavior. We used the AFM nanorobotics to investigate the mechanical properties in real-time of cells that are stimulated by different chemical species. These reagents could result in similar ion channel responses but distinctive mechanical behaviors. We applied these measurement results to establish a model that describes the cellular stimulation and the mechanical property change, a "two-hit" model that comprises the loss of cell adhesion and the initiation of cell apoptosis. The first hit was verified by functional experiments: depletion of Calcium and nanosurgery to disrupt the cellular adhesion. The second hit was tested by a labeling of apoptotic markers that

  4. Quantitative phase measurement for wafer-level optics

    NASA Astrophysics Data System (ADS)

    Qu, Weijuan; Wen, Yongfu; Wang, Zhaomin; Yang, Fang; Huang, Lei; Zuo, Chao

    2015-07-01

    Wafer-level-optics now is widely used in smart phone camera, mobile video conferencing or in medical equipment that require tiny cameras. Extracting quantitative phase information has received increased interest in order to quantify the quality of manufactured wafer-level-optics, detect defective devices before packaging, and provide feedback for manufacturing process control, all at the wafer-level for high-throughput microfabrication. We demonstrate two phase imaging methods, digital holographic microscopy (DHM) and Transport-of-Intensity Equation (TIE) to measure the phase of the wafer-level lenses. DHM is a laser-based interferometric method based on interference of two wavefronts. It can perform a phase measurement in a single shot. While a minimum of two measurements of the spatial intensity of the optical wave in closely spaced planes perpendicular to the direction of propagation are needed to do the direct phase retrieval by solving a second-order differential equation, i.e., with a non-iterative deterministic algorithm from intensity measurements using the Transport-of-Intensity Equation (TIE). But TIE is a non-interferometric method, thus can be applied to partial-coherence light. We demonstrated the capability and disability for the two phase measurement methods for wafer-level optics inspection.

  5. TISSUE-TO-CELLULAR LEVEL DEFORMATION COUPLING IN CELL-MICROINTEGRATED ELASTOMERIC SCAFFOLDS

    PubMed Central

    Stella, John A.; Liao, Jun; Hong, Yi; Merryman, W. David; Wagner, William R.; Sacks, Michael S.

    2008-01-01

    In engineered tissues we are challenged to reproduce extracellular matrix and cellular deformation coupling that occurs within native tissues, which is a meso-micro scale phenomenon that profoundly affects tissue growth and remodeling. With our ability to electrospin polymer fiber scaffolds while simultaneously electrospraying viable cells, we are provided with a unique platform to investigate cellular deformations within a three dimensional elastomeric fibrous scaffold. Scaffold specimens micro-integrated with vascular smooth muscle cells were subjected to controlled biaxial stretch with 3D cellular deformations and local fiber micro-architecture simultaneously quantified. We demonstrated that the local fiber geometry followed an affine behavior, so that it could be predicted by macro scaffold deformations. However, local cellular deformations depended non-linearly on changes in fiber microarchitecture and ceased at large strains where the scaffold fibers completely straightened. Thus, local scaffold microstructural changes induced by macro-level applied strain dominated cellular deformations, so that monotonic increases in scaffold strain do not necessitate similar levels of cellular deformation. This result has fundamental implications when attempting to elucidate the events of de-novo tissue development and remodeling in engineered tissues, which are thought to depend substantially on cellular deformations. PMID:18472154

  6. A Quantitative Microscopy Technique for Determining the Number of Specific Proteins in Cellular Compartments

    PubMed Central

    Mutch, Sarah A.; Gadd, Jennifer C.; Fujimoto, Bryant S.; Kensel-Hammes, Patricia; Schiro, Perry G.; Bajjalieh, Sandra M.; Chiu, Daniel T.

    2013-01-01

    This protocol describes a method to determine both the average number and variance of proteins in the few to tens of copies in isolated cellular compartments, such as organelles and protein complexes. Other currently available protein quantification techniques either provide an average number but lack information on the variance or are not suitable for reliably counting proteins present in the few to tens of copies. This protocol entails labeling the cellular compartment with fluorescent primary-secondary antibody complexes, TIRF (total internal reflection fluorescence) microscopy imaging of the cellular compartment, digital image analysis, and deconvolution of the fluorescence intensity data. A minimum of 2.5 days is required to complete the labeling, imaging, and analysis of a set of samples. As an illustrative example, we describe in detail the procedure used to determine the copy number of proteins in synaptic vesicles. The same procedure can be applied to other organelles or signaling complexes. PMID:22094731

  7. A Nanogram Level Colloidal Gold Single Reagent Quantitative Protein Assay

    PubMed Central

    Harrison, Gerald; Haffey, Patrick; Golub, Ellis E.

    2008-01-01

    We have developed a nanogram level quantitative protein assay based on the binding of colloidal gold to proteins adhered to nitrocellulose paper. The protein-gold complex produces a purple color proportional to the amount of protein present, and the intensity of the stain is quantified by densitometry. Typical assays require minimal starting material (10 – 20 μl) containing 2 – 5 μg protein. A small volume (2 μl) of protein solution is applied to nitrocellulose paper in a grid array, and dried. The nitrocellulose is incubated in colloidal gold suspension with gentle agitation (4–16 h), rinsed with water and scanned. Densitometric analysis of the scanned images allows quantitation of the unknown sample protein concentration by comparison with protein standards placed on the same nitrocellulose grid. The assay requires significantly less sample than conventional protein assays. In this report, the Golddots assay is calibrated against weighed protein samples, and compared with the Pierce Micro BCA protein assay kit. In addition, the assay is evaluated with several known proteins with different physical properties. PMID:18539124

  8. A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption

    PubMed Central

    Sherry, David M; Parks, Eileen E; Bullen, Elizabeth C; Updike, Dawn L; Howard, Eric W

    2013-01-01

    Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic “scratch” assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the “wound” is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell–substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling. PMID:24430202

  9. Interferometric time-stretch microscopy for ultrafast quantitative cellular and tissue imaging at 1 μm

    NASA Astrophysics Data System (ADS)

    Lau, Andy K. S.; Wong, Terence T. W.; Ho, Kenneth K. Y.; Tang, Matthew T. H.; Chan, Antony C. S.; Wei, Xiaoming; Lam, Edmund Y.; Shum, Ho Cheung; Wong, Kenneth K. Y.; Tsia, Kevin K.

    2014-07-01

    Quantitative phase imaging (QPI) has been proven to be a powerful tool for label-free characterization of biological specimens. However, the imaging speed, largely limited by the image sensor technology, impedes its utility in applications where high-throughput screening and efficient big-data analysis are mandated. We here demonstrate interferometric time-stretch (iTS) microscopy for delivering ultrafast quantitative phase cellular and tissue imaging at an imaging line-scan rate >20 MHz-orders-of-magnitude faster than conventional QPI. Enabling an efficient time-stretch operation in the 1-μm wavelength window, we present an iTS microscope system for practical ultrafast QPI of fixed cells and tissue sections, as well as ultrafast flowing cells (at a flow speed of up to 8 m/s). To the best of our knowledge, this is the first time that time-stretch imaging could reveal quantitative morphological information of cells and tissues with nanometer precision. As many parameters can be further extracted from the phase and can serve as the intrinsic biomarkers for disease diagnosis, iTS microscopy could find its niche in high-throughput and high-content cellular assays (e.g., imaging flow cytometry) as well as tissue refractometric imaging (e.g., whole-slide imaging for digital pathology).

  10. AMPK: Regulating Energy Balance at the Cellular and Whole Body Levels

    PubMed Central

    Hardie, D. Grahame; Ashford, Michael L. J.

    2014-01-01

    AMP-activated protein kinase appears to have evolved in single-celled eukaryotes as an adenine nucleotide sensor that maintains energy homeostasis at the cellular level. However, during evolution of more complex multicellular organisms, the system has adapted to interact with hormones so that it also plays a key role in balancing energy intake and expenditure at the whole body level. PMID:24583766

  11. Short Communication: High Cellular Iron Levels Are Associated with Increased HIV Infection and Replication

    PubMed Central

    Chang, Hsiang-Chun; Bayeva, Marina; Taiwo, Babafemi; Palella, Frank J.; Hope, Thomas J.

    2015-01-01

    Abstract HIV is a pandemic disease, and many cellular and systemic factors are known to alter its infectivity and replication. Earlier studies had suggested that anemia is common in HIV-infected patients; however, higher iron was also observed in AIDS patients prior to the introduction of antiretroviral therapy (ART). Therefore, the relationship between iron and viral infection is not well delineated. To address this issue, we altered the levels of cellular iron in primary CD4+ T cells and showed that higher iron is associated with increased HIV infection and replication. In addition, HIV infection alone leads to increased cellular iron, and several ART drugs increase cellular iron independent of HIV infection. Finally, HIV infection is associated with increased serum iron in HIV-positive patients regardless of treatment with ART. These results establish a relationship between iron and HIV infection and suggest that iron homeostasis may be a viable therapeutic target for HIV. PMID:25291189

  12. Short communication: high cellular iron levels are associated with increased HIV infection and replication.

    PubMed

    Chang, Hsiang-Chun; Bayeva, Marina; Taiwo, Babafemi; Palella, Frank J; Hope, Thomas J; Ardehali, Hossein

    2015-03-01

    HIV is a pandemic disease, and many cellular and systemic factors are known to alter its infectivity and replication. Earlier studies had suggested that anemia is common in HIV-infected patients; however, higher iron was also observed in AIDS patients prior to the introduction of antiretroviral therapy (ART). Therefore, the relationship between iron and viral infection is not well delineated. To address this issue, we altered the levels of cellular iron in primary CD4(+) T cells and showed that higher iron is associated with increased HIV infection and replication. In addition, HIV infection alone leads to increased cellular iron, and several ART drugs increase cellular iron independent of HIV infection. Finally, HIV infection is associated with increased serum iron in HIV-positive patients regardless of treatment with ART. These results establish a relationship between iron and HIV infection and suggest that iron homeostasis may be a viable therapeutic target for HIV.

  13. KDM5 Interacts with Foxo to Modulate Cellular Levels of Oxidative Stress

    PubMed Central

    Liu, Xingyin; Greer, Christina; Secombe, Julie

    2014-01-01

    Increased cellular levels of oxidative stress are implicated in a large number of human diseases. Here we describe the transcription co-factor KDM5 (also known as Lid) as a new critical regulator of cellular redox state. Moreover, this occurs through a novel KDM5 activity whereby it alters the ability of the transcription factor Foxo to bind to DNA. Our microarray analyses of kdm5 mutants revealed a striking enrichment for genes required to regulate cellular levels of oxidative stress. Consistent with this, loss of kdm5 results in increased sensitivity to treatment with oxidizers, elevated levels of oxidized proteins, and increased mutation load. KDM5 activates oxidative stress resistance genes by interacting with Foxo to facilitate its recruitment to KDM5-Foxo co-regulated genes. Significantly, this occurs independently of KDM5's well-characterized demethylase activity. Instead, KDM5 interacts with the lysine deacetylase HDAC4 to promote Foxo deacetylation, which affects Foxo DNA binding. PMID:25329053

  14. Quantitative imaging of cellular adhesion by total internal reflection holographic microscopy.

    PubMed

    Ash, William M; Krzewina, Leo; Kim, Myung K

    2009-12-01

    Total internal reflection (TIR) holographic microscopy uses a prism in TIR as a near-field imager to perform quantitative phase microscopy of cell-substrate interfaces. The presence of microscopic organisms, cell-substrate interfaces, adhesions, and tissue structures on the prism's TIR face causes relative index of refraction and frustrated TIR to modulate the object beam's evanescent wave phase front. We present quantitative phase images of test specimens such as Amoeba proteus and cells such as SKOV-3 and 3T3 fibroblasts.

  15. Insights into the cellular response triggered by silver nanoparticles using quantitative proteomics.

    PubMed

    Verano-Braga, Thiago; Miethling-Graff, Rona; Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina; Brewer, Jonathan R; Erdmann, Helmut; Kjeldsen, Frank

    2014-03-25

    The use of nanoparticles in foods, materials, and clinical treatments has increased dramatically in the past decade. Because of the possibility of human exposure to nanoparticles, there is an urgent need to investigate the molecular mechanisms underlying the cellular responses that might be triggered. Such information is necessary to assess potential health risks arising from the use of nanoparticles, and for developing new formulations of next generation nanoparticles for clinical treatments. Using mass spectrometry-based proteomic technologies and complementary techniques (e.g., Western blotting and confocal laser scanning microscopy), we present insights into the silver nanoparticle-protein interaction in the human LoVo cell line. Our data indicate that some unique cellular processes are driven by the size. The 100 nm nanoparticles exerted indirect effects via serine/threonine protein kinase (PAK), mitogen-activated protein kinase (MAPK), and phosphatase 2A pathways, and the 20 nm nanoparticles induced direct effects on cellular stress, including generation of reactive oxygen species and protein carbonylation. In addition, we report that proteins involved in SUMOylation were up-regulated after exposure to 20 nm silver nanoparticles. These results were further substantiated by the observation of silver nanoparticles entering the cells; however, data indicate that this was determined by the size of the nanoparticles, since 20 nm particles entered the cells while 100 nm particles did not.

  16. Quantitative aspects of digital microscopy applied to cellular localization of heparin in smooth muscle cells

    NASA Astrophysics Data System (ADS)

    Johnston, Richard F.; Hanzel, David K.; Stack, Bob; Brandley, Brian; Castellot, John

    1995-04-01

    High Resolution digital acquisition allows a great deal of flexibility in the types of questions that can be directed to microscopic samples. To eliminate subjective bias and provide quantitative results we have approached microscopy with an automated digital format. This mode can return quantitative data at high resolution over large fields. The digital format makes accessible data including [data segmentation]: multispectral colocalization, seeding and connectivity, particle size and shape distribution and population analysis. We have begun a program to investigate this approach using the confocal microscope. Scanning larger fields-of-view at lower spatial resolutions (e.g., low magnification objective) defines large maps that allow alignment of high spatial resolution (diffraction limited) sampling. The [objective] selection of the field-of-view with low spatial resolution reduces the subjective nature of the selection of a 'typical staining pattern'. High resolution digital scanning in three dimensions contribute both to the 'objective' nature of the analysis and allow for quantitation of characteristics not historically available/accessible. The complex carbohydrate heparin is implicated in tumor growth and wound healing by affecting angiogenesis, cell proliferation and motility. The internal localization of heparin within vascular cells appears to be a good predictor of the sensitivity of those cells to the action of heparin. Cells resistant to the antiproliferative action of heparin are able to sequester the heparin in large vacuoles whereas those cells sensitive to the carbohydrate do not exhibit these structures. We have applied our approach to QUANTITATIVE DIGITAL MICROSCOPY to the analysis of intracellular heparin distribution.

  17. Predicting in vivo cardiovascular properties of β-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses

    PubMed Central

    Baker, Jillian G.; Kemp, Philip; March, Julie; Fretwell, Laurice; Hill, Stephen J.; Gardiner, Sheila M.

    2011-01-01

    β-Adrenoceptor antagonists differ in their degree of partial agonism. In vitro assays have provided information on ligand affinity, selectivity, and intrinsic efficacy. However, the extent to which these properties are manifest in vivo is less clear. Conscious freely moving rats, instrumented for measurement of heart rate (β1; HR) and hindquarters vascular conductance (β2; HVC) were used to measure receptor selectivity and ligand efficacy in vivo. CGP 20712A caused a dose-dependent decrease in basal HR (P<0.05, ANOVA) at 5 doses between 6.7 and 670 μg/kg (i.v.) and shifted the dose-response curve for isoprenaline to higher agonist concentrations without altering HVC responses. In contrast, at doses of 67 μg/kg (i.v.) and above, ICI 118551 substantially reduced the HVC response to isoprenaline without affecting HR responses. ZD 7114, xamoterol, and bucindolol significantly increased basal HR (ΔHR: +122±12, +129±11, and +59±11 beats/min, respectively; n=6), whereas other β-blockers caused significant reductions (all at 2 mg/kg i.v.). The agonist effects of xamoterol and ZD 7114 were equivalent to that of the highest dose of isoprenaline. Bucindolol, however, significantly antagonized the response to the highest doses isoprenaline. An excellent correlation was obtained between in vivo and in vitro measures of β1-adrenoceptor efficacy (R2=0.93; P<0.0001).—Baker, J. G., Kemp, P., March, J., Fretwell, L., Hill, S. J., Gardiner, S. M. Predicting in vivo cardiovascular properties of β-blockers from cellular assays: a quantitative comparison of cellular and cardiovascular pharmacological responses. PMID:21865315

  18. Cellular phone-based image acquisition and quantitative ratiometric method for detecting cocaine and benzoylecgonine for biological and forensic applications.

    PubMed

    Cadle, Brian A; Rasmus, Kristin C; Varela, Juan A; Leverich, Leah S; O'Neill, Casey E; Bachtell, Ryan K; Cooper, Donald C

    2010-01-01

    Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new point-of-care diagnostic and forensics tool with health applications. Quantitative ratiometric pixel density analysis (QRPDA) is an automated method requiring end-users to utilize inexpensive (∼ $1 USD/each) immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service. A model is described whereby a central computer server and freely available IMAGEJ image analysis software records and analyzes the incoming image data with time-stamp and geo-tag information and performs the QRPDA using custom JAVA based macros (http://www.neurocloud.org). To demonstrate QRPDA we developed a standardized method using rapid immunotest strips directed against cocaine and its major metabolite, benzoylecgonine. Images from standardized samples were acquired using several devices, including a mobile phone camera, web cam, and scanner. We performed image analysis of three brands of commercially available dye-conjugated anti-cocaine/benzoylecgonine (COC/BE) antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml. This data was then used to create standard curves to allow quantification of COC/BE in biological samples. Across all devices, QRPDA quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography. The limit of detection was determined to be between 0.1 and 5 ng/ml. To simulate conditions in the field, QRPDA was found to be robust under a variety of image acquisition and testing conditions that varied temperature, lighting, resolution, magnification and concentrations of biological fluid in a sample. To

  19. Cellular Phone-Based Image Acquisition and Quantitative Ratiometric Method for Detecting Cocaine and Benzoylecgonine for Biological and Forensic Applications

    PubMed Central

    Cadle, Brian A.; Rasmus, Kristin C.; Varela, Juan A.; Leverich, Leah S.; O’Neill, Casey E.; Bachtell, Ryan K.; Cooper, Donald C.

    2010-01-01

    Here we describe the first report of using low-cost cellular or web-based digital cameras to image and quantify standardized rapid immunoassay strips as a new point-of-care diagnostic and forensics tool with health applications. Quantitative ratiometric pixel density analysis (QRPDA) is an automated method requiring end-users to utilize inexpensive (∼ $1 USD/each) immunotest strips, a commonly available web or mobile phone camera or scanner, and internet or cellular service. A model is described whereby a central computer server and freely available IMAGEJ image analysis software records and analyzes the incoming image data with time-stamp and geo-tag information and performs the QRPDA using custom JAVA based macros (http://www.neurocloud.org). To demonstrate QRPDA we developed a standardized method using rapid immunotest strips directed against cocaine and its major metabolite, benzoylecgonine. Images from standardized samples were acquired using several devices, including a mobile phone camera, web cam, and scanner. We performed image analysis of three brands of commercially available dye-conjugated anti-cocaine/benzoylecgonine (COC/BE) antibody test strips in response to three different series of cocaine concentrations ranging from 0.1 to 300 ng/ml and BE concentrations ranging from 0.003 to 0.1 ng/ml. This data was then used to create standard curves to allow quantification of COC/BE in biological samples. Across all devices, QRPDA quantification of COC and BE proved to be a sensitive, economical, and faster alternative to more costly methods, such as gas chromatography-mass spectrometry, tandem mass spectrometry, or high pressure liquid chromatography. The limit of detection was determined to be between 0.1 and 5 ng/ml. To simulate conditions in the field, QRPDA was found to be robust under a variety of image acquisition and testing conditions that varied temperature, lighting, resolution, magnification and concentrations of biological fluid in a sample. To

  20. Contaminant effects on cellular metabolic differential pressure curve: a quantitative analysis

    NASA Astrophysics Data System (ADS)

    Milani, Marziale; Ballerini, Monica; Ferraro, Lorenzo; Zabeo, Matteo; Barberis, Massimo; Cannone, Maria; Faraone, Venera

    2002-06-01

    The possibility of using a pressure monitoring system based on differential pressure sensors to detect contaminant effects on cellular cultures metabolic activity is discussed using Saccharomyces cerevisiae cell cultures: differential pressure curves' shape, starting slope and maximum are affected both by physical and chemical contamination. Aim of the present study is the investigation of the effects generated by a 72h exposition of Saccharomyces cerevisiae, human lymphocytes and AHH1 cellular line cultures to 50Hz, 60(mu) T electromagnetic field. No significant differences have been recorded between irradiated and control yeast samples. On other hand irradiated lymphocytes samples, cultures in a PHA medium, grow less than control ones, but exhibit a greater metabolic activity: changes in the exposure system configuration influence neither sample growth differences nor metabolic response variations between control and irradiated samples. Control and irradiated lymphocyte samples, without PHA in culture medium, show the same behavior both during irradiation and metabolic test. AHH1 control and irradiated samples show no difference both in growth percentage during irradiation and in metabolic test. Different cell cultures respond to the same stimulus in different manners.

  1. A SIMPLE CELLULAR AUTOMATON MODEL FOR HIGH-LEVEL VEGETATION DYNAMICS

    EPA Science Inventory

    We have produced a simple two-dimensional (ground-plan) cellular automata model of vegetation dynamics specifically to investigate high-level community processes. The model is probabilistic, with individual plant behavior determined by physiologically-based rules derived from a w...

  2. A SIMPLE CELLULAR AUTOMATON MODEL FOR HIGH-LEVEL VEGETATION DYNAMICS

    EPA Science Inventory

    We have produced a simple two-dimensional (ground-plan) cellular automata model of vegetation dynamics specifically to investigate high-level community processes. The model is probabilistic, with individual plant behavior determined by physiologically-based rules derived from a w...

  3. A `Clicked' Tetrameric Hydroxamic Acid Glycopeptidomimetic Antagonizes Sugar-Lectin Interactions On The Cellular Level

    NASA Astrophysics Data System (ADS)

    Zhang, Hai-Lin; Zang, Yi; Xie, Juan; Li, Jia; Chen, Guo-Rong; He, Xiao-Peng; Tian, He

    2014-07-01

    A tetrameric N-acetyl galactosaminyl (GalNAc) peptidomimetic was constructed by N-acetylation of repeating proline-based hydroxamic acid units, followed by a convergent `click chemistry' coupling. This novel glycopeptidomimetic was determined to effectively antagonize the interaction between a transmembrane hepatic lectin and GalNAc on the cellular level.

  4. Quantitative assessment of barriers to the clinical development and adoption of cellular therapies: A pilot study

    PubMed Central

    Rikabi, Sarah; French, Anna; Pinedo-Villanueva, Rafael; Morrey, Mark E; Wartolowska, Karolina; Judge, Andrew; MacLaren, Robert E; Mathur, Anthony; Williams, David J; Wall, Ivan; Birchall, Martin; Reeve, Brock; Atala, Anthony; Barker, Richard W; Cui, Zhanfeng; Furniss, Dominic; Bure, Kim; Snyder, Evan Y; Karp, Jeffrey M; Price, Andrew; Carr, Andrew; Brindley, David A

    2014-01-01

    There has been a large increase in basic science activity in cell therapy and a growing portfolio of cell therapy trials. However, the number of industry products available for widespread clinical use does not match this magnitude of activity. We hypothesize that the paucity of engagement with the clinical community is a key contributor to the lack of commercially successful cell therapy products. To investigate this, we launched a pilot study to survey clinicians from five specialities and to determine what they believe to be the most significant barriers to cellular therapy clinical development and adoption. Our study shows that the main concerns among this group are cost-effectiveness, efficacy, reimbursement, and regulation. Addressing these concerns can best be achieved by ensuring that future clinical trials are conducted to adequately answer the questions of both regulators and the broader clinical community. PMID:25383173

  5. Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity

    PubMed Central

    Mortimer, Nathan T.; Goecks, Jeremy; Kacsoh, Balint Z.; Mobley, James A.; Bowersock, Gregory J.; Taylor, James; Schlenke, Todd A.

    2013-01-01

    Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity. PMID:23690612

  6. Parasitoid wasp venom SERCA regulates Drosophila calcium levels and inhibits cellular immunity.

    PubMed

    Mortimer, Nathan T; Goecks, Jeremy; Kacsoh, Balint Z; Mobley, James A; Bowersock, Gregory J; Taylor, James; Schlenke, Todd A

    2013-06-04

    Because parasite virulence factors target host immune responses, identification and functional characterization of these factors can provide insight into poorly understood host immune mechanisms. The fruit fly Drosophila melanogaster is a model system for understanding humoral innate immunity, but Drosophila cellular innate immune responses remain incompletely characterized. Fruit flies are regularly infected by parasitoid wasps in nature and, following infection, flies mount a cellular immune response culminating in the cellular encapsulation of the wasp egg. The mechanistic basis of this response is largely unknown, but wasps use a mixture of virulence proteins derived from the venom gland to suppress cellular encapsulation. To gain insight into the mechanisms underlying wasp virulence and fly cellular immunity, we used a joint transcriptomic/proteomic approach to identify venom genes from Ganaspis sp.1 (G1), a previously uncharacterized Drosophila parasitoid species, and found that G1 venom contains a highly abundant sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. Accordingly, we found that fly immune cells termed plasmatocytes normally undergo a cytoplasmic calcium burst following infection, and that this calcium burst is required for activation of the cellular immune response. We further found that the plasmatocyte calcium burst is suppressed by G1 venom in a SERCA-dependent manner, leading to the failure of plasmatocytes to become activated and migrate toward G1 eggs. Finally, by genetically manipulating plasmatocyte calcium levels, we were able to alter fly immune success against G1 and other parasitoid species. Our characterization of parasitoid wasp venom proteins led us to identify plasmatocyte cytoplasmic calcium bursts as an important aspect of fly cellular immunity.

  7. ELF (extremely-low-frequency) field interactions at the animal, tissue and cellular levels

    SciTech Connect

    Tenforde, T.S.

    1990-10-01

    A description is given of the fundamental physical properties of extremely-low-frequency (ELF) electromagnetic fields, and the mechanisms through which these fields interact with the human body at a macroscopic level. Biological responses to ELF fields at the tissue, cellular and molecular levels are summarized, including new evidence that ELF field exposure produces alterations in gene expression and the cytoplasmic concentrations of specific proteins.

  8. Physiological enzymology: The next frontier in understanding protein structure and function at the cellular level.

    PubMed

    Lee, Irene; Berdis, Anthony J

    2016-01-01

    Historically, the study of proteins has relied heavily on characterizing the activity of a single purified protein isolated from other cellular components. This classic approach allowed scientists to unambiguously define the intrinsic kinetic and chemical properties of that protein. The ultimate hope was to extrapolate this information toward understanding how the enzyme or receptor behaves within its native cellular context. These types of detailed in vitro analyses were necessary to reduce the innate complexities of measuring the singular activity and biochemical properties of a specific enzyme without interference from other enzymes and potential competing substrates. However, recent developments in fields encompassing cell biology, molecular imaging, and chemical biology now provide the unique chemical tools and instrumentation to study protein structure, function, and regulation in their native cellular environment. These advancements provide the foundation for a new field, coined physiological enzymology, which quantifies the function and regulation of enzymes and proteins at the cellular level. In this Special Edition, we explore the area of Physiological Enzymology and Protein Function through a series of review articles that focus on the tools and techniques used to measure the cellular activity of proteins inside living cells. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Upregulation of cellular glutathione levels in human ABCB5- and murine Abcb5-transfected cells.

    PubMed

    Kondo, Shingo; Hongama, Keita; Hanaya, Kengo; Yoshida, Ryota; Kawanobe, Takaaki; Katayama, Kazuhiro; Noguchi, Kohji; Sugimoto, Yoshikazu

    2015-12-15

    Previously, we have demonstrated that human ABCB5 is a full-sized ATP-binding cassette transporter that shares strong homology with ABCB1/P-glycoprotein. ABCB5-transfected cells showed resistance to taxanes and anthracyclines. Herein, we further screened ABCB5 substrates, and explored the mechanism of resistance. Sensitivity of the cells to test compounds was evaluated using cell growth inhibition assay. Cellular levels of buthionine sulfoximine (BSO), glutathione and amino acids were measured using HPLC and an enzyme-based assay. Cellular and vesicular transport of glutathione was evaluated by a radiolabeled substrate. Expression levels of glutathione-metabolizing enzymes were assessed by RT-PCR. Human ABCB5-transfected 293/B5-11 cells and murine Abcb5-transfected 293/mb5-8 cells showed 6.5- and 14-fold higher resistance to BSO than the mock-transfected 293/mock cells, respectively. BSO is an inhibitor of gamma-glutamylcysteine ligase (GCL), which is a key enzyme of glutathione synthesis. 293/B5-11 and 293/mb5-8 cells also showed resistance to methionine sulfoximine, another GCL inhibitor. A cellular uptake experiment revealed that BSO accumulation in 293/B5-11 and 293/mb5-8 cells was similar to that in 293/mock cells, suggesting that BSO is not an ABCB5 substrate. The cellular glutathione content in 293/B5-11 and 293/mb5-8 cells was significantly higher than that in 293/mock cells. Evaluation of the BSO effect on the cellular glutathione content showed that compared with 293/mock cells the BSO concentration required for a 50 % reduction in glutathione content in 293/B5-11 and 293/mb5-8 cells was approximately 2- to 3-fold higher. This result suggests that the BSO resistance of the ABCB5- and Abcb5-transfected cells can be attributed to the reduced effect of BSO on the transfectants. Cellular and vesicular transport assays showed that the transport of radiolabeled glutathione in 293/B5-11 cells was similar to that in 293/mock cells. The mRNA expression of genes

  10. An Image Analysis Method for the Precise Selection and Quantitation of Fluorescently Labeled Cellular Constituents

    PubMed Central

    Agley, Chibeza C.; Velloso, Cristiana P.; Lazarus, Norman R.

    2012-01-01

    The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored. PMID:22511600

  11. Quantitative analysis of the cellular inflammatory response against biofilm bacteria in chronic wounds.

    PubMed

    Fazli, Mustafa; Bjarnsholt, Thomas; Kirketerp-Møller, Klaus; Jørgensen, Anne; Andersen, Claus Bøgelund; Givskov, Michael; Tolker-Nielsen, Tim

    2011-01-01

    Chronic wounds are an important problem worldwide. These wounds are characterized by a persistent inflammatory stage associated with excessive accumulation and elevated cell activity of neutrophils, suggesting that there must be a persistent stimulus that attracts and recruits neutrophils to the wound. One such stimulus might be the presence of bacterial biofilms in chronic wounds. In the present study, biopsy specimens from chronic venous leg ulcers were investigated for the detection of bacteria using peptide nucleic acid-based fluorescence in situ hybridization (PNA-FISH) and confocal laser scanning microscopy. The bacteria in the wounds were often situated in large aggregates. To obtain a measure of the cellular inflammatory response against the bacteria in the chronic wounds, the amount of neutrophils accumulated at the site of infection was evaluated through differential neutrophil counting on the tissue sections from wounds containing either Pseudomonas aeruginosa or Staphylococcus aureus. The P. aeruginosa-containing wounds had significantly higher numbers of neutrophils accumulated compared with the S. aureus-containing wounds. These results are discussed in relation to the hypothesis that the presence of P. aeruginosa biofilms in chronic wounds may be one of the main factors leading to a persistent inflammatory response and impaired wound healing.

  12. Raf/MEK/ERK can regulate cellular levels of LC3B and SQSTM1/p62 at expression levels

    PubMed Central

    Kim, Jin-Hwan; Hong, Seung-Keun; Wu, Pui-Kei; Richards, Alexsia L; Jackson, William T; Park, Jong-In

    2014-01-01

    While cellular LC3B and SQSTM1 levels serve as key autophagy markers, their regulation by different signaling pathways requires better understanding. Here, we report the mechanisms by which the Raf/MEK/ERK pathway regulates cellular LC3B and SQSTM1 levels. In different cell types, δRaf-1:ER- or B-RafV600E-mediated MEK/ERK activation increased LC3B-I, LC3B-II, and SQSTM1/p62 levels, which was accompanied by increased BiP/GRP78 expression. Use of the autophagy inhibitors chloroquine and bafilomycin A1, or RNA interference of ATG7, suggested that these increases in LC3B and SQSTM1 levels were in part attributed to altered autophagic flux. However, intriguingly, these increases were also attributed to their increased expression. Upon Raf/MEK/ERK activation, mRNA levels of LC3B and SQSTM1 were also increased, and subsequent luciferase reporter analyses suggested that SQSTM1 upregulation was mediated at transcription level. Under this condition, transcription of BiP/GRP78 was also increased, which was necessary for Raf/MEK/ERK to regulate LC3B at the protein, but not mRNA, level. This suggests that BiP has a role in regulating autophagy machinery when Raf/MEK/ERK is activated. In conclusion, these results suggest that, under a Raf/MEK/ERK-activated condition, the steady-state cellular levels of LC3B and SQSTM1 can also be determined by their altered expression wherein BiP is utilized as an effector of the signaling. PMID:25128814

  13. Near-infrared quantitative phase imaging of cellular manipulation under different physio-chemical environments

    NASA Astrophysics Data System (ADS)

    Joshi, Bipin

    Quantitative phase imaging using Digital Holographic Microscopy (DHM) is emerging as a label-free and wide-field method of characterizing cells with high spatio-temporal resolution. In parallel, silicon based micromechanical and electronic devices are allowing both manipulation (e.g. electrical stimulation, mechanical actuation) as well as characterization (electrical and mechanical) of micro and nano-scopic samples. This has revolutionized development of lab-on-a-chip devices for high throughput analysis of cells and molecules for diagnosis of disease and screening of drug-effects. However, very little progress has been made in optical (e.g. fluorescence, Raman etc) characterization of samples on these silicon-based devices. Especially, wide-field high-resolution optical imaging and characterization of samples under silicon environment has not been possible owing to the opacity of silicon to visible light. This thesis reports high resolution near-infrared quantitative phase imaging of cells through silicon, in isotonic as well as hypotonic environment using DHM. Further, several microscopic (AFM, laser manipulation) methods are being developed for characterization of mechanical properties (e.g. elasticity) of cells so as to determine changes during physiological stress. In particular, optical tweezers are used for transverse-stretching cells by actuating anchored-beads as handles and imaging using phase-contrast microscopy. While this method is constantly gaining more attention due to non-contact nature of actuation, it is very time consuming and has low working distance. The thesis describes development of a weakly-focused laser beam for axial-stretching of cell by scattering force, which can be easily extended for wide-area stretching. Application of DHM allowed cell imaging with nm-resolution when stretched axially. Development of an empirical formula for force exerted by defocused light beam on cell surface led to measurement of elastic property of cell. In

  14. Research of epidermal cellular vegetal cycle of intravascular low level laser irradiation in treatment of psoriasis

    NASA Astrophysics Data System (ADS)

    Zhu, Jing; Bao, Xiaoqing; Zhang, Mei-Jue

    2005-07-01

    Objective: To research epidermal cellular vegetal cycle and the difference of DNA content between pre and post Intravascular Low Level Laser Irradiation treatment of psoriasis. Method: 15 patients suffered from psoriasis were treated by intravascular low level laser irradiation (output power: 4-5mw, 1 hour per day, a course of treatment is 10 days). We checked the different DNA content of epidermal cell between pre and post treatment of psoriasis and 8 natural human. Then the percentage of each phase among the whole cellular cycle was calculated and the statistical analysis was made. Results: The mean value of G1/S phase is obviously down while G2+M phase increased obviously. T test P<0.05.The related statistical analysis showed significant difference between pre and post treatments. Conclusions: The Intravascular Low Level Laser Irradiation (ILLLI) in treatment of psoriasis is effective according to the research of epidermal cellular vegetal cycle and the difference DNA content of Intravascular Low Level Laser Irradiation between pre and post treatment of psoriasis

  15. Quantitative microspectroscopic imaging reveals viral and cellular RNA helicase interactions in live cells.

    PubMed

    Corby, M J; Stoneman, Michael R; Biener, Gabriel; Paprocki, Joel D; Kolli, Rajesh; Raicu, Valerica; Frick, David N

    2017-07-07

    Human cells detect RNA viruses through a set of helicases called RIG-I-like receptors (RLRs) that initiate the interferon response via a mitochondrial signaling complex. Many RNA viruses also encode helicases, which are sometimes covalently linked to proteases that cleave signaling proteins. One unresolved question is how RLRs interact with each other and with viral proteins in cells. This study examined the interactions among the hepatitis C virus (HCV) helicase and RLR helicases in live cells with quantitative microspectroscopic imaging (Q-MSI), a technique that determines FRET efficiency and subcellular donor and acceptor concentrations. HEK293T cells were transfected with various vector combinations to express cyan fluorescent protein (CFP) or YFP fused to either biologically active HCV helicase or one RLR (i.e. RIG-I, MDA5, or LGP2), expressed in the presence or absence of polyinosinic-polycytidylic acid (poly(I:C)), which elicits RLR accumulation at mitochondria. Q-MSI confirmed previously reported RLR interactions and revealed an interaction between HCV helicase and LGP2. Mitochondria in CFP-RIG-I:YFP-RIG-I cells, CFP-MDA5:YFP-MDA5 cells, and CFP-MDA5:YFP-LGP2 cells had higher FRET efficiencies in the presence of poly(I:C), indicating that RNA causes these proteins to accumulate at mitochondria in higher-order complexes than those formed in the absence of poly(I:C). However, mitochondria in CFP-LGP2:YFP-LGP2 cells had lower FRET signal in the presence of poly(I:C), suggesting that LGP2 oligomers disperse so that LGP2 can bind MDA5. Data support a new model where an LGP2-MDA5 oligomer shuttles NS3 to the mitochondria to block antiviral signaling. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Quantitative proteomics and transcriptomics of anaerobic and aerobic yeast cultures reveals post-transcriptional regulation of key cellular processes.

    PubMed

    de Groot, Marco J L; Daran-Lapujade, Pascale; van Breukelen, Bas; Knijnenburg, Theo A; de Hulster, Erik A F; Reinders, Marcel J T; Pronk, Jack T; Heck, Albert J R; Slijper, Monique

    2007-11-01

    Saccharomyces cerevisiae is unique among yeasts in its ability to grow rapidly in the complete absence of oxygen. S. cerevisiae is therefore an ideal eukaryotic model to study physiological adaptation to anaerobiosis. Recent transcriptome analyses have identified hundreds of genes that are transcriptionally regulated by oxygen availability but the relevance of this cellular response has not been systematically investigated at the key control level of the proteome. Therefore, the proteomic response of S. cerevisiae to anaerobiosis was investigated using metabolic stable-isotope labelling in aerobic and anaerobic glucose-limited chemostat cultures, followed by relative quantification of protein expression. Using independent replicate cultures and stringent statistical filtering, a robust dataset of 474 quantified proteins was generated, of which 249 showed differential expression levels. While some of these changes were consistent with previous transcriptome studies, many of the responses of S. cerevisiae to oxygen availability were, to our knowledge, previously unreported. Comparison of transcriptomes and proteomes from identical cultivations yielded strong evidence for post-transcriptional regulation of key cellular processes, including glycolysis, amino-acyl-tRNA synthesis, purine nucleotide synthesis and amino acid biosynthesis. The use of chemostat cultures provided well-controlled and reproducible culture conditions, which are essential for generating robust datasets at different cellular information levels. Integration of transcriptome and proteome data led to new insights into the physiology of anaerobically growing yeast that would not have been apparent from differential analyses at either the mRNA or protein level alone, thus illustrating the power of multi-level studies in yeast systems biology.

  17. Possible cellular regulation schemes of isoprene synthesis and emission under different ambient carbon dioxide levels. (Invited)

    NASA Astrophysics Data System (ADS)

    Noe, S. M.; Schnitzler, J.; Arneth, A.; Monson, R. K.; Niinemets, U.

    2010-12-01

    Research on the effects of higher atmospheric carbon dioxide (CO2) levels on isoprene synthesis and emission leaded to several newly proposed regulation schemes. They can be classified as substrate level control on one side and as energetic cofactor control of the plastidic 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway on the other one. Viewed on a whole cell scale, the precursors of isoprene, such as dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP), can be found in several cellular compartments such as chloroplasts, cytosol and mitochondria. Furthermore, necessary entry points into the isoprene synthesis pathway like phosphoenolpyruvate (PEP) and pyruvate are provided by two processes, photosynthesis and glycolysis, which are as well located in different cellular compartments. These findings imply, that the effect of modulating the isoprene emission under high levels of atmospheric CO2 have to take transport over membranes, possible concurrent pathways, i.e. Shikimi acid pathway or anaplerotic metabolism reactions and the availability of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) on a cellular scale into account. In this modeling study we applied box models that include several facets of the proposed regulation and transport schemes. The models have been set up such that at least two cellular compartments, chloroplast and cytosol are taken into account. The boxes itself represent metabolites and several possible regulation schemes have been realized by the formulation of rate equations between those metabolite pools. As many intermediates are not readily available as measured values, the models aim to build a set of tools to simulate possible regulatory schemes and provide parameter estimations for key processes. Inverse modeling techniques allow to assess certain parameter ranges within the proposed regulation schemes by fitting the models to data on isoprene emission and photosynthesis under

  18. Regulation of SIRT2-dependent α-tubulin deacetylation by cellular NAD levels.

    PubMed

    Skoge, Renate Hvidsten; Dölle, Christian; Ziegler, Mathias

    2014-11-01

    Acetylation of α-tubulin on lysine 40 is one of the major posttranslational modifications of microtubules. The acetylation reaction is catalyzed by alpha-tubulin N-acetyltransferase and the modification can be reversed by either the NAD-independent class II histone deacetylase HDAC6 or the NAD-dependent deacetylase SIRT2. In this study, we assessed to what extent cellular NAD levels are involved in the regulation of the α-tubulin acetylation state. Cells were subjected to different treatments known to influence cellular NAD content. In response to NAD depletion caused by inhibition of NAD synthesis from nicotinamide, α-tubulin was hyperacetylated. Under these conditions, the normal tubulin acetylation state could be restored by providing the cells with alternative NAD precursors. Likewise, decreasing the rate of endogenous NAD consumption using an inhibitor of poly-ADP-ribosylation also stabilized the acetylation of α-tubulin. Conversely, the level of acetylated α-tubulin decreased when NAD synthesis was enhanced by overexpression of an NAD biosynthetic enzyme. Combined, these results show that the tubulin acetylation status is reciprocally regulated by cellular NAD levels. Furthermore, we provide evidence confirming that the NAD-dependent regulation of tubulin acetylation is mediated by SIRT2.

  19. Monitoring Cellular Phosphorylation Signaling Pathways into Chromatin and Down to the Gene Level*

    PubMed Central

    Han, Yumiao; Yuan, Zuo-Fei; Molden, Rosalynn C.; Garcia, Benjamin A.

    2016-01-01

    Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood. Here, we describe the optimization and comparison of methods based on thiophosphorylation affinity enrichment, which can be utilized to monitor phosphorylation signaling into chromatin by isolation of phosphoprotein containing nucleosomes, a method we term phosphorylation-specific chromatin affinity purification (PS-ChAP). We utilized this PS-ChAP1 approach in combination with quantitative proteomics to identify changes in the phosphorylation status of chromatin-bound proteins on nucleosomes following perturbation of transcriptional processes. We also demonstrate that this method can be employed to map phosphoprotein signaling into chromatin containing nucleosomes through identifying the genes those phosphorylated proteins are found on via thiophosphate PS-ChAP-qPCR. Thus, our results showed that PS-ChAP offers a new strategy for studying cellular signaling and chromatin biology, allowing us to directly and comprehensively investigate phosphorylation signaling into chromatin to investigate if these pathways are involved in altering gene expression. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set

  20. A detailed view on sulphur metabolism at the cellular and whole-plant level illustrates challenges in metabolite flux analyses.

    PubMed

    Rennenberg, Heinz; Herschbach, Cornelia

    2014-11-01

    Understanding the dynamics of physiological process in the systems biology era requires approaches at the genome, transcriptome, proteome, and metabolome levels. In this context, metabolite flux experiments have been used in mapping metabolite pathways and analysing metabolic control. In the present review, sulphur metabolism was taken to illustrate current challenges of metabolic flux analyses. At the cellular level, restrictions in metabolite flux analyses originate from incomplete knowledge of the compartmentation network of metabolic pathways. Transport of metabolites through membranes is usually not considered in flux experiments but may be involved in controlling the whole pathway. Hence, steady-state and snapshot readings need to be expanded to time-course studies in combination with compartment-specific metabolite analyses. Because of species-specific differences, differences between tissues, and stress-related responses, the quantitative significance of different sulphur sinks has to be elucidated; this requires the development of methods for whole-sulphur metabolome approaches. Different cell types can contribute to metabolite fluxes to different extents at the tissue and organ level. Cell type-specific analyses are needed to characterize these contributions. Based on such approaches, metabolite flux analyses can be expanded to the whole-plant level by considering long-distance transport and, thus, the interaction of roots and the shoot in metabolite fluxes. However, whole-plant studies need detailed empirical and mathematical modelling that have to be validated by experimental analyses.

  1. One-micron resolution optical coherence tomography (OCT) in vivo for cellular level imaging

    NASA Astrophysics Data System (ADS)

    Cui, Dongyao; Liu, Xinyu; Zhang, Jing; Yu, Xiaojun; Sun, Ding; Luo, Yuemei; Gu, Jun; Shum, Ping; Liu, Linbo

    2015-03-01

    We developed a spectral domain OCT system combining two NIR, CW light sources of different spectral range. Its resolving power is validated by visualizing the cellular structures of zebra fish larvae in vivo. An NIR extended illumination from 755-1100 nm is achieved. The axial resolution is 1.27 μm in air, corresponding to 0.93μm in tissue (n=1.36), which is the highest axial resolution using NIR, CW laser sources up to date to the best of our knowledge. In vivo imaging is conducted to demonstrate the resolving power of proposed one-micron resolution OCT system. The top and bottom surfaces of individual disk-like red blood cell is reliably visualized, as well as flat, spindle shaped endothelial cells lining along the luminal surface of the blood vessel wall. This study provides a viable solution for cellular and subcellular level OCT imaging system which is also very competitive in cost.

  2. Quantitative phase imaging of cellular and subcellular structures for non-invasive screening diagnostics of socially significant diseases

    NASA Astrophysics Data System (ADS)

    Vasilenko, Irina; Metelin, Vladislav; Nasyrov, Marat; Belyakov, Vladimir; Kuznetsov, Alexander; Sukhenko, Evgeniy

    2015-03-01

    The objective of the present study is to increase the quality of the early diagnosis using cytological differential-diagnostic criteria for reactive changes in the nuclear structures of the immunocompetent cells. The morphofunctional status of living cells were estimated in the real time using new technologic platform of the hardware-software complex for phase cell imaging. The level of functional activity for lymphocyte subpopulations was determined on the base of modification of nuclear structures and decreasing of nuclear phase thickness. The dynamics of nuclear parameters was used as the quantitative measuring for cell activating level and increasing of proliferative potential.

  3. Within-Host Spatiotemporal Dynamics of Plant Virus Infection at the Cellular Level

    PubMed Central

    Lafforgue, Guillaume; Elena, Santiago F.

    2014-01-01

    A multicellular organism is not a monolayer of cells in a flask; it is a complex, spatially structured environment, offering both challenges and opportunities for viruses to thrive. Whereas virus infection dynamics at the host and within-cell levels have been documented, the intermediate between-cell level remains poorly understood. Here, we used flow cytometry to measure the infection status of thousands of individual cells in virus-infected plants. This approach allowed us to determine accurately the number of cells infected by two virus variants in the same host, over space and time as the virus colonizes the host. We found a low overall frequency of cellular infection (<0.3), and few cells were coinfected by both virus variants (<0.1). We then estimated the cellular contagion rate (R), the number of secondary infections per infected cell per day. R ranged from 2.43 to values not significantly different from zero, and generally decreased over time. Estimates of the cellular multiplicity of infection (MOI), the number of virions infecting a cell, were low (<1.5). Variance of virus-genotype frequencies increased strongly from leaf to cell levels, in agreement with a low MOI. Finally, there were leaf-dependent differences in the ease with which a leaf could be colonized, and the number of virions effectively colonizing a leaf. The modeling of infection patterns suggests that the aggregation of virus-infected cells plays a key role in limiting spread; matching the observation that cell-to-cell movement of plant viruses can result in patches of infection. Our results show that virus expansion at the between-cell level is restricted, probably due to the host environment and virus infection itself. PMID:24586207

  4. Within-host spatiotemporal dynamics of plant virus infection at the cellular level.

    PubMed

    Tromas, Nicolas; Zwart, Mark P; Lafforgue, Guillaume; Elena, Santiago F

    2014-02-01

    A multicellular organism is not a monolayer of cells in a flask; it is a complex, spatially structured environment, offering both challenges and opportunities for viruses to thrive. Whereas virus infection dynamics at the host and within-cell levels have been documented, the intermediate between-cell level remains poorly understood. Here, we used flow cytometry to measure the infection status of thousands of individual cells in virus-infected plants. This approach allowed us to determine accurately the number of cells infected by two virus variants in the same host, over space and time as the virus colonizes the host. We found a low overall frequency of cellular infection (<0.3), and few cells were coinfected by both virus variants (<0.1). We then estimated the cellular contagion rate (R), the number of secondary infections per infected cell per day. R ranged from 2.43 to values not significantly different from zero, and generally decreased over time. Estimates of the cellular multiplicity of infection (MOI), the number of virions infecting a cell, were low (<1.5). Variance of virus-genotype frequencies increased strongly from leaf to cell levels, in agreement with a low MOI. Finally, there were leaf-dependent differences in the ease with which a leaf could be colonized, and the number of virions effectively colonizing a leaf. The modeling of infection patterns suggests that the aggregation of virus-infected cells plays a key role in limiting spread; matching the observation that cell-to-cell movement of plant viruses can result in patches of infection. Our results show that virus expansion at the between-cell level is restricted, probably due to the host environment and virus infection itself.

  5. High content analysis at single cell level identifies different cellular responses dependent on nanomaterial concentrations.

    PubMed

    Manshian, Bella B; Munck, Sebastian; Agostinis, Patrizia; Himmelreich, Uwe; Soenen, Stefaan J

    2015-09-08

    A mechanistic understanding of nanomaterial (NM) interaction with biological environments is pivotal for the safe transition from basic science to applied nanomedicine. NM exposure results in varying levels of internalized NM in different neighboring cells, due to variances in cell size, cell cycle phase and NM agglomeration. Using high-content analysis, we investigated the cytotoxic effects of fluorescent quantum dots on cultured cells, where all effects were correlated with the concentration of NMs at the single cell level. Upon binning the single cell data into different categories related to NM concentration, this study demonstrates, for the first time, that quantum dots activate both cytoprotective and cytotoxic mechanisms, resulting in a zero net result on the overall cell population, yet with significant effects in cells with higher cellular NM levels. Our results suggest that future NM cytotoxicity studies should correlate NM toxicity with cellular NM numbers on the single cell level, as conflicting mechanisms in particular cell subpopulations are commonly overlooked using classical toxicological methods.

  6. Comparison of cellular responses induced by low level light in different cell types

    NASA Astrophysics Data System (ADS)

    Huang, Ying-Ying; Chen, Aaron C.-H.; Sharma, Sulbha K.; Wu, Qiuhe; Hamblin, Michael R.

    2010-02-01

    Discoveries are rapidly being made in multiple laboratories that shed "light" on the fundamental molecular and cellular mechanisms underlying the use of low level light therapy (LLLT) in vitro, in animal models and in clinical practice. Increases in cellular levels of respiration, in cytochrome c oxidase activity, in ATP levels and in cyclic AMP have been found. Increased expression of reactive oxygen species and release of nitric oxide have also been shown. In order for these molecular changes to have a major effect on cell behavior, it is likely that various transcription factors will be activated, possibly via different signal transduction pathways. In this report we compare and contrast the effects of LLLT in vitro on murine embryonic fibroblasts, primary cortical neurons, cardiomyocytes and bone-marrow derived dendritic cells. We also examined two human cell lines, HeLa cancer cells and HaCaT keratinocytes. The effects of 810-nm near-infra-red light delivered at low and high fluences were addressed. Reactive oxygen species generation, transcription factor activation and ATP increases are reported. The data has led to the hypothesis that cells with a high level of mitochondrial activity (mitochondrial membrane potential) have a higher response to light than cells with low mitochondrial activity.

  7. Ubiquitin Ligase Substrate Identification through Quantitative Proteomics at Both the Protein and Peptide Levels

    PubMed Central

    Lee, Kimberly A.; Hammerle, Lisa P.; Andrews, Paul S.; Stokes, Matthew P.; Mustelin, Tomas; Silva, Jeffrey C.; Black, Roy A.; Doedens, John R.

    2011-01-01

    Protein ubiquitination is a key regulatory process essential to life at a cellular level; significant efforts have been made to identify ubiquitinated proteins through proteomics studies, but the level of success has not reached that of heavily studied post-translational modifications, such as phosphorylation. HRD1, an E3 ubiquitin ligase, has been implicated in rheumatoid arthritis, but no disease-relevant substrates have been identified. To identify these substrates, we have taken both peptide and protein level approaches to enrich for ubiquitinated proteins in the presence and absence of HRD1. At the protein level, a two-step strategy was taken using cells expressing His6-tagged ubiquitin, enriching proteins first based on their ubiquitination and second based on the His tag with protein identification by LC-MS/MS. Application of this method resulted in identification and quantification of more than 400 ubiquitinated proteins, a fraction of which were found to be sensitive to HRD1 and were therefore deemed candidate substrates. In a second approach, ubiquitinated peptides were enriched after tryptic digestion by peptide immunoprecipitation using an antibody specific for the diglycine-labeled internal lysine residue indicative of protein ubiquitination, with peptides and ubiquitination sites identified by LC-MS/MS. Peptide immunoprecipitation resulted in identification of over 1800 ubiquitinated peptides on over 900 proteins in each study, with several proteins emerging as sensitive to HRD1 levels. Notably, significant overlap exists between the HRD1 substrates identified by the protein-based and the peptide-based strategies, with clear cross-validation apparent both qualitatively and quantitatively, demonstrating the effectiveness of both strategies and furthering our understanding of HRD1 biology. PMID:21987572

  8. An overview of the regulation of bone remodelling at the cellular level.

    PubMed

    Kular, Jasreen; Tickner, Jennifer; Chim, Shek Man; Xu, Jiake

    2012-08-01

    To review the current literature on the regulation of bone remodelling at the cellular level. The cellular activities of the cells in the basic multicellular unit (BMU) were evaluated. Bone remodelling requires an intimate cross-talk between osteoclasts and osteoblasts and is tightly coordinated by regulatory proteins that interact through complex autocrine/paracrine mechanisms. Osteocytes, bone lining cells, osteomacs, and vascular endothelial cells also regulate bone remodelling in the BMU via cell signalling networks of ligand-receptor complexes. In addition, through secreted and membrane-bound factors in the bone microenvironment, T and B lymphocytes mediate bone homeostasis in osteoimmunology. Osteoporosis and other bone diseases occur because multicellular communication within the BMU is disrupted. Understanding the cellular and molecular basis of bone remodelling and the discovery of novel paracrine or coupling factors, such as RANKL, sclerostin, EGFL6 and semaphorin 4D, will lay the foundation for drug development against bone diseases. Copyright © 2012 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  9. Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

    PubMed Central

    Shi, Bin; Keough, Ed; Matter, Andrea; Leander, Karen; Young, Stephanie; Carlini, Ed; Sachs, Alan B.; Tao, Weikang; Abrams, Marc; Howell, Bonnie; Sepp-Lorenzino, Laura

    2011-01-01

    Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles. PMID:21804077

  10. X-ray micro-tomography for investigations of brain tissues on cellular level

    NASA Astrophysics Data System (ADS)

    Khimchenko, Anna; Schulz, Georg; Deyhle, Hans; Thalmann, Peter; Zanette, Irene; Zdora, Marie-Christine; Bikis, Christos; Hipp, Alexander; Hieber, Simone E.; Schweighauser, Gabriel; Hench, Jürgen; Müller, Bert

    2016-10-01

    X-ray imaging in absorption contrast mode is well established for hard tissue visualization. However, performance for lower density materials is limited due to a reduced contrast. Our aim is three-dimensional (3D) characterization of micro-morphology of human brain tissues down to (sub-)cellular resolution within a laboratory environment. Using the laboratory-based microtomography (μCT) system nanotom m (GE Sensing and Inspection Technologies GmbH, Wunstorf, Germany) and synchrotron radiation at the Diamond-Manchester Imaging Branchline I13-2 (Diamond Light Source, Didcot, UK), we have acquired 3D data with a resolution down to 0.45 μm for visualization of a human cerebellum specimen down to cellular level. We have shown that all selected modalities, namely laboratory-based absorption contrast micro-tomography (LBμCT), synchrotron radiation based in-line single distance phase contrast tomography (SDPR) and synchrotron radiation based single-grating interferometry (GI), can reach cellular resolution for tissue samples with a size in the mm-range. The results are discussed qualitatively in comparison to optical microscopy of haematoxylin and eosin (HE) stained sections. As phase contrast yields to a better data quality for soft tissues and in order to overcome restrictions of limited beamline access for phase contrast measurements, we have equipped the μCT system nanotom m with a double-grating phase contrast set-up. Preliminary experimental results of a knee sample consisting of a bony part and a cartilage demonstrate that phase contrast data exhibits better quality compared to absorption contrast. Currently, the set-up is under adjustment. It is expected that cellular resolution would also be achieved. The questions arise (1) what would be the quality gain of laboratory-based phase contrast in comparison to laboratory-based absorption contrast tomography and (2) could laboratory-based phase contrast data provide comparable results to synchrotron radiation based

  11. Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

    PubMed Central

    2017-01-01

    The effects of crowding in biological environments on biomolecular structure, dynamics, and function remain not well understood. Computer simulations of atomistic models of concentrated peptide and protein systems at different levels of complexity are beginning to provide new insights. Crowding, weak interactions with other macromolecules and metabolites, and altered solvent properties within cellular environments appear to remodel the energy landscape of peptides and proteins in significant ways including the possibility of native state destabilization. Crowding is also seen to affect dynamic properties, both conformational dynamics and diffusional properties of macromolecules. Recent simulations that address these questions are reviewed here and discussed in the context of relevant experiments. PMID:28666087

  12. Cellular Automata as a Computational Model for Low-Level Vision

    NASA Astrophysics Data System (ADS)

    Broggi, Alberto; D'Andrea, Vincenzo; Destri, Giulio

    In this paper we discuss the use of the Cellular Automata (CA) computational model in computer vision applications on massively parallel architectures. Motivations and guidelines of this approach to low-level vision in the frame of the PROMETHEUS project are discussed. The hard real-time requirement of actual application can be only satisfied using an ad hoc VLSI massively parallel architecture (PAPRICA). The hardware solutions and the specific algorithms can be efficiently verified and tested only using, as a simulator, a general purpose machine with a parent architecture (CM-2). An example of application related to feature extraction is discussed.

  13. A theoretical study of bone remodelling under PEMF at cellular level.

    PubMed

    Wang, Yanan; Qin, Qing-Hua

    2012-01-01

    Pulsed electromagnetic field (PEMF) devices have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. However, the underlying mechanism by which bone remodelling under PEMF is regulated remains poorly understood. In this paper, a mathematical model of bone cell population of bone remodelling under PEMF at cellular level is developed to address this issue for the first time. On the basis of this model and control theory, parametric study of control mechanisms is carried out and a number of possible control mechanisms are identified. These findings will help further the understanding of bone remodelling under PEMF and advance therapies and pharmacological developments in clinical trials.

  14. Cellular Quantitative Structure–Activity Relationship (Cell-QSAR): Conceptual Dissection of Receptor Binding and Intracellular Disposition in Antifilarial Activities of Selwood Antimycins

    PubMed Central

    2012-01-01

    We present the cellular quantitative structure–activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug’s lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition. PMID:22468611

  15. Association of plasma adiponectin levels with cellular hydration state measured using bioelectrical impedance analysis in patients with COPD.

    PubMed

    Yoshikawa, Takahiro; Kanazawa, Hiroshi

    2012-01-01

    It is widely recognized that chronic obstructive pulmonary disease (COPD) includes a variety of extra pulmonary complications and comorbidities. Recently, adiponectin was shown to regulate cellular metabolism in humans. Cellular hydration state is affected by a variety of hormonal factors and regulates cellular metabolic state. Therefore, this study was designed to determine whether adiponectin is a possible factor involved in cellular hydration state in COPD. Thirty patients with COPD and 41 age-matched controls participated in the study. Plasma levels of total and high molecular weight (HMW) adiponectin were measured and anthropometry and pulmonary function tests were conducted. Intracellular water (ICW), extracellular water (ECW), and ECW/ICW ratio, which are parameters of cellular hydration state, were measured using bioelectrical impedance analysis. Higher levels of total and HMW adiponectin in plasma were found in patients with COPD compared with levels in controls. A significant inverse correlation was observed between body mass index and plasma levels of total and HMW adiponectin in the control group. However, this significant correlation was not observed in patients with COPD. The plasma levels of total and HMW adiponectin were also not significantly correlated with any pulmonary function parameters in patients with COPD. Regarding the state of cellular hydration, the plasma levels of total adiponectin were inversely correlated with the ECW/ICW ratio and positively with ICW values in patients with COPD. Moreover, closer correlations were found between these parameters and plasma HMW adiponectin levels. The results of the present study suggest a novel association of the plasma adiponectin with cellular hydration state in patients with COPD. Accordingly, lower adiponectin levels may result in cellular shrinkage, leading to metabolic malfunction at a cellular level. Thus, our findings provide new insights regarding the preventive roles of adiponectin in the

  16. Quantum biology at the cellular level--elements of the research program.

    PubMed

    Bordonaro, Michael; Ogryzko, Vasily

    2013-04-01

    Quantum biology is emerging as a new field at the intersection between fundamental physics and biology, promising novel insights into the nature and origin of biological order. We discuss several elements of QBCL (quantum biology at cellular level) - a research program designed to extend the reach of quantum concepts to higher than molecular levels of biological organization. We propose a new general way to address the issue of environmentally induced decoherence and macroscopic superpositions in biological systems, emphasizing the 'basis-dependent' nature of these concepts. We introduce the notion of 'formal superposition' and distinguish it from that of Schroedinger's cat (i.e., a superposition of macroscopically distinct states). Whereas the latter notion presents a genuine foundational problem, the former one contradicts neither common sense nor observation, and may be used to describe cellular 'decision-making' and adaptation. We stress that the interpretation of the notion of 'formal superposition' should involve non-classical correlations between molecular events in a cell. Further, we describe how better understanding of the physics of Life can shed new light on the mechanism driving evolutionary adaptation (viz., 'Basis-Dependent Selection', BDS). Experimental tests of BDS and the potential role of synthetic biology in closing the 'evolvability mechanism' loophole are also discussed.

  17. Cellular Antioxidant and Anti-Inflammatory Effects of Coffee Extracts with Different Roasting Levels.

    PubMed

    Jung, Soohan; Kim, Min Hyung; Park, Jae Hee; Jeong, Yoonhwa; Ko, Kwang Suk

    2017-06-01

    During roasting, major changes occur in the composition and physiological effects of coffee beans. In this study, in vitro antioxidant effects and anti-inflammatory effects of Coffea arabica green coffee extracts were investigated at different roasting levels corresponding to Light, Medium, City, and French roast. Total caffeine did not show huge difference according to roasting level, but total chlorogenic acid contents were higher in light roasted coffee extract than other roasted groups. In addition, light roasted coffee extract had the highest antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. To determine the in vitro antioxidant property, coffee extracts were used to treat AML-12 cells. Intracellular glutathione (GSH) concentration and mRNA expression levels of genes related to GSH synthesis were negatively related to roasting levels. The anti-inflammatory effects of coffee extracts were investigated in lipopolysaccharide-treated RAW 264.7 macrophage cells. The cellular antioxidant activity of coffee extracts exhibited similar patterns as the AML-12 cells. The expression of mRNA for tumor necrosis factor-alpha and interleukin-6 was decreased in cells treated with the coffee extracts and the expression decreased with increasing roasting levels. These data suggest that coffee has physiological antioxidant and anti-inflammatory activities and these effects are negatively correlated with roasting levels in the cell models.

  18. Chronobiology at the cellular and molecular levels: models and mechanisms for circadian timekeeping.

    PubMed

    Edmunds, L N

    1983-12-01

    This review considers cellular chronobiology and examines, at least in a superficial way, several classes of models and mechanisms that have been proposed for circadian rhythmicity and some of the experimental approaches that have appeared to be most productive. After a brief discussion of temporal organization and the metabolic, epigenetic, and circadian time domains, the general properties of circadian rhythms are enumerated. A survey of independent oscillations in isolated organs, tissues, and cells is followed by a review of selected circadian rhythms in eukaryotic microorganisms, with particular emphasis placed on the rhythm of cell division in the algal flagellate Euglena as a model system illustrating temporal differentiation. In the ensuing section, experimental approaches to circadian clock mechanisms are considered. The dissection of the clock by the use of chemical inhibitors is illustrated for the rhythm of bioluminescence in the marine dinoflagellate Gonyaulax and for the rhythm of photosynthetic capacity in the unicellular green alga Acetabularia. Alternatively, genetic analysis of circadian oscillators is considered in the green alga Chlamydomonas and in the bread mold Neurospora, both of which have yielded clock mutants and mutants having biochemical lesions that exhibit altered clock properties. On the basis of the evidence generated by these experimental approaches, several classes of biochemical and molecular models for circadian clocks have been proposed. These include strictly molecular models, feedback loop (network) models, transcriptional (tape-reading) models, and membrane models; some of their key elements and predictions are discussed. Finally, a number of general unsolved problems at the cellular level are briefly mentioned: cell cycle interfaces, the evolution of circadian rhythmicity, the possibility of multiple cellular oscillators, chronopharmacology and chronotherapy, and cell-cycle clocks in development and aging.

  19. Linking cellular zinc status to body weight and fat mass: mapping quantitative trait loci in Znt7 knockout mice.

    PubMed

    Tepaamorndech, Surapun; Kirschke, Catherine P; Huang, Liping

    2014-08-01

    Zinc transporter 7 (Znt7, Slc30a7) knockout (KO) mice display abnormalities in body weight gain and body adiposity. Regulation of body weight and body fat accumulation is complex, involving multiple genetic and environmental factors. To understand how zinc homeostasis influences body weight and fat deposit and to identify quantitative trait loci (QTLs) that link zinc metabolism to growth and adiposity, we conducted a genome-wide mapping study using male F2 Znt7 KO mice and wild-type (WT) littermates with a mixed 129P1/ReJ and C57BL/6J genetic background. The mice were fed a semi-purified diet containing 30-mg Zn/kg diet at weaning. Body weights and fat pad weights including epididymal, retroperitoneal, and femoral subcutaneous fat pads were measured at 16 weeks of age. We detected two significant QTLs (p < 0.05) for body weight and fat deposit. One was in the F2 Znt7 KO population and the other in the F2 WT population. In Znt7 KO mice, the body weight and fat deposit was significantly linked to a locus on chromosome 7 ranging from 64.3 to 78.3 Mb. In WT mice, a significant linkage of retroperitoneal fat mass was found on chromosome 8 between 14.5 and 63.5 Mb. In addition, several other suggestive QTLs (p < 0.63) for body weight and fat accumulation were detected in Znt7 KO and WT mice. In conclusion, the QTLs identified in this study may provide new hints to uncover the genes linking cellular zinc status to growth and body fat accumulation.

  20. Quantitative proteomic analysis of cellular protein modulation upon inhibition of the NEDD8-activating enzyme by MLN4924.

    PubMed

    Liao, Hua; Liu, Xiaozhen J; Blank, Jonathan L; Bouck, David C; Bernard, Hugues; Garcia, Khristofer; Lightcap, Eric S

    2011-11-01

    Cullin-RING ubiquitin ligases (CRLs) are responsible for the ubiquitination of many cellular proteins, thereby targeting them for proteasomal degradation. In most cases the substrates of the CRLs have not been identified, although many of those that are known have cancer relevance. MLN4924, an investigational small molecule that is a potent and selective inhibitor of the Nedd8-activating enzyme (NAE), is currently being explored in Phase I clinical trials. Inhibition of Nedd8-activating enzyme by MLN4924 prevents the conjugation of cullin proteins with NEDD8, resulting in inactivation of the entire family of CRLs. We have performed stable isotope labeling with amino acids in cell culture analysis of A375 melanoma cells treated with MLN4924 to identify new CRL substrates, confidently identifying and quantitating 5122-6012 proteins per time point. Proteins such as MLX, EID1, KLF5, ORC6L, MAGEA6, MORF4L2, MRFAP1, MORF4L1, and TAX1BP1 are rapidly stabilized by MLN4924, suggesting that they are novel CRL substrates. Proteins up-regulated at later times were also identified and siRNA against their corresponding genes were used to evaluate their influence on MLN4924-induced cell death. Thirty-eight proteins were identified as being particularly important for the cytotoxicity of MLN4924. Strikingly, these proteins had roles in cell cycle, DNA damage repair, and ubiquitin transfer. Therefore, the combination of RNAi with stable isotope labeling with amino acids in cell culture provides a paradigm for understanding the mechanism of action of novel agents affecting the ubiquitin proteasome system and a path to identifying mechanistic biomarkers.

  1. Improved Quantitative Structure-Activity Relationship Models to Predict Antioxidant Activity of Flavonoids in Chemical, Enzymatic, and Cellular Systems

    PubMed Central

    Khlebnikov, Andrei I.; Schepetkin, Igor A.; Domina, Nina G.; Kirpotina, Liliya N.; Quinn, Mark T.

    2007-01-01

    Quantitative structure-activity relationship (QSAR) models are useful in understanding how chemical structure relates to the biological activity of natural and synthetic chemicals and for design of newer and better therapeutics. In the present study, 46 flavonoids and related polyphenols were evaluated for direct/indirect antioxidant activity in three different assay systems of increasing complexity (chemical, enzymatic, and intact phagocytes). Based on these data, two different QSAR models were developed using i) physicochemical and structural (PC&S) descriptors to generate multiparameter partial least squares (PLS) regression equations derived from optimized molecular structures of the tested compounds and ii) a partial 3D comparison of the 46 compounds with local fingerprints obtained from fragments of the molecules by the frontal polygon (FP) method. We obtained much higher QSAR correlation coefficients (r) for flavonoid end-point antioxidant activity in all 3 assay systems using the FP method (0.966, 0.948, and 0.965 for datasets in evaluated in the biochemical, enzymatic, and whole cells assay systems, respectively). Furthermore, high leave-one-out cross-validation coefficients (q2) of 0.907, 0.821, and 0.897 for these datasets, respectively, indicated enhanced predictive ability and robustness of the model. Using the FP method, structural fragments (submolecules) responsible for the end-point antioxidant activity in the three assay systems were also identified. To our knowledge, this is the first QSAR model derived for description of flavonoid direct/indirect antioxidant effects in a cellular system, and this model could form the basis for further drug development of flavonoid-like antioxidant compounds with therapeutic potential. PMID:17166721

  2. Maintenance of cellular ATP level by caloric restriction correlates chronological survival of budding yeast

    SciTech Connect

    Choi, Joon-Seok; Lee, Cheol-Koo

    2013-09-13

    Highlights: •CR decreases total ROS and mitochondrial superoxide during the chronological aging. •CR does not affect the levels of oxidative damage on protein and DNA. •CR contributes extension of chronological lifespan by maintenance of ATP level -- Abstract: The free radical theory of aging emphasizes cumulative oxidative damage in the genome and intracellular proteins due to reactive oxygen species (ROS), which is a major cause for aging. Caloric restriction (CR) has been known as a representative treatment that prevents aging; however, its mechanism of action remains elusive. Here, we show that CR extends the chronological lifespan (CLS) of budding yeast by maintaining cellular energy levels. CR reduced the generation of total ROS and mitochondrial superoxide; however, CR did not reduce the oxidative damage in proteins and DNA. Subsequently, calorie-restricted yeast had higher mitochondrial membrane potential (MMP), and it sustained consistent ATP levels during the process of chronological aging. Our results suggest that CR extends the survival of the chronologically aged cells by improving the efficiency of energy metabolism for the maintenance of the ATP level rather than reducing the global oxidative damage of proteins and DNA.

  3. Level of receptor-associated protein moderates cellular susceptibility to pseudomonas exotoxin A.

    PubMed Central

    Mucci, D; Forristal, J; Strickland, D; Morris, R; Fitzgerald, D; Saelinger, C B

    1995-01-01

    Pseudomonas exotoxin A (PE) enters mammalian cells via a receptor-mediated endocytic pathway. The initial step in this pathway is binding to the multiligand receptor termed the alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (LRP). Binding of toxin, and of the many other ligands that bind to LRP, is blocked by the addition of a 39-kDa receptor-associated protein (RAP). Here we show that approximately 40% of the cell-associated LRP is on the surface of toxin-sensitive mouse LM fibroblasts and thus accessible for toxin internalization. The remainder is located intracellularly, primarily in the Golgi region. Mammalian cells exhibit a wide range of sensitivity to PE. To investigate possible reasons for this, we examined the expression levels of both LRP and RAP. Results from a variety of cell lines indicated that there was a positive correlation between LRP expression and toxin sensitivity. In the absence of LRP, cells were as much as 200-fold more resistant to PE compared with sensitive cells. A second group of resistant cells expressed LRP but had a high level of RAP. Thus, a toxin-resistant phenotype would be expected when cells expressed either low levels of LRP or high levels of LRP in the presence of high levels of RAP. We hypothesize that RAP has a pivotal role in moderating cellular susceptibility to PE. PMID:7622212

  4. A High-throughput Screening Assay for Determining Cellular Levels of Total Tau Protein

    PubMed Central

    Dehdashti, Seameen J.; Zheng, Wei; Gever, Joel R.; Wilhelm, Robert; Nguyen, Dac-Trung; Sittampalam, Gurusingham; McKew, John C.; Austin, Christopher P.; Prusiner, Stanley B.

    2014-01-01

    The microtubule-associated protein (MAP) tau has been implicated in the pathology of numerous neurodegenerative diseases. In the past decade, the hyperphosphorylated and aggregated states of tau protein have been important targets in the drug discovery field for the potential treatment of Alzheimer’s disease. Although several compounds have been reported to reduce the hyperphosphorylated state of tau or impact the stabilization of tau, their therapeutic activities are still to be validated. Recently, reduction of total cellular tau protein has emerged as an alternate intervention point for drug development and a potential treatment of tauopathies. We have developed and optimized a homogenous assay, using the AlphaLISA and HTRF assay technologies, for the quantification of total cellular tau protein levels in the SH-SY5Y neuroblastoma cell line. The signal-to-basal ratios were 375 and 5.3, and the Z’ factors were 0.67 and 0.60 for the AlphaLISA and HTRF tau assays, respectively. The clear advantages of this homogeneous tau assay over conventional total tau assays, such as ELISA and Western blot, are the elimination of plate wash steps and miniaturization of the assay into 1536-well plate format for the ultra–high-throughput screening of large compound libraries. PMID:23905996

  5. A scientific role for Space Station Freedom: Research at the cellular level

    NASA Technical Reports Server (NTRS)

    Johnson, Terry C.; Brady, John N.

    1993-01-01

    The scientific importance of Space Station Freedom is discussed in light of the valuable information that can be gained in cellular and developmental biology with regard to the microgravity environment on the cellular cytoskeleton, cellular responses to extracellular signal molecules, morphology, events associated with cell division, and cellular physiology. Examples of studies in basic cell biology, as well as their potential importance to concerns for future enabling strategies, are presented.

  6. Quantitative Research Attitudes and Research Training Perceptions among Master's-Level Students

    ERIC Educational Resources Information Center

    Steele, Janeé M.; Rawls, Glinda J.

    2015-01-01

    This study explored master's-level counseling students' (N = 804) perceptions of training in the Council for Accreditation of Counseling and Related Educational Programs (2009) Research and Program Evaluation standard, and their attitudes toward quantitative research. Training perceptions and quantitative research attitudes were low to moderate,…

  7. Quantitative Research Attitudes and Research Training Perceptions among Master's-Level Students

    ERIC Educational Resources Information Center

    Steele, Janeé M.; Rawls, Glinda J.

    2015-01-01

    This study explored master's-level counseling students' (N = 804) perceptions of training in the Council for Accreditation of Counseling and Related Educational Programs (2009) Research and Program Evaluation standard, and their attitudes toward quantitative research. Training perceptions and quantitative research attitudes were low to moderate,…

  8. Effects of electromagnetic radiation from a cellular telephone on the oxidant and antioxidant levels in rabbits.

    PubMed

    Irmak, M Kemal; Fadillioğlu, Ersin; Güleç, Mukaddes; Erdoğan, Hasan; Yağmurca, Murat; Akyol, Omer

    2002-12-01

    The number of reports on the effects induced by electromagnetic radiation (EMR) in various cellular systems is still increasing. Until now no satisfactory mechanism has been proposed to explain the biological effects of this radiation. Oxygen free radicals may play a role in mechanisms of adverse effects of EMR. This study was undertaken to investigate the influence of electromagnetic radiation of a digital GSM mobile telephone (900 MHz) on oxidant and antioxidant levels in rabbits. Adenosine deaminase, xanthine oxidase, catalase, myeloperoxidase, superoxide dismutase (SOD) and glutathione peroxidase activities as well as nitric oxide (NO) and malondialdehyde levels were measured in sera and brains of EMR-exposed and sham-exposed rabbits. Serum SOD activity increased, and serum NO levels decreased in EMR-exposed animals compared to the sham group. Other parameters were not changed in either group. This finding may indicate the possible role of increased oxidative stress in the pathophysiology of adverse effect of EMR. Decreased NO levels may also suggest a probable role of NO in the adverse effect. Copyright 2002 John Wiley & Sons, Ltd.

  9. Finding modulators of stochasticity levels by quantitative genetics.

    PubMed

    Fehrmann, Steffen; Yvert, Gaël

    2011-01-01

    Although bakers and wine makers constantly select, compare, and hunt for new wild strains of Saccharomyces cerevisiae, yeast geneticists have long focused on a few "standard" strains to ensure reproducibility and easiness of experimentation. And so far, the wonderful natural resource of wild genetic variation has been poorly exploited in most academic laboratories. We describe here how one can use this resource to investigate the molecular sources of stochasticity in a gene regulatory network. The approach is general enough to be applied to any network of interest, as long as the experimental read-out offers robust statistics. For a given network, a typical study first identifies two backgrounds A and B displaying different levels of stochasticity and then study the network in A × B progeny. Taking advantage of microarrays or resequencing technologies, genotyping of appropriate segregants can then lead to the genomic regions housing modulators of stochasticity. The powerful toolbox available to manipulate the yeast genome offers several ways to narrow these regions further and to unambiguously demonstrate the regulatory consequences of DNA polymorphisms.

  10. How the Venus flytrap actively snaps: hydrodynamic measurements at the cellular level

    NASA Astrophysics Data System (ADS)

    Colombani, Mathieu; Forterre, Yoel; GEP Team

    2012-11-01

    Although they lack muscle, plants have evolved a remarkable range of mechanisms to create rapid motion, from the rapid folding of sensitive plants to seed dispersal. Of these spectacular examples that have long fascinated scientists, the carnivorous plant Venus flytrap, whose leaves snap together in a fraction of second to capture insects, has long been a paradigm for study. Recently, we have shown that this motion involves a snap-buckling instability due to the shell-like geometry of the leaves of the trap. However, the origin of the movement that allows the plant to cross the instability threshold and actively bend remains largely unknown. In this study, we investigate this active motion using a micro-fluidic pressure probe that gives direct hydraulic and mechanical measurements at the cellular level (osmotic pressure, cell membrane permeability, cell wall elasticity). Our results challenge the role of osmotically-driven water flows usually put forward to explain Venus flytrap's active closure.

  11. Human Metapneumovirus Inhibits IFN-β Signaling by Downregulating Jak1 and Tyk2 Cellular Levels

    PubMed Central

    Ren, Junping; Kolli, Deepthi; Liu, Tianshuang; Xu, Renling; Garofalo, Roberto P.; Casola, Antonella; Bao, Xiaoyong

    2011-01-01

    Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN) signaling by an unidentified mechanism. In this study, we showed that infection of airway epithelial cells with hMPV decreased cellular level of Janus tyrosine kinase (Jak1) and tyrosine kinase 2 (Tyk2), due to enhanced proteosomal degradation and reduced gene transcription. In addition, hMPV infection also reduced the surface expression of type I IFN receptor (IFNAR). These inhibitory mechanisms are different from the ones employed by respiratory syncytial virus (RSV), which does not affect Jak1, Tyk2 or IFNAR expression, but degrades downstream signal transducer and activator of transcription proteins 2 (STAT2), although both viruses are pneumoviruses belonging to the Paramyxoviridae family. Our study identifies a novel mechanism by which hMPV inhibits STAT1 and 2 activation, ultimately leading to viral evasion of host IFN responses. PMID:21949722

  12. Modeling of trophospheric ozone concentrations using genetically trained multi-level cellular neural networks

    NASA Astrophysics Data System (ADS)

    Ozcan, H. Kurtulus; Bilgili, Erdem; Sahin, Ulku; Ucan, O. Nuri; Bayat, Cuma

    2007-09-01

    Tropospheric ozone concentrations, which are an important air pollutant, are modeled by the use of an artificial intelligence structure. Data obtained from air pollution measurement stations in the city of Istanbul are utilized in constituting the model. A supervised algorithm for the evaluation of ozone concentration using a genetically trained multi-level cellular neural network (ML-CNN) is introduced, developed, and applied to real data. A genetic algorithm is used in the optimization of CNN templates. The model results and the actual measurement results are compared and statistically evaluated. It is observed that seasonal changes in ozone concentrations are reflected effectively by the concentrations estimated by the multilevel-CNN model structure, with a correlation value of 0.57 ascertained between actual and model results. It is shown that the multilevel-CNN modeling technique is as satisfactory as other modeling techniques in associating the data in a complex medium in air pollution applications.

  13. Lethal and mutagenic interactions between γ-rays, cisplatin and etoposide at the cellular and molecular levels.

    PubMed

    Lillo, Olga; Bracesco, Nelson; Nunes, Elia

    2011-02-01

    We analysed the lethal and mutagenic interactions between γ-rays, cisplatin (Pt) and etoposide (E), three agents used in tumour chemoradiotherapy. Corresponding results at cellular and molecular levels could provide additional elements on involved mechanisms and, on antitumour activity and toxicity in combined cancer treatments. The yeast Saccharomyces cerevisiae SC7K(lys2-3) (auxotrophic for lysine) was used as eukaryotic model. Exponential growing cells were exposed to the mentioned agents, as single and combined treatments. Lethal and mutation interaction equations were determined as a function of doses according to quantitative models. DNA double-strand breaks were evaluated immediately after treatments, through pulsed-field electrophoresis and laser densitometry. All three agents induced significant mutant frequency. The γ +Pt + E combination determined maximal lethal and mutagenic synergism, followed by γ + Pt and γ + E combinations. Meanwhile, Pt + E combination showed lethal additivity and very low mutagenic synergism. Pt + E double combination determined moderate DNA degradation. DNA degradation after γ-exposure, was similar to that of γ + Pt, γ + E and γ + Pt + E combinations. Synergistic lethal and mutagenic interactions indicate crosstalk between non-homologous end joining, homologous recombination and postreplicative repair pathways. Pt + E additivity indicate independence of involved repair pathways. Furthermore, the quantification of interactive events may be an additional suitable tool in tumour therapy planning.

  14. Cellular-level characterization of B cells infiltrating pulmonary MALT lymphoma tissues.

    PubMed

    Fujii, Keiichiro; Ishibashi, Ken-Ichiro; Kato, Junki; Kan, Jushin; Fujii, Kana; Ito, Yohei; Takino, Hisashi; Masaki, Ayako; Murase, Takayuki; Inagaki, Hiroshi

    2016-11-01

    Mucosa-associated lymphoid tissue (MALT) lymphoma mainly consists of three types of tumor B cells, small (centrocyte-like), scattered large transformed, and intraepithelial. However, it is difficult to differentiate tumor B cells from reactive B cells at the cellular level. We examined five cases of API2-MALT1 fusion-positive MALT lymphoma of the lung. A single paraffin section for each case was subjected to sequential retrieval of whole-slide imaging (WSI) data of hematoxylin and eosin (HE) staining, immunofluorescence staining for CD79a, and fluorescence in situ hybridization (FISH) for the MALT1 split. We counted the number of MALT1 split-positive or MALT1 split-negative cells among CD79a-positive cells. The MALT1 split was detected in 59, 46, and 76 % of small, large, and intraepithelial B cells, respectively. A review of the HE-WSI data showed that cytomorphological distinction between the MALT1 split-positive and MALT1 split-negative B cells was virtually impossible. None of CD79a-negative lymphoid cells, epithelial cells, and microvascular endothelial cells was positive for MALT1 splits. As API2-MALT1 fusion is an early and critical event in the lymphomatogenesis, our findings are best interpreted as that a considerable number of B cells, either small, large, or intraepithelial, are reactive cells and that it is difficult to distinguish cytomorphologically between tumor B cells and reactive B cells. These findings suggest that the tumor architecture may be the central factor for making a correct histopathological diagnosis of MALT lymphoma. The sequential WSI of HE staining, immunofluorescence staining, and FISH as described here is a useful tool for pathological analysis at the cellular level.

  15. Protection of low density lipoprotein oxidation at chemical and cellular level by the antioxidant drug dipyridamole.

    PubMed Central

    Iuliano, L.; Colavita, A. R.; Camastra, C.; Bello, V.; Quintarelli, C.; Alessandroni, M.; Piovella, F.; Violi, F.

    1996-01-01

    1. The oxidative modification of low density lipoprotein (LDL) is thought to be an important factor in the initiation and development of atherosclerosis. Natural and synthetic antioxidants have been shown to protect LDL from oxidation and to inhibit atherosclerosis development in animals. Synthetic antioxidants are currently being tested, by they are not necessarily safe for human use. 2. We have previously reported that dipyridamole, currently used in clinical practice, is a potent scavenger of free radicals. Thus, we tested whether dipyridamole could affect LDL oxidation at chemical and cellular level. 3. Chemically induced LDL oxidation was made by Cu(II), Cu(II) plus hydrogen peroxide or peroxyl radicals generated by thermolysis of 2,2'-azo-bis(2-amidino propane). Dipyridamole, (1-10 microM), inhibited LDL oxidation as monitored by diene formation, evolution of hydroperoxides and thiobarbituric acid reactive substances, apoprotein modification and by the fluorescence of cis-parinaric acid. 4. The physiological relevance of the antioxidant activity was validated by experiments at the cellular level where dipyridamole inhibited endothelial cell-mediated LDL oxidation, their degradation by monocytes, and cytotoxicity. 5. In comparison with ascorbic acid, alpha-tocopherol and probucol, dipyridamole was the more efficient antioxidant with the following order of activity: dipyridamole > probucol > ascorbic acid > alpha-tocopherol. The present study shows that dipyridamole inhibits oxidation of LDL at pharmacologically relevant concentrations. The inhibition of LDL oxidation is unequivocally confirmed by use of three different methods of chemical oxidation, by several methods of oxidation monitoring, and the pharmacological relevance is demonstrated by the superiority of dipyridamole over the naturally occurring antioxidants, ascorbic acid and alpha-tocopherol and the synthetic antioxidant probucol. Images Figure 6 PMID:8968553

  16. Axial level-dependent molecular and cellular mechanisms underlying the genesis of the embryonic neural plate.

    PubMed

    Kondoh, Hisato; Takada, Shinji; Takemoto, Tatsuya

    2016-06-01

    The transcription factor gene Sox2, centrally involved in neural primordial regulation, is activated by many enhancers. During the early stages of embryonic development, Sox2 is regulated by the enhancers N2 and N1 in the anterior neural plate (ANP) and posterior neural plate (PNP), respectively. This differential use of the enhancers reflects distinct regulatory mechanisms underlying the genesis of ANP and PNP. The ANP develops directly from the epiblast, triggered by nodal signal inhibition, and via the combined action of TFs SOX2, OTX2, POU3F1, and ZIC2, which promotes the the ANP development and inhibits other cell lineages. In contrast, the PNP is derived from neuromesodermal bipotential axial stem cells that develop into the neural plate when Sox2 is activated by the N1 enhancer, whereas they develop into the paraxial mesoderm when the N1 enhancer is repressed by the action of TBX6. The axial stem cells are maintained by the activity of WNT3a and T (Brachyury). However, at axial levels more anterior to the 8th somites (cervical levels), the development of both the neural plate and somite proceeds in the absence of WNT3a, T, or TBX6. These observations indicate that distinct molecular and cellular mechanisms determine neural plate genesis based on the axial level, and contradict the classical concept of the term "neural induction," which assumes a pan-neural plate mechanism.

  17. Energy-Efficient Crowdsensing of Human Mobility and Signal Levels in Cellular Networks

    PubMed Central

    Foremski, Paweł; Gorawski, Michał; Grochla, Krzysztof; Polys, Konrad

    2015-01-01

    The paper presents a practical application of the crowdsensing idea to measure human mobility and signal coverage in cellular networks. Currently, virtually everyone is carrying a mobile phone, which may be used as a sensor to gather research data by measuring, e.g., human mobility and radio signal levels. However, many users are unwilling to participate in crowdsensing experiments. This work begins with the analysis of the barriers for engaging people in crowdsensing. A survey showed that people who agree to participate in crowdsensing expect a minimum impact on their battery lifetime and phone usage habits. To address these requirements, this paper proposes an application for measuring the location and signal strength data based on energy-efficient GPS tracking, which allows one to perform the measurements of human mobility and radio signal levels with minimum energy utilization and without any engagement of the user. The method described combines measurements from the accelerometer with effective management of the GPS to monitor the user mobility with the decrease in battery lifetime by approximately 20%. To show the applicability of the proposed platform, the sample results of signal level distribution and coverage maps gathered for an LTE network and representing human mobility are shown. PMID:26340633

  18. Auxin transport at cellular level: new insights supported by mathematical modelling

    PubMed Central

    Hošek, Petr; Kubeš, Martin; Laňková, Martina; Dobrev, Petre I.; Klíma, Petr; Kohoutová, Milada; Petrášek, Jan; Hoyerová, Klára; Jiřina, Marcel; Zažímalová, Eva

    2012-01-01

    The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport. PMID:22438304

  19. Auxin transport at cellular level: new insights supported by mathematical modelling.

    PubMed

    Hosek, Petr; Kubes, Martin; Lanková, Martina; Dobrev, Petre I; Klíma, Petr; Kohoutová, Milada; Petrásek, Jan; Hoyerová, Klára; Jirina, Marcel; Zazímalová, Eva

    2012-06-01

    The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.

  20. Lectin-Mediated Resistance Impairs Plant Virus Infection at the Cellular Level[C][W][OA

    PubMed Central

    Yamaji, Yasuyuki; Maejima, Kensaku; Komatsu, Ken; Shiraishi, Takuya; Okano, Yukari; Himeno, Misako; Sugawara, Kyoko; Neriya, Yutaro; Minato, Nami; Miura, Chihiro; Hashimoto, Masayoshi; Namba, Shigetou

    2012-01-01

    Plants possess a multilayered defense response, known as plant innate immunity, to infection by a wide variety of pathogens. Lectins, sugar binding proteins, play essential roles in the innate immunity of animal cells, but the role of lectins in plant defense is not clear. This study analyzed the resistance of certain Arabidopsis thaliana ecotypes to a potexvirus, plantago asiatica mosaic virus (PlAMV). Map-based positional cloning revealed that the lectin gene JACALIN-TYPE LECTIN REQUIRED FOR POTEXVIRUS RESISTANCE1 (JAX1) is responsible for the resistance. JAX1-mediated resistance did not show the properties of conventional resistance (R) protein–mediated resistance and was independent of plant defense hormone signaling. Heterologous expression of JAX1 in Nicotiana benthamiana showed that JAX1 interferes with infection by other tested potexviruses but not with plant viruses from different genera, indicating the broad but specific resistance to potexviruses conferred by JAX1. In contrast with the lectin gene RESTRICTED TEV MOVEMENT1, which inhibits the systemic movement of potyviruses, which are distantly related to potexviruses, JAX1 impairs the accumulation of PlAMV RNA at the cellular level. The existence of lectin genes that show a variety of levels of virus resistance, their targets, and their properties, which are distinct from those of known R genes, suggests the generality of lectin-mediated resistance in plant innate immunity. PMID:22307853

  1. Multimodal imaging of the human knee down to the cellular level

    NASA Astrophysics Data System (ADS)

    Schulz, G.; Götz, C.; Müller-Gerbl, M.; Zanette, I.; Zdora, M.-C.; Khimchenko, A.; Deyhle, H.; Thalmann, P.; Müller, B.

    2017-06-01

    Computed tomography reaches the best spatial resolution for the three-dimensional visualization of human tissues among the available nondestructive clinical imaging techniques. Nowadays, sub-millimeter voxel sizes are regularly obtained. Regarding investigations on true micrometer level, lab-based micro-CT (μCT) has become gold standard. The aim of the present study is firstly the hierarchical investigation of a human knee post mortem using hard X-ray μCT and secondly a multimodal imaging using absorption and phase contrast modes in order to investigate hard (bone) and soft (cartilage) tissues on the cellular level. After the visualization of the entire knee using a clinical CT, a hierarchical imaging study was performed using the lab-system nanotom® m. First, the entire knee was measured with a pixel length of 65 μm. The highest resolution with a pixel length of 3 μm could be achieved after extracting cylindrically shaped plugs from the femoral bones. For the visualization of the cartilage, grating-based phase contrast μCT (I13-2, Diamond Light Source) was performed. With an effective voxel size of 2.3 μm it was possible to visualize individual chondrocytes within the cartilage.

  2. Characterization of cellular traction forces at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Dunn, Alexander

    2013-03-01

    The ability of cells to generate and respond to mechanical cues is an essential aspect of stem cell differentiation, embryonic development, and our senses of touch and hearing. However, our understanding of the roles of mechanical force in cell biology remains in its infancy, due largely to a lack of tools that measure the forces generated by living cells at the molecular scale. Here we describe a new technique termed Molecular Force Microscopy (MFM) that visualizes the forces exerted by single cellular adhesion molecules with nm, pN, and sub-second resolutions. MFM uses novel FRET-based molecular tension sensors that bind to a glass coverslip and present a binding site for integrins, a ubiquitous class of cell adhesion proteins. Cell-generated forces stretch the MFM sensor molecules, resulting in decreased FRET with increasing load that can be imaged at the single-molecule level. Human foreskin fibroblasts adhere to surfaces functionalized with the MFM probes and develop robust focal adhesions. FRET values measured using MFM indicate forces of between 1 and 4 pN per integrin, thus providing the first direct measurement of the tension per integrin molecule necessary to form stable adhesions. The relatively narrow force distribution suggests that mechanical tension is subject to exquisite feedback and control at the molecular level.

  3. Periodic forcing in a three-level cellular automata model for a vector-transmitted disease

    NASA Astrophysics Data System (ADS)

    Santos, L. B. L.; Costa, M. C.; Pinho, S. T. R.; Andrade, R. F. S.; Barreto, F. R.; Teixeira, M. G.; Barreto, M. L.

    2009-07-01

    A periodically forced two-dimensional cellular automata model is used to reproduce and analyze the complex spatiotemporal patterns observed in the transmission of vector infectious diseases. The system, which comprises three population levels, is introduced to describe complex features of the dynamics of the vector-transmitted dengue epidemics, known to be very sensitive to seasonal variables. The three coupled levels represent the human, the adult, and immature vector populations. The dynamics includes external seasonality forcing, human and mosquito mobility, and vector control effects. The model parameters, even if bounded to well-defined intervals obtained from reported data, can be selected to reproduce specific epidemic outbursts. In the current study, explicit results are obtained by comparison with actual data retrieved from the time series of dengue epidemics in two cities in Brazil. The results show fluctuations that are not captured by mean-field models. It also reveals the qualitative behavior of the spatiotemporal patterns of the epidemics. In the extreme situation of the absence of external periodic drive, the model predicts a completely distinct long-time evolution. The model is robust in the sense that it is able to reproduce the time series of dengue epidemics of different cities, provided that the forcing term takes into account the local rainfall modulation. Finally, an analysis is provided of the effect of the dependence between epidemics threshold and vector control actions, both in the presence and absence of human mobility factor.

  4. System-Level Performance of Antenna Arrays in CDMA-Based Cellular Mobile Radio Systems

    NASA Astrophysics Data System (ADS)

    Czylwik, Andreas; Dekorsy, Armin

    2004-12-01

    Smart antennas exploit the inherent spatial diversity of the mobile radio channel, provide an antenna gain, and also enable spatial interference suppression leading to reduced intracell as well as intercell interference. Especially, for the downlink of future CDMA-based mobile communications systems, transmit beamforming is seen as a well-promising smart antenna technique. The main objective of this paper is to study the performance of diverse antenna array topologies when applied for transmit beamforming in the downlink of CDMA-based networks. In this paper, we focus on uniform linear array (ULA) and uniform circular array (UCA) topologies. For the ULA, we consider three-sector base stations with one linear array per sector. While recent research on downlink beamforming is often restricted to one single cell, this study takes into account the important impact of intercell interference on the performance by evaluating complete networks. Especially, from the operator perspective, system capacity and system coverage are very essential parameters of a cellular system so that there is a clear necessity of intensive system level investigations. Apart from delivering assessments on the performance of the diverse antenna array topologies, in the paper also different antenna array parameters, such as element spacing and beamwidth of the sector antennas, are optimized. Although we focus on the network level, fast channel fluctuations are taken into account by including them analytically into the signal-to-interference calculation.

  5. Low levels of graphene and graphene oxide inhibit cellular xenobiotic defense system mediated by efflux transporters.

    PubMed

    Liu, Su; Jiang, Wei; Wu, Bing; Yu, Jing; Yu, Haiyan; Zhang, Xu-Xiang; Torres-Duarte, Cristina; Cherr, Gary N

    2016-01-01

    Low levels of graphene and graphene oxide (GO) are considered to be environmentally safe. In this study, we analyzed the potential effects of graphene and GO at relatively low concentrations on cellular xenobiotic defense system mediated by efflux transporters. The results showed that graphene (<0.5 μg/mL) and GO (<20 μg/mL) did not decrease cell viability, generate reactive oxygen species, or disrupt mitochondrial function. However, graphene and GO at the nontoxic concentrations could increase calcein-AM (CAM, an indicator of membrane ATP-binding cassette (ABC) transporter) activity) accumulation, indicating inhibition of ABC transporters' efflux capabilities. This inhibition was observed even at 0.005 μg/mL graphene and 0.05 μg/mL GO, which are 100 times and 400 times lower than their lowest toxic concentration from cytotoxicity experiments, respectively. The inhibition of ABC transporters significantly increased the toxicity of paraquat and arsenic, known substrates of ABC transporters. The inhibition of ABC transporters was found to be based on graphene and GO damaging the plasma membrane structure and fluidity, thus altering functions of transmembrane ABC transporters. This study demonstrates that low levels of graphene and GO are not environmentally safe since they can significantly make cell more susceptible to other xenobiotics, and this chemosensitizing activity should be considered in the risk assessment of graphene and GO.

  6. Implementing online quantitative support modules in an intermediate-level course

    NASA Astrophysics Data System (ADS)

    Daly, J.

    2011-12-01

    While instructors typically anticipate that students in introductory geology courses enter a class with a wide range of quantitative ability, we often overlook the fact that this may also be true in upper-level courses. Some students are drawn to the subject and experience success in early courses with an emphasis on descriptive geology, then experience frustration and disappointment in mid- and upper-level courses that are more quantitative. To bolster student confidence in quantitative skills and enhance their performance in an upper-level course, I implemented several modules from The Math You Need (TMYN) online resource with a 200-level geomorphology class. Student facility with basic quantitative skills (rearranging equations, manipulating units, and graphing) was assessed with an online pre- and post-test. During the semester, modules were assigned to complement existing course activities (for example, the module on manipulating units was assigned prior to a lab on measurement of channel area and water velocity, then calculation of discharge). The implementation was designed to be a concise review of relevant skills for students with higher confidence in their quantitative abilities, and to provide a self-paced opportunity for students with less quantitative facility to build skills. This course already includes a strong emphasis on quantitative data collection, analysis, and presentation; in the past, student performance in the course has been strongly influenced by their individual quantitative ability. I anticipate that giving students the opportunity to improve mastery of fundamental quantitative skills will improve their performance on higher-stakes assignments and exams, and will enhance their sense of accomplishment in the course.

  7. Towards quantitative ecological risk assessment of elevated carbon dioxide levels in the marine environment.

    PubMed

    de Vries, Pepijn; Tamis, Jacqueline E; Foekema, Edwin M; Klok, Chris; Murk, Albertinka J

    2013-08-30

    The environmental impact of elevated carbon dioxide (CO2) levels has become of more interest in recent years. This, in relation to globally rising CO2 levels and related considerations of geological CO2 storage as a mitigating measure. In the present study effect data from literature were collected in order to conduct a marine ecological risk assessment of elevated CO2 levels, using a Species Sensitivity Distribution (SSD). It became evident that information currently available from the literature is mostly insufficient for such a quantitative approach. Most studies focus on effects of expected future CO2 levels, testing only one or two elevated concentrations. A full dose-response relationship, a uniform measure of exposure, and standardized test protocols are essential for conducting a proper quantitative risk assessment of elevated CO2 levels. Improvements are proposed to make future tests more valuable and usable for quantitative risk assessment. Copyright © 2013 Elsevier Ltd. All rights reserved.

  8. Serum levels, tissue expression and cellular secretion of macrophage migration inhibitory factor in limited and diffuse systemic sclerosis.

    PubMed

    Corallo, Claudio; Paulesu, Luana; Cutolo, Maurizio; Ietta, Francesca; Carotenuto, Claudio; Mannelli, Chiara; Romagnoli, Roberta; Nuti, Ranuccio; Giordano, Nicola

    2015-01-01

    To investigate serum levels, tissue/cellular expression of macrophage migration inhibitory factor (MIF) in patients with limited (lSSc) and diffuse (dSSc) systemic sclerosis. 10 lSSc-patients, 10 dSSc-patients and 10 controls were enrolled. MIF serum levels were assayed by ELISA. MIF and its receptors CD74/CD44 were evaluated by immunohistochemistry on skin biopsies from patients with dSSc, lSSc (affected and not-affected skin) and controls. MIF levels were assessed (ELISA) in supernatants of healthy dermal microvascular endothelial cells (MVECs) and in control (CTR), non-affected SSc (NA) and affected (SSc) fibroblasts treated for 48 h with 10% control serum and 10% SSc-serum. MIF supernatant (ELISA) and mRNA (quantitative real-time PCR) levels were determined in SSc dermal fibroblasts and in control dermal fibroblasts untreated or stimulated at 6 h-24 h-48 h with bleomycin (50 mU/ml). Serum MIF was significantly higher in dSSc (18.7±4.1 ng/ml, p<0.001) and in lSSc (10.4±4.4 ng/ml, p<0.001) patients respect to controls (2.6±1.4 ng/ml). Enhanced MIF immunoreactivity was found in keratinocytes, fibroblasts, endothelium, sebaceous/sweat glands from lSSc/dSSc affected skin. Faint MIF immunoreactivity was found in control skin and not-affected skin of lSSc patients. No differences were found in CD74/CD44 receptors' analysis among control and dSSc/lSSc affected and non-affected skin. MVECs and fibroblasts (CTR, NA and SSc) produced significantly more MIF, when stimulated with SSc serum respect to control-serum (p<0.001). Finally, MIF mRNA levels significantly increased at 6h (p<0.001) and decreased at 48 h (p<0.001) in control fibroblasts treated with bleomycin compared to control untreated. Simultaneously, MIF supernatant protein levels increased after 48 h (p<0.01) in bleomycin-treated fibroblasts respect to untreated ones. These results suggest that MIF could be implicated in the pathogenesis of SSc, probably acting as protective factor against the SSc stressful

  9. Sulforaphane restores cellular glutathione levels and reduces chronic periodontitis neutrophil hyperactivity in vitro.

    PubMed

    Dias, Irundika H K; Chapple, Ian L C; Milward, Mike; Grant, Melissa M; Hill, Eric; Brown, James; Griffiths, Helen R

    2013-01-01

    The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 (. -) by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients' neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 (. -) production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis.

  10. Review of quantitative phase-digital holographic microscopy: promising novel imaging technique to resolve neuronal network activity and identify cellular biomarkers of psychiatric disorders

    PubMed Central

    Marquet, Pierre; Depeursinge, Christian; Magistretti, Pierre J.

    2014-01-01

    Abstract. Quantitative phase microscopy (QPM) has recently emerged as a new powerful quantitative imaging technique well suited to noninvasively explore a transparent specimen with a nanometric axial sensitivity. In this review, we expose the recent developments of quantitative phase-digital holographic microscopy (QP-DHM). Quantitative phase-digital holographic microscopy (QP-DHM) represents an important and efficient quantitative phase method to explore cell structure and dynamics. In a second part, the most relevant QPM applications in the field of cell biology are summarized. A particular emphasis is placed on the original biological information, which can be derived from the quantitative phase signal. In a third part, recent applications obtained, with QP-DHM in the field of cellular neuroscience, namely the possibility to optically resolve neuronal network activity and spine dynamics, are presented. Furthermore, potential applications of QPM related to psychiatry through the identification of new and original cell biomarkers that, when combined with a range of other biomarkers, could significantly contribute to the determination of high risk developmental trajectories for psychiatric disorders, are discussed. PMID:26157976

  11. Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia.

    PubMed

    Alikhani, Mehdi; Mirzaei, Mehdi; Sabbaghian, Marjan; Parsamatin, Pouria; Karamzadeh, Razieh; Adib, Samane; Sodeifi, Niloofar; Gilani, Mohammad Ali Sadighi; Zabet-Moghaddam, Masoud; Parker, Lindsay; Wu, Yunqi; Gupta, Vivek; Haynes, Paul A; Gourabi, Hamid; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

    2017-02-15

    Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction.

  12. MicroRNA-15b Modulates Cellular ATP Levels and Degenerates Mitochondria via Arl2 in Neonatal Rat Cardiac Myocytes*

    PubMed Central

    Nishi, Hitoo; Ono, Koh; Iwanaga, Yoshitaka; Horie, Takahiro; Nagao, Kazuya; Takemura, Genzou; Kinoshita, Minako; Kuwabara, Yasuhide; Mori, Rieko Takanabe; Hasegawa, Koji; Kita, Toru; Kimura, Takeshi

    2010-01-01

    MicroRNAs (miRNAs or miRs) are small, non-coding RNAs that modulate mRNA stability and post-transcriptional translation. A growing body of evidence indicates that specific miRNAs can affect the cellular function of cardiomyocytes. In the present study, miRNAs that are highly expressed in the heart were overexpressed in neonatal rat ventricular myocytes, and cellular ATP levels were assessed. As a result, miR-15b, -16, -195, and -424, which have the same seed sequence, the most critical determinant of miRNA targeting, decreased cellular ATP levels. These results suggest that these miRNAs could specifically down-regulate the same target genes and consequently decrease cellular ATP levels. Through a bioinformatics approach, ADP-ribosylation factor-like 2 (Arl2) was identified as a potential target of miR-15b. It has already been shown that Arl2 localizes to adenine nucleotide transporter 1, the exchanger of ADP/ATP in mitochondria. Overexpression of miR-15b, -16, -195, and -424 suppressed the activity of a luciferase reporter construct fused with the 3′-untranslated region of Arl2. In addition, miR-15b overexpression decreased Arl2 mRNA and protein expression levels. The effects of Arl2 siRNA on cellular ATP levels were the same as those of miR-15b, and the expression of Arl2 could restore ATP levels reduced by miR-15b. A loss-of-function study of miR-15b resulted in increased Arl2 protein and cellular ATP levels. Electron microscopic analysis revealed that mitochondria became degenerated in cardiomyocytes that had been transduced with miR-15b and Arl2 siRNA. The present results suggest that miR-15b may decrease mitochondrial integrity by targeting Arl2 in the heart. PMID:20007690

  13. High-throughput quantitation of inorganic nanoparticle biodistribution at the single-cell level using mass cytometry

    PubMed Central

    Yang, Yu-Sang Sabrina; Atukorale, Prabhani U.; Moynihan, Kelly D.; Bekdemir, Ahmet; Rakhra, Kavya; Tang, Li; Stellacci, Francesco; Irvine, Darrell J.

    2017-01-01

    Inorganic nanoparticles (NPs) are studied as drug carriers, radiosensitizers and imaging agents, and characterizing nanoparticle biodistribution is essential for evaluating their efficacy and safety. Tracking NPs at the single-cell level with current technologies is complicated by the lack of reliable methods to stably label particles over extended durations in vivo. Here we demonstrate that mass cytometry by time-of-flight provides a label-free approach for inorganic nanoparticle quantitation in cells. Furthermore, mass cytometry can enumerate AuNPs with a lower detection limit of ∼10 AuNPs (3 nm core size) in a single cell with tandem multiparameter cellular phenotyping. Using the cellular distribution insights, we selected an amphiphilic surface ligand-coated AuNP that targeted myeloid dendritic cells in lymph nodes as a peptide antigen carrier, substantially increasing the efficacy of a model vaccine in a B16-OVA melanoma mouse model. This technology provides a powerful new level of insight into nanoparticle fate in vivo. PMID:28094297

  14. Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation.

    PubMed

    Rea, Matthew; Jiang, Tingting; Eleazer, Rebekah; Eckstein, Meredith; Marshall, Alan G; Fondufe-Mittendorf, Yvonne N

    2016-07-01

    Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Quantitative Mass Spectrometry Reveals Changes in Histone H2B Variants as Cells Undergo Inorganic Arsenic-Mediated Cellular Transformation*

    PubMed Central

    Rea, Matthew; Jiang, Tingting; Eleazer, Rebekah; Eckstein, Meredith; Marshall, Alan G.; Fondufe-Mittendorf, Yvonne N.

    2016-01-01

    Exposure to inorganic arsenic, a ubiquitous environmental toxic metalloid, leads to carcinogenesis. However, the mechanism is unknown. Several studies have shown that inorganic arsenic exposure alters specific gene expression patterns, possibly through alterations in chromatin structure. While most studies on understanding the mechanism of chromatin-mediated gene regulation have focused on histone post-translational modifications, the role of histone variants remains largely unknown. Incorporation of histone variants alters the functional properties of chromatin. To understand the global dynamics of chromatin structure and function in arsenic-mediated carcinogenesis, analysis of the histone variants incorporated into the nucleosome and their covalent modifications is required. Here we report the first global mass spectrometric analysis of histone H2B variants as cells undergo arsenic-mediated epithelial to mesenchymal transition. We used electron capture dissociation-based top-down tandem mass spectrometry analysis validated with quantitative reverse transcription real-time polymerase chain reaction to identify changes in the expression levels of H2B variants in inorganic arsenic-mediated epithelial-mesenchymal transition. We identified changes in the expression levels of specific histone H2B variants in two cell types, which are dependent on dose and length of exposure of inorganic arsenic. In particular, we found increases in H2B variants H2B1H/1K/1C/1J/1O and H2B2E/2F, and significant decreases in H2B1N/1D/1B as cells undergo inorganic arsenic-mediated epithelial-mesenchymal transition. The analysis of these histone variants provides a first step toward an understanding of the functional significance of the diversity of histone structures, especially in inorganic arsenic-mediated gene expression and carcinogenesis. PMID:27169413

  16. Leptin Levels Are Higher in Whole Compared to Skim Human Milk, Supporting a Cellular Contribution.

    PubMed

    Kugananthan, Sambavi; Lai, Ching Tat; Gridneva, Zoya; Mark, Peter J; Geddes, Donna T; Kakulas, Foteini

    2016-11-08

    Human milk (HM) contains a plethora of metabolic hormones, including leptin, which is thought to participate in the regulation of the appetite of the developing infant. Leptin in HM is derived from a combination of de novo mammary synthesis and transfer from the maternal serum. Moreover, leptin is partially lipophilic and is also present in HM cells. However, leptin has predominately been measured in skim HM, which contains neither fat nor cells. We optimised an enzyme-linked immunosorbent assay for leptin measurement in both whole and skim HM and compared leptin levels between both HM preparations collected from 61 lactating mothers. Whole HM leptin ranged from 0.2 to 1.47 ng/mL, whilst skim HM leptin ranged from 0.19 to 0.9 ng/mL. Whole HM contained, on average, 0.24 ± 0.01 ng/mL more leptin than skim HM (p < 0.0001, n = 287). No association was found between whole HM leptin and fat content (p = 0.17, n = 287), supporting a cellular contribution to HM leptin. No difference was found between pre- and post-feed samples (whole HM: p = 0.29, skim HM: p = 0.89). These findings highlight the importance of optimising HM leptin measurement and assaying it in whole HM to accurately examine the amount of leptin received by the infant during breastfeeding.

  17. Tracking and quantifying polymer therapeutic distribution on a cellular level using 3D dSTORM

    PubMed Central

    Hartley, Jonathan M.; Zhang, Rui; Gudheti, Manasa; Yang, Jiyuan; Kopeček, Jindřich

    2016-01-01

    We used a single-molecule localization technique called direct stochastic optical reconstruction microscopy (dSTORM) to quantify both colocalization and spatial distribution on a cellular level for two conceptually different N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates. Microscopy images were acquired of entire cells with resolutions as high as 25 nm revealing the nanoscale distribution of the fluorescently labeled therapeutic components. Drug-free macromolecular therapeutics consisting of two self-assembling nanoconjugates showed slight increase in nanoclusters on the cell surface with time. Additionally, dSTORM provided high resolution images of the nanoscale organization of the self-assembling conjugates at the interface between two cells. A conjugate designed for treating ovarian cancer showed that the model drug (Cy3) and polymer bound to Cy5 were colocalized at an early time point before the model drug was enzymatically cleaved from the polymer. Using spatial descriptive statistics it was found that the drug was randomly distributed after 24 h while the polymer bound dye remained in clusters. Four different fluorescent dyes were used and two different therapeutic systems were tested to demonstrate the versatility and possible general applicability of dSTORM for use in studying drug delivery systems. PMID:26855050

  18. Distribution of elements in individual blood cells in metabolic disorders at the cellular level

    NASA Astrophysics Data System (ADS)

    Johansson, Erland; Lindh, Ulf

    1985-08-01

    In comparison with controls neutrophil granulocytes from Rheumatoid arthritis (RA), Infantile Neuronal Ceroid Lipofuscinosis (INCL), Chronic Lymphatic Leukemia (L) and Aplastic Anemia (AA) displayed significant alterations in essential and non-essential elements which might be interpreted as fingerprints of these deseases. The neutrophils from RA patients displayed alterations in the concentrations of iron, calcium, strontium, manganese, zinc and copper. INCL displayed alterations in the concentrations of iron and copper but in the INCL disease the iron concentration was about 2 times higher than in RA. In leukemia, aluminium was observed but not in the controls (< 0.5 μg/ g). The zinc concentration was lowered in leukemia. Aplastic anemia displayed alterations in zirconium, arsenic, molybdenum, iron and zinc. The platelets from RA, INCL, L and AA patients also displayed alterations in the elemental profiles. The platelets from AA patients displayed a unique elemental distribution of arsenic, zirconium and molybdenum. The elemental profiles of the thrombocytes and neutrophils might be used as a complement in the diagnosis of the examined diseases and in therapy the elemental profile might be used to monitor drugs at the cellular level.

  19. A high-precision micropipette sensor for cellular-level real-time thermal characterization.

    PubMed

    Shrestha, Ramesh; Choi, Tae-Youl; Chang, Wonseok; Kim, Donsik

    2011-01-01

    We report herein development of a novel glass micropipette thermal sensor fabricated in a cost-effective manner, which is capable of measuring steady thermal fluctuation at spatial resolution of ∼2 μm with an accuracy of ±0.01 °C. We produced and tested various micrometer-sized sensors, ranging from 2 μm to 30 μm. The sensor comprises unleaded low-melting-point solder alloy (Sn-based) as a core metal inside a pulled borosilicate glass pipette and a thin film of nickel coating outside, creating a thermocouple junction at the tip. The sensor was calibrated using a thermally insulated calibration chamber, the temperature of which can be controlled with an accuracy of ±0.01 °C, and the thermoelectric power (Seebeck coefficient) of the sensor was recorded from 8.46 to 8.86 μV/°C. We have demonstrated the capability of measuring temperatures at a cellular level by inserting our temperature sensor into the membrane of a live retinal pigment epithelium cell subjected to a laser beam with a focal spot of 6 μm. We measured transient temperature profiles and the maximum temperatures were in the range of 38-55 ± 0.5 °C.

  20. Leptin Levels Are Higher in Whole Compared to Skim Human Milk, Supporting a Cellular Contribution

    PubMed Central

    Kugananthan, Sambavi; Lai, Ching Tat; Gridneva, Zoya; Mark, Peter J.; Geddes, Donna T.; Kakulas, Foteini

    2016-01-01

    Human milk (HM) contains a plethora of metabolic hormones, including leptin, which is thought to participate in the regulation of the appetite of the developing infant. Leptin in HM is derived from a combination of de novo mammary synthesis and transfer from the maternal serum. Moreover, leptin is partially lipophilic and is also present in HM cells. However, leptin has predominately been measured in skim HM, which contains neither fat nor cells. We optimised an enzyme-linked immunosorbent assay for leptin measurement in both whole and skim HM and compared leptin levels between both HM preparations collected from 61 lactating mothers. Whole HM leptin ranged from 0.2 to 1.47 ng/mL, whilst skim HM leptin ranged from 0.19 to 0.9 ng/mL. Whole HM contained, on average, 0.24 ± 0.01 ng/mL more leptin than skim HM (p < 0.0001, n = 287). No association was found between whole HM leptin and fat content (p = 0.17, n = 287), supporting a cellular contribution to HM leptin. No difference was found between pre- and post-feed samples (whole HM: p = 0.29, skim HM: p = 0.89). These findings highlight the importance of optimising HM leptin measurement and assaying it in whole HM to accurately examine the amount of leptin received by the infant during breastfeeding. PMID:27834797

  1. Interrelationship among muscle, fat, and bone: connecting the dots on cellular, hormonal, and whole body levels.

    PubMed

    Ilich, Jasminka Z; Kelly, Owen J; Inglis, Julia E; Panton, Lynn B; Duque, Gustavo; Ormsbee, Michael J

    2014-05-01

    While sarcopenia and sarcopenic obesity have been recognized in the last decade, a combined concept to include decreased muscle mass and strength, as well as decreased bone mass with coexistence of adiposity is discussed here. We introduce a new term, osteopenic obesity, and operationalize its meaning within the context of osteopenia and obesity. Next, we consolidate osteopenic obesity with the already existing and more familiar term, sarcopenic obesity, and delineate the resulting combined condition assigning it the term osteosarcopenic obesity. Identification and possible diagnosis of each condition are discussed, as well as the interactions of muscle, fat and bone tissues on cellular level, considering their endocrine features. Special emphasis is placed on the mesenchymal stem cell commitment into osteoblastogenic, adipogenic and myogenic lineages and causes of its deregulation. Based on the presented evidence and as expounded within the text, it is reasonable to say that under certain conditions, osteoporosis and sarcopenia could be the obesity of bone and muscle, respectively, with the term osteosarcopenic obesity as an encompassment for all. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. High-resolution CMOS MEA platform to study neurons at subcellular, cellular, and network levels.

    PubMed

    Müller, Jan; Ballini, Marco; Livi, Paolo; Chen, Yihui; Radivojevic, Milos; Shadmani, Amir; Viswam, Vijay; Jones, Ian L; Fiscella, Michele; Diggelmann, Roland; Stettler, Alexander; Frey, Urs; Bakkum, Douglas J; Hierlemann, Andreas

    2015-07-07

    Studies on information processing and learning properties of neuronal networks would benefit from simultaneous and parallel access to the activity of a large fraction of all neurons in such networks. Here, we present a CMOS-based device, capable of simultaneously recording the electrical activity of over a thousand cells in in vitro neuronal networks. The device provides sufficiently high spatiotemporal resolution to enable, at the same time, access to neuronal preparations on subcellular, cellular, and network level. The key feature is a rapidly reconfigurable array of 26 400 microelectrodes arranged at low pitch (17.5 μm) within a large overall sensing area (3.85 × 2.10 mm(2)). An arbitrary subset of the electrodes can be simultaneously connected to 1024 low-noise readout channels as well as 32 stimulation units. Each electrode or electrode subset can be used to electrically stimulate or record the signals of virtually any neuron on the array. We demonstrate the applicability and potential of this device for various different experimental paradigms: large-scale recordings from whole networks of neurons as well as investigations of axonal properties of individual neurons.

  3. In vivo reflectance confocal microscopy detects pigmentary changes in melasma at a cellular level resolution.

    PubMed

    Kang, Hee Young; Bahadoran, Philippe; Suzuki, Itaru; Zugaj, Didier; Khemis, Abdallah; Passeron, Thierry; Andres, Philippe; Ortonne, Jean-Paul

    2010-08-01

    Melasma is a frequent pigmentary disorder caused by abnormal melanin deposits in the skin. In vivo reflectance confocal microscopy (RCM) is a repetitive imaging tool that provides real-time images of the skin at nearly histological resolution. As melanin is the strongest endogenous contrast in human skin, pigmentary disorders are the most suitable candidates for RCM examination but RCM features of melasma have never been reported. This study investigates the pilot use of RCM in melasma to provide a set of well-described morphological criteria with histological correlations. RCM images were acquired from melasma skin and compared to adjacent control skin in 26 patients. Skin biopsies were obtained from eight patients. In the epidermis, RCM showed in all patients a significant increase in hyperrefractile cobblestoning cells. These cells corresponded to hyperpigmented basal keratinocytes in histology. In six patients, dendritic cells corresponding to activated melanocytes were also found in the epidermis. In the dermis, RCM identified in nine patients plump bright cells corresponding to melanophages. Interestingly, for a given patient, the topographic distribution of melanophages in melasma lesions was very heterogeneous. RCM also showed a significant increase in solar elastosis and blood vessels in the dermis. RCM is a non-invasive technique that detects pigmentary changes in melasma at a cellular level resolution. Therefore, RCM provides an innovative way to classify melasma by pigment changes.

  4. A High-Precision Micropipette Sensor for Cellular-Level Real-Time Thermal Characterization

    PubMed Central

    Shrestha, Ramesh; Choi, Tae-Youl; Chang, Wonseok; Kim, Donsik

    2011-01-01

    We report herein development of a novel glass micropipette thermal sensor fabricated in a cost-effective manner, which is capable of measuring steady thermal fluctuation at spatial resolution of ∼2 μm with an accuracy of ±0.01 °C. We produced and tested various micrometer-sized sensors, ranging from 2 μm to 30 μm. The sensor comprises unleaded low-melting-point solder alloy (Sn-based) as a core metal inside a pulled borosilicate glass pipette and a thin film of nickel coating outside, creating a thermocouple junction at the tip. The sensor was calibrated using a thermally insulated calibration chamber, the temperature of which can be controlled with an accuracy of ±0.01 °C, and the thermoelectric power (Seebeck coefficient) of the sensor was recorded from 8.46 to 8.86 μV/°C. We have demonstrated the capability of measuring temperatures at a cellular level by inserting our temperature sensor into the membrane of a live retinal pigment epithelium cell subjected to a laser beam with a focal spot of 6 μm. We measured transient temperature profiles and the maximum temperatures were in the range of 38–55 ± 0.5 °C. PMID:22164108

  5. Cellular levels of feedback regulator of adenylate cyclase and the effect of epinephrine and insulin.

    PubMed Central

    Ho, R j; Russell, T R; Asakawa, T; Sutherland, E W

    1975-01-01

    We have obtained direct evidence that shows the cellular formation and subsequent release of a potent inhibitor (feedback regulator) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] by adipocytes, upon stimulation with epinephrine. The appearance of such a feedback regulator in adipocytes preceded its release into the medium. During a 30 min incubation, intracellular regulator levels rose rapidly and reached 39-61 units/g of adipocyte at 10 min. Release of inhibitor into the medium increased slowly and was 11-16 units/g of adipocyte at 10 min. Upon continued incubation, the cells at 30 min contained 30-41 units/g of ingibitor, slightly less than the content at 30 min; meanwhile, the medium content rose more than 3-fold. The inhibitor from both locations appeared to have the same characteristics, judging from the purification procedures and the biological activities on hormone-stimulated adenylate cyclase. Adenylate cyclase was inhibited by the feedback regulator in vitro when either epinephrine, corticotropin (ACTH), or glucagon was used as activator. The site of action of this inhibitor is therefore most likely beyond the specific hormone receptors. A new in vitro action of insulin has been found. Insulin, 50-500 microunits/ml, inhibited the formation and release of this factor from isolated rat or hamster adipocytes by 29-81% after these cells were stimulated by hormones that raise intracellular adenosine 3':5'-cyclic monophosphate. This factor enhaced the effect of insulin in lowering the adenosine 3':5'-cyclic monophosphate levels in fresh rat adipocytes. A reduced formation of such a factor may modify the metabolic events in adipocytes, and some as yet unexplained effects of insulin could therefore be linked to the metabolic effects of this factor. PMID:174073

  6. SINGLE-CELL LEVEL INVESTIGATION OF CYTOSKELETAL/CELLULAR RESPONSE TO EXTERNAL STIMULI

    SciTech Connect

    Hiddessen, A L

    2007-02-26

    A detailed understanding of the molecular mechanisms by which chemical signals control cell behavior is needed if the complex biological processes of embryogenesis, development, health and disease are to be completely understood. Yet, if we are to fully understand the molecular mechanisms controlling cell behavior, measurements at the single cell level are needed to supplement information gained from population level studies. One of the major challenges to accomplishing studies at the single cell level has been a lack of physical tools to complement the powerful molecular biological assays which have provided much of what we currently know about cell behavior. The goal of this exploratory project is the development of an experimental platform that facilitates integrated observation, tracking and analysis of the responses of many individual cells to controlled environmental factors (e.g. extracellular signals). Toward this goal, we developed chemically-patterned microarrays of both adherent and suspension mammalian cell types. A novel chemical patterning methodology, based on photocatalytic lithography, was developed to construct biomolecule and cell arrays that facilitate analysis of biological function. Our patterning techniques rely on inexpensive stamp materials and visible light, and do not necessitate mass transport or specified substrates. Patterned silicon and glass substrates are modified such that there is a non-biofouling polymer matrix surrounding the adhesive regions that target biomolecules and cells. Fluorescence and reflectance microscopy reveal successful patterning of proteins and single to small clusters of mammalian cells. In vitro assays conducted upon cells on the patterned arrays demonstrate the viability of cells interfacing with this synthetic system. Hence, we have successfully established a versatile cell measurement platform which can be used to characterize the molecular regulators of cellular behavior in a variety of important

  7. Computational Approaches to Analyze and Predict Small Molecule Transport and Distribution at Cellular and Subcellular Levels

    PubMed Central

    Ah Min, Kyoung; Zhang, Xinyuan; Yu, Jing-yu; Rosania, Gus R.

    2013-01-01

    Quantitative structure-activity relationship (QSAR) studies and mechanistic mathematical modeling approaches have been independently employed for analyzing and predicting the transport and distribution of small molecule chemical agents in living organisms. Both of these computational approaches have been useful to interpret experiments measuring the transport properties of small molecule chemical agents, in vitro and in vivo. Nevertheless, mechanistic cell-based pharmacokinetic models have been especially useful to guide the design of experiments probing the molecular pathways underlying small molecule transport phenomena. Unlike QSAR models, mechanistic models can be integrated from microscopic to macroscopic levels, to analyze the spatiotemporal dynamics of small molecule chemical agents from intracellular organelles to whole organs, well beyond the experiments and training data sets upon which the models are based. Based on differential equations, mechanistic models can also be integrated with other differential equations-based systems biology models of biochemical networks or signaling pathways. Although the origin and evolution of mathematical modeling approaches aimed at predicting drug transport and distribution has occurred independently from systems biology, we propose that the incorporation of mechanistic cell-based computational models of drug transport and distribution into a systems biology modeling framework is a logical next-step for the advancement of systems pharmacology research. PMID:24218242

  8. Computational approaches to analyse and predict small molecule transport and distribution at cellular and subcellular levels.

    PubMed

    Min, Kyoung Ah; Zhang, Xinyuan; Yu, Jing-yu; Rosania, Gus R

    2014-01-01

    Quantitative structure-activity relationship (QSAR) studies and mechanistic mathematical modeling approaches have been independently employed for analysing and predicting the transport and distribution of small molecule chemical agents in living organisms. Both of these computational approaches have been useful for interpreting experiments measuring the transport properties of small molecule chemical agents, in vitro and in vivo. Nevertheless, mechanistic cell-based pharmacokinetic models have been especially useful to guide the design of experiments probing the molecular pathways underlying small molecule transport phenomena. Unlike QSAR models, mechanistic models can be integrated from microscopic to macroscopic levels, to analyse the spatiotemporal dynamics of small molecule chemical agents from intracellular organelles to whole organs, well beyond the experiments and training data sets upon which the models are based. Based on differential equations, mechanistic models can also be integrated with other differential equations-based systems biology models of biochemical networks or signaling pathways. Although the origin and evolution of mathematical modeling approaches aimed at predicting drug transport and distribution has occurred independently from systems biology, we propose that the incorporation of mechanistic cell-based computational models of drug transport and distribution into a systems biology modeling framework is a logical next step for the advancement of systems pharmacology research. Copyright © 2013 John Wiley & Sons, Ltd.

  9. Morphine Produces Immunosuppressive Effects in Non-human Primates at the Proteomic and Cellular Levels

    SciTech Connect

    Brown, Joseph N.; Ortiz, Gabriel M.; Angel, Thomas E.; Jacobs, Jon M.; Gritsenko, Marina A.; Chan, Eric Y.; Purdy, David E.; Murnane, Robert D.; Larsen, Kay; Palermo, Robert E.; Shukla, Anil K.; Clauss, Therese RW; Katze, Michael G.; McCune, Joseph M.; Smith, Richard D.

    2012-05-11

    Morphine has long been known to have immunosuppressive properties in vivo, but the molecular and immunologic changes induced by it are incompletely understood. As a prelude to understanding how these changes might interact with lentiviral infection in vivo, animals from two non-human primate (NHP) species [African green monkey (AGMs) and pigtailed macaque (PTs)] were provided morphine and studied using a systems biology approach. Biological specimens were obtained from multiple sources (e.g., lymph node, colon, cerebrospinal fluid (CSF), and peripheral blood) before and after the administration of morphine (titrated up to a maximum dose of 5 mg/kg over a period of 20 days). Cellular immune, plasma cytokine, and proteome changes were measured and morphine-induced changes in these parameters were assessed on an inter-organ, inter-individual, and inter-species basis. In both species, morphine was associated with decreased levels of (Ki-67+) T cell activation but with only minimal changes in overall T cell counts, neutrophil counts, and NK cells counts. While changes in T cell maturation were observed, these varied across the various tissue/fluid compartments studied. Proteomic analysis revealed a morphine-induced suppressive effect in the lymph node, with decreased abundance of protein mediators involved in the functional categories of energy metabolism, signaling, and maintenance of cell structure. These findings have relevance for understanding the impact of heroin addiction and the opioids used to treat addiction as well as on the interplay between opioid abuse and the response to infection with agents such as the human immunodeficiency virus, type 1 (HIV).

  10. Effects of UV and visible light on cyanobacteria at the cellular level.

    PubMed

    Sinha, Rajeshwar P; Richter, Peter; Faddoul, Jaoudat; Braun, Markus; Häder, Donat P

    2002-08-01

    The effect of UV and visible light on cyanobacteria was determined at the cellular level by means of epifluorescence and confocal microscopy techniques. These methods allow the examination of light effects in spatial resolution. Series of measurements were performed to determine the effect of different light qualities and quantities on cyanobacteria. To analyze the effect of the light quality, samples of Anabaena and Scytonema sp. were exposed to intense blue, green or red light applied from the epifluorescence path of the microscope. The fluorescence of the phycobiliproteins was recorded by means of epifluorescence (excitation 550 nm, 20 nm half band width (HBW), emission above 635 nm) or by confocal microscopy (560 nm laser line). Upon exposure to blue or green light the cells showed an increase in fluorescence followed by a sudden and complete loss of fluorescence. Blue light was more effective (bleaching of phycobiliproteins within 45 min) than green light (bleaching within 120 min). Red light was not as effective, and bleaching of the cells took at least 24 h. Initially the cells showed an increase in fluorescence followed by fast bleaching of the fluorescence signal. Cells exposed to UV plus PAR were bleached within 60 min, while cells exposed to photosynthetically active radiation (PAR) only were totally bleached after about 120 min. FL-DHP (dihydropyridine) labeling was performed in two cyanobacteria, Anabaena sp. and Nostoc commune, to visualize L-type calcium channels. Both cyanobacterial strains showed a pronounced FL-DHP signal of the heterocysts and akinetes but only a weak signal from the vegetative cells. The results clearly indicate the presence of calcium channels in these cells. UV radiation decreased the amount of chlorophyll and phycocyanin as could be seen from a decline in the autofluorescence of the cells. In contrast, the FL-DHP signal was not affected by UV.

  11. Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria.

    PubMed

    Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L

    2016-02-01

    The second messenger cyclic dimeric (3'→5') GMP (cyclic di-GMP or c-di-GMP) has been implicated in the transition between motile and sessile lifestyles in bacteria. In this study, we demonstrate that biofilm formation, cellular aggregation or flocculation, and cellular buoyancy are under the control of c-di-GMP in Synechocystis sp. PCC 6803 (Synechocystis) and Fremyella diplosiphon. Synechocystis is a unicellular cyanobacterium and displays lower levels of c-di-GMP; F. diplosiphon is filamentous and displays higher intracellular c-di-GMP levels. We transformed Synechocystis and F. diplosiphon with a plasmid for constitutive expression of genes encoding diguanylate cylase (DGC) and phosphodiesterase (PDE) proteins from Vibrio cholerae or Escherichia coli, respectively. These engineered strains allowed us to modulate intracellular c-di-GMP levels. Biofilm formation and cellular deposition were induced in the DGC-expressing Synechocystis strain which exhibited high intracellular levels of c-di-GMP; whereas strains expressing PDE in Synechocystis and F. diplosiphon to drive low intracellular levels of c-di-GMP exhibited enhanced cellular buoyancy. In addition, the PDE-expressing F. diplosiphon strain showed elevated chlorophyll levels. These results imply roles for coordinating c-di-GMP homeostasis in regulating native cyanobacterial phenotypes. Engineering exogenous DGC or PDE proteins to regulate intracellular c-di-GMP levels represents an effective tool for uncovering cryptic phenotypes or modulating phenotypes in cyanobacteria for practical applications in biotechnology applicable in photobioreactors and in green biotechnologies, such as energy-efficient harvesting of cellular biomass or the treatment of metal-containing wastewaters.

  12. Elevation of cellular NAD levels by nicotinic acid and involvement of nicotinic acid phosphoribosyltransferase in human cells.

    PubMed

    Hara, Nobumasa; Yamada, Kazuo; Shibata, Tomoko; Osago, Harumi; Hashimoto, Tatsuya; Tsuchiya, Mikako

    2007-08-24

    NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels. The enzyme was abundantly expressed in the small intestine, liver, and kidney in mice and mediated [(14)C]NAD synthesis from [(14)C]NA in human cells. In cells expressing endogenous NAPRT, the addition of NA but not Nam almost doubled cellular NAD contents and decreased cytotoxicity by H(2)O(2). Both effects were reversed by knockdown of NAPRT expression. These results indicate that NAPRT is essential for NA to increase cellular NAD levels and, thus, to prevent oxidative stress of the cells. Kinetic analyses revealed that NAPRT, but not Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor or visfatin), is insensitive to the physiological concentration of NAD. Together, we conclude that NA elevates cellular NAD levels through NAPRT function and, thus, protects the cells against stress, partly due to lack of feedback inhibition of NAPRT but not NamPRT by NAD. The ability of NA to increase cellular NAD contents may account for some of the clinically observed effects of the vitamin and further implies a novel application of the vitamin to treat diseases such as those associated with the depletion of cellular NAD pools.

  13. Monitoring Astronaut Health at the Nanoscale Cellular Level Through the Eye

    NASA Technical Reports Server (NTRS)

    Ansari, Rafat R.; Singh, Bhim S.; Rovati, Luigi; Docchio, Franco; Sebag, Jerry

    2000-01-01

    A user friendly goggles-like head-mounted device equipped with a suite of instruments for several non-invasive and quantitative medical evaluation of the eye, skin, and brain is desired for monitoring the health of astronauts during space travel and exploration of neighboring and distant planets. Real-time non-invasive evaluation of the different structures within the above organs can provide indices of the health of not just these organs, but the entire body. The techniques such as dynamic light scattering (for the early detection of uveitis, cholesterol levels, cataract, changes in the vitreous and possibly Alzheimer's disease), corneal autofluorescence (to assess extracellular matrix biology e.g., in diabetes), optical activity measurements (of anterior ocular fluid to evaluate blood-glucose levels), laser Doppler velocimetry (to assess retinal, optic nerve, and choroidal blood flow), reflectometry/oximetry (for assessing ocular and central nervous system oxygen metabolism), optical coherence tomography (to determine retinal tissue microstructure) and possibly scanning laser technology (for intraocular tissue imaging and scanning) will he integrated into this compact device. Skin sensors will also be mounted on the portion of the device in contact with the periocular region. This will enable monitoring of body temperature, EEG, and electrolyte status. This device will monitor astronaut health during long-duration space travel by detecting aberrations from pre-established "nonns", enabling prompt diagnosis and possibly the initiation of early preventative/curative therapy. The non-invasive nature of the device technologies permits frequent repetition of tests, enabling real-time complete crew health monitoring. This device may ultimately be useful in tele-medicine to bring modern healthcare to under-served areas on Earth as well as in so-called "advanced" care settings (e.g. diabetes in the USA).

  14. Cellular Levels of Signaling Factors Are Sensed by β-actin Alleles to Modulate Transcriptional Pulse Intensity.

    PubMed

    Kalo, Alon; Kanter, Itamar; Shraga, Amit; Sheinberger, Jonathan; Tzemach, Hadar; Kinor, Noa; Singer, Robert H; Lionnet, Timothée; Shav-Tal, Yaron

    2015-04-21

    The transcriptional response of β-actin to extra-cellular stimuli is a paradigm for transcription factor complex assembly and regulation. Serum induction leads to a precisely timed pulse of β-actin transcription in the cell population. Actin protein is proposed to be involved in this response, but it is not known whether cellular actin levels affect nuclear β-actin transcription. We perturbed the levels of key signaling factors and examined the effect on the induced transcriptional pulse by following endogenous β-actin alleles in single living cells. Lowering serum response factor (SRF) protein levels leads to loss of pulse integrity, whereas reducing actin protein levels reveals positive feedback regulation, resulting in elevated gene activation and a prolonged transcriptional response. Thus, transcriptional pulse fidelity requires regulated amounts of signaling proteins, and perturbations in factor levels eliminate the physiological response, resulting in either tuning down or exaggeration of the transcriptional pulse.

  15. A Viral Protein Mediates Superinfection Exclusion at the Whole-Organism Level but Is Not Required for Exclusion at the Cellular Level

    PubMed Central

    Bergua, María; Zwart, Mark P.; El-Mohtar, Choaa; Shilts, Turksen; Elena, Santiago F.

    2014-01-01

    ABSTRACT Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels—the cellular and the whole-organism levels—by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host

  16. Neighborhood disorder and telomeres: connecting children's exposure to community level stress and cellular response.

    PubMed

    Theall, Katherine P; Brett, Zoë H; Shirtcliff, Elizabeth A; Dunn, Erin C; Drury, Stacy S

    2013-05-01

    Our objective was to explore the utility of salivary telomere length (sTL) as an early indicator of neighborhood-level social environmental risk during child development. We therefore tested the hypothesis that sTL would be associated with markers of social stress exposure in children. Children age 4-14 from 87 neighborhoods were recruited through five urban schools in New Orleans, Louisiana, U.S. Data were collected at the level of the child, family/household, and neighborhood. DNA was obtained from saliva using commercially available kits and sTL was determined for 104 children using quantitative PCR. Analysis was performed on 99 children who had complete data including sTL, social environmental stress, and additional covariates. The mean sTL value was 7.4 T/S (telomere signal/single-copy signal) ratio units (±2.4, range = 2.5-18.0), and 4.7% of the variance in sTL was attributed to differences across neighborhoods. Children living in neighborhoods characterized by high disorder had an sTL value 3.2 units lower than children not living in high disordered environments (p < 0.05) and their odds of having low relative sTL (defined as <1 standard deviation below standardized Z-score mean) values was 3.43 times that of children not living in high disorder environments (adjusted OR = 3.43, 95% CI = 1.22, 9.62). Our findings are consistent with previous studies in adults demonstrating a strong link between psychosocial stress and sTL obtained from peripheral blood, consistent with previous studies in youth demonstrating an association between early life stress and sTL obtained from buccal cell DNA and offer increased support for the hypothesis that sTL represents a non-invasive biological indicator of psychosocial stress exposure (i.e., neighborhood disorder) able to reflect differences in stress exposure levels even in young children.

  17. Dissecting Analogical Leveling Quantitatively: The Case of the Innovative Potential Suffix in Tokyo Japanese.

    ERIC Educational Resources Information Center

    Matsuda, Kenjiro

    1993-01-01

    Analogical leveling in progress of a potential suffix in Tokyo Japanese is analyzed within a quantitative model. The phenomenon is shown to be controlled by five factors: sociological variable complex, verb stem length, verb conjugation pattern, the following inflectional form, and embeddedness of the clause containing the suffix. (Contains 70…

  18. A Quantitative Study of Cohesion in Chinese Graduate Students' Writing: Variations across Genres and Proficiency Levels.

    ERIC Educational Resources Information Center

    Jin, Wenjun

    This study presents a quantitative analysis of cohesion of the academic writing of Chinese English-as-a-Second-Language (ESL) graduate students by applying Halliday and Hansen's (1976) model. Six Chinese graduate students from a Midwestern university were selected for the study, representing two proficiency levels in written English--advanced and…

  19. Corequisite Model: An Effective Strategy for Remediation in Freshmen Level Quantitative Reasoning Course

    ERIC Educational Resources Information Center

    Kashyap, Upasana; Mathew, Santhosh

    2017-01-01

    The purpose of this study was to compare students' performances in a freshmen level quantitative reasoning course (QR) under three different instructional models. A cohort of 155 freshmen students was placed in one of the three models: needing a prerequisite course, corequisite (students enroll simultaneously in QR course and a course that…

  20. Transmural heterogeneity of cellular level power output is reduced in human heart failure

    PubMed Central

    Haynes, Premi; Nava, Kristofer E.; Lawson, Benjamin A.; Chung, Charles S.; Mitov, Mihail I.; Campbell, Stuart G.; Stromberg, Arnold J.; Sadayappan, Sakthivel; Bonnell, Mark R.; Hoopes, Charles W.; Campbell, Kenneth S.

    2014-01-01

    Heart failure is associated with pump dysfunction and remodeling but it is not yet known if the condition affects different transmural regions of the heart in the same way. We tested the hypotheses that the left ventricles of non-failing human hearts exhibit transmural heterogeneity of cellular level contractile properties, and that heart failure produces transmural region-specific changes in contractile function. Permeabilized samples were prepared from the sub-epicardial, mid-myocardial, and sub-endocardial regions of the left ventricular free wall of non-failing (n=6) and failing (n=10) human hearts. Power, an in vitro index of systolic function, was higher in non-failing mid-myocardial samples (0.59±0.06 μW mg−1) than in samples from the sub-epicardium (p=0.021) and the sub-endocardium (p=0.015). Non-failing mid-myocardial samples also produced more isometric force (14.3±1.33 kN m−2) than samples from the sub-epicardium (p=0.008) and the sub-endocardium (p=0.026). Heart failure reduced power (p=0.009) and force (p=0.042) but affected the mid-myocardium more than the other transmural regions. Fibrosis increased with heart failure (p=0.021) and mid-myocardial tissue from failing hearts contained more collagen than matched sub-epicardial (p<0.001) and sub-endocardial (p=0.043) samples. Power output was correlated with the relative content of actin and troponin I, and was also statistically linked to the relative content and phosphorylation of desmin and myosin light chain- 1. Non-failing human hearts exhibit transmural heterogeneity of contractile properties. In failing organs, region-specific fibrosis produces the greatest contractile deficits in the mid-myocardium. Targeting fibrosis and sarcomeric proteins in the mid-myocardium may be particularly effective therapies for heart failure. PMID:24560668

  1. How do tissues respond to damage at the cellular level? The role of cytokines in irradiated tissues

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1998-01-01

    The capacity of ionizing radiation to affect tissue function, control tumor growth and elicit pathological sequelae has been attributed in great part to its effects on cellular DNA, which, as the transmitter of genetic information, can both register damage and perpetuate it. Nonetheless, multicellular organisms function as the result of the cooperation of many cell types. What then occurs when individual cells are damaged by ionizing radiation? Is tissue response a sum of cellular effects such as cell death and DNA damage? Or does the tissue respond as a coherent unit to the damage of its parts? In this paper, data in support of the latter model that indicate a role for cytokines, in particular transforming growth factor beta1, as critical components of extracellular signaling pathways that mediate tissue response to radiation will be reviewed. The key to manipulating the consequences of radiation exposure lies in understanding the complex interplay of events initiated at the cellular level, but acting on the tissue.

  2. How do tissues respond to damage at the cellular level? The role of cytokines in irradiated tissues

    NASA Technical Reports Server (NTRS)

    Barcellos-Hoff, M. H.; Chatterjee, A. (Principal Investigator)

    1998-01-01

    The capacity of ionizing radiation to affect tissue function, control tumor growth and elicit pathological sequelae has been attributed in great part to its effects on cellular DNA, which, as the transmitter of genetic information, can both register damage and perpetuate it. Nonetheless, multicellular organisms function as the result of the cooperation of many cell types. What then occurs when individual cells are damaged by ionizing radiation? Is tissue response a sum of cellular effects such as cell death and DNA damage? Or does the tissue respond as a coherent unit to the damage of its parts? In this paper, data in support of the latter model that indicate a role for cytokines, in particular transforming growth factor beta1, as critical components of extracellular signaling pathways that mediate tissue response to radiation will be reviewed. The key to manipulating the consequences of radiation exposure lies in understanding the complex interplay of events initiated at the cellular level, but acting on the tissue.

  3. Complex I Disorders: Causes, Mechanisms, and Development of Treatment Strategies at the Cellular Level

    ERIC Educational Resources Information Center

    Valsecchi, Federica; Koopman, Werner J. H.; Manjeri, Ganesh R.; Rodenburg, Richard J.; Smeitink, Jan A. M.; Willems, Peter H. G. M.

    2010-01-01

    Mitochondrial oxidative phosphorylation (OXPHOS) represents the final step in the conversion of nutrients into cellular energy. Genetic defects in the OXPHOS system have an incidence between 1:5,000 and 1:10,000 live births. Inherited isolated deficiency of the first complex (CI) of this system, a multisubunit assembly of 45 different proteins,…

  4. Complex I Disorders: Causes, Mechanisms, and Development of Treatment Strategies at the Cellular Level

    ERIC Educational Resources Information Center

    Valsecchi, Federica; Koopman, Werner J. H.; Manjeri, Ganesh R.; Rodenburg, Richard J.; Smeitink, Jan A. M.; Willems, Peter H. G. M.

    2010-01-01

    Mitochondrial oxidative phosphorylation (OXPHOS) represents the final step in the conversion of nutrients into cellular energy. Genetic defects in the OXPHOS system have an incidence between 1:5,000 and 1:10,000 live births. Inherited isolated deficiency of the first complex (CI) of this system, a multisubunit assembly of 45 different proteins,…

  5. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay.

    PubMed

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3-4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

    SciTech Connect

    Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil; Kim, Dong-Myung; Yoo, Tae Hyeon; Kim, Yong-Sung

    2015-11-27

    Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

  7. Using Nano-mechanics and Surface Acoustic Wave (SAW) for Disease Monitoring and Diagnostics at a Cellular Level in Red Blood Cells

    NASA Astrophysics Data System (ADS)

    Sivanantha, Ninnuja; Ma, Charles; Collins, David J.; Sesen, Muhsincan; Brenker, Jason; Coppel, Ross L.; Neild, Adrian; Alan, Tuncay

    A popular approach to monitoring diseases and their diagnosis is through biological, pathological or immunological characterization. However, at a cellular level progression of certain diseases manifests itself through mechanical effects as well. Here, we present a method which exploits localised flow; surface acoustic wave (SAW) induced acoustic streaming in a 9 μL droplet to characterize the adhesive properties of red blood cells (healthy, gluteraldehyde treated and malaria infected) in approximately 50 seconds. Our results show a 79% difference in cell mobilization between healthy malaria infected RBCs (and a 39% difference between healthy and treated ones), indicating that the method can serve as a platform for rapid clinical diagnosis; where separation of two or more different cell populations in a mixed solution is desirable. It can also act as a key biomarker for monitoring some diseases offering quantitative measures of disease progression and response to therapy.

  8. Baseline Cellular HIV DNA Load Predicts HIV DNA Decline and Residual HIV Plasma Levels during Effective Antiretroviral Therapy

    PubMed Central

    Andreis, Samantha; Mengoli, Carlo; Scaggiante, Renzo; Ferretto, Roberto; Manfrin, Vinicio; Cruciani, Mario; Giobbia, Mario; Boldrin, Caterina; Basso, Monica; Andreoni, Massimo; Palù, Giorgio; Sarmati, Loredana

    2012-01-01

    Cellular human immunodeficiency virus type 1 (HIV-1) DNA may be considered a marker of disease progression with significant predictive power, but published data on its correlation with plasma HIV RNA levels and CD4 counts in acute and chronic patients are not conclusive. We evaluated a cohort of 180 patients naïve for antiretroviral therapy before the beginning of treatment and after a virological response in order to define the indicators correlated with HIV DNA load decrease until undetectability. The following variables were evaluated as continuous variables: age, CD4 cell count and log10 HIV DNA level at baseline and follow-up, and baseline log10 HIV RNA level. Primary HIV infection at the start of therapy, an HIV RNA level at follow-up of <2.5 copies/ml, origin, gender, and transmission risk were evaluated as binary variables. The decline of HIV DNA values during effective therapy was directly related to baseline HIV DNA and HIV RNA values, to an increase in the number of CD4 cells, and to the achievement of an HIV RNA load of <2.5 copies/ml. An undetectable cellular HIV DNA load was achieved by 21.6% of patients at the follow-up time point and correlated significantly with lower baseline cellular HIV DNA values and with being in the primary stage of infection when therapy started. In conclusion, early treatment facilitated the achievement of undetectable levels of plasma viremia and cellular HIV DNA and a better recovery of CD4 lymphocytes. HIV DNA levels before and during highly active antiretroviral therapy may be used as a new tool for monitoring treatment efficacy. PMID:22135262

  9. "Pumping iron"-how macrophages handle iron at the systemic, microenvironmental, and cellular levels.

    PubMed

    Nairz, Manfred; Theurl, Igor; Swirski, Filip K; Weiss, Guenter

    2017-04-01

    Macrophages reside in virtually every organ. First arising during embryogenesis, macrophages replenish themselves in the adult through a combination of self-renewal and influx of bone marrow-derived monocytes. As large phagocytic cells, macrophages participate in innate immunity while contributing to tissue-specific homeostatic functions. Among the key metabolic tasks are senescent red blood cell recycling, free heme detoxification, and provision of iron for de novo hemoglobin synthesis. While this systemic mechanism involves the shuttling of iron between spleen, liver, and bone marrow through the concerted function of defined macrophage populations, similar circuits appear to exist within the microenvironment of other organs. The high turnover of iron is the prerequisite for continuous erythropoiesis and tissue integrity but challenges macrophages' ability to maintain cellular iron homeostasis and immune function.This review provides a brief overview of systemic, microenvironmental, and cellular aspects of macrophage iron handling with a focus on exciting and unresolved questions in the field.

  10. Fusion of mitochondria in tobacco suspension cultured cells is dependent on the cellular ATP level but not on actin polymerization.

    PubMed

    Wakamatsu, Kairo; Fujimoto, Masaru; Nakazono, Mikio; Arimura, Shin-ichi; Tsutsumi, Nobuhiro

    2010-10-01

    Mitochondria in plant cells undergo fusion and fission frequently. Although the mechanisms and proteins of mitochondrial fusion are well known in yeast and mammalian cells, they remain poorly understood in plant cells. To clarify the physiological requirements for plant mitochondrial fusion, we investigated the fusion frequency of mitochondria in tobacco cultured cells using the photoconvertible fluorescent protein Kaede and some physiological inhibitors. The latter included two uncouplers, 2,4-dinitrophenol (DNP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP), an inhibitor of mitochondrial ATP synthase, oligomycin, and an actin polymerization inhibitor, latrunculin B (Lat B). The frequency of mitochondrial fusion was clearly reduced by DNP, CCCP and oligomycin, but not by Lat B, although Lat B severely inhibited mitochondrial movement. Moreover, DNP, CCCP and oligomycin evidently lowered the cellular ATP levels. These results indicate that plant mitochondrial fusion depends on the cellular ATP level, but not on actin polymerization.

  11. BmNHR96 participate BV entry of BmN-SWU1 cells via affecting the cellular cholesterol level.

    PubMed

    Dong, Xiao-Long; Liu, Tai-Hang; Wang, Wei; Pan, Cai-Xia; Du, Guo-Yu; Wu, Yun-Fei; Pan, Min-Hui; Lu, Cheng

    2017-01-22

    B.mori nucleopolyhedrovirus (BmNPV), which produces BV and ODV two virion phenotypes in its life cycle, caused the amount of economic loss in sericulture. But the mechanism of its infection was still unclear. In this study we characterized B.mori nuclear hormone receptor 96 (BmNHR96) as a NHR96 family member, which was localized in the nucleus. We also found BmNHR96 over-expression could enhance the entry of BV as well as cellular cholesterol level. Furthermore, we validated that BmNHR96 increased membrane fusion mediated by GP64, which could probably promote BV-infection. In summary, our study suggested that BmNHR96 plays an important role in BV infection and this function probably actualized by affecting cellular cholesterol level, and our results provided insights to the mechanisms of BV-infection of B.mori.

  12. Aging and the Immune Response to Tetanus Toxoid: Diminished Frequency and Level of Cellular Immune Reactivity to Antigenic Stimulation

    PubMed Central

    Schatz, Desmond; Ellis, Tamir; Ottendorfer, Eric; Jodoin, Eric; Barrett, Douglas; Atkinson, Mark

    1998-01-01

    The period of efficacious immune reactivity afforded by tetanus immunization and the need for continuing some forms of tetanus vaccination programs have been the subjects of recent debates. Our studies demonstrate that the level of antitetanus immunity based on immunological memory (i.e., cellular immune responsiveness) varies dramatically as a function of age, with older individuals constituting a population which is increasingly susceptible to tetanus infection. PMID:9801353

  13. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation.

    PubMed

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2.

  14. MicroRNAs Regulate Cellular ATP Levels by Targeting Mitochondrial Energy Metabolism Genes during C2C12 Myoblast Differentiation

    PubMed Central

    Siengdee, Puntita; Trakooljul, Nares; Murani, Eduard; Schwerin, Manfred; Wimmers, Klaus; Ponsuksili, Siriluck

    2015-01-01

    In our previous study, we identified an miRNA regulatory network involved in energy metabolism in porcine muscle. To better understand the involvement of miRNAs in cellular ATP production and energy metabolism, here we used C2C12 myoblasts, in which ATP levels increase during differentiation, to identify miRNAs modulating these processes. ATP level, miRNA and mRNA microarray expression profiles during C2C12 differentiation into myotubes were assessed. The results suggest 14 miRNAs (miR-423-3p, miR-17, miR-130b, miR-301a/b, miR-345, miR-15a, miR-16a, miR-128, miR-615, miR-1968, miR-1a/b, and miR-194) as cellular ATP regulators targeting genes involved in mitochondrial energy metabolism (Cox4i2, Cox6a2, Ndufb7, Ndufs4, Ndufs5, and Ndufv1) during C2C12 differentiation. Among these, miR-423-3p showed a high inverse correlation with increasing ATP levels. Besides having implications in promoting cell growth and cell cycle progression, its function in cellular ATP regulation is yet unknown. Therefore, miR-423-3p was selected and validated for the function together with its potential target, Cox6a2. Overexpression of miR-423-3p in C2C12 myogenic differentiation lead to decreased cellular ATP level and decreased expression of Cox6a2 compared to the negative control. These results suggest miR-423-3p as a novel regulator of ATP/energy metabolism by targeting Cox6a2. PMID:26010876

  15. Supraphysiological androgen levels induce cellular senescence in human prostate cancer cells through the Src-Akt pathway.

    PubMed

    Roediger, Julia; Hessenkemper, Wiebke; Bartsch, Sophie; Manvelyan, Marina; Huettner, Soeren S; Liehr, Thomas; Esmaeili, Mohsen; Foller, Susan; Petersen, Iver; Grimm, Marc-Oliver; Baniahmad, Aria

    2014-09-12

    Prostate cancer (PCa) is the second leading cause of cancer mortality of men in Western countries. The androgen receptor (AR) and AR-agonists (androgens) are required for the development and progression of the normal prostate as well as PCa. However, it is discussed that in addition to their tumor promoting activity, androgens may also exhibit tumor suppressive effects. A biphasic growth response to androgens a growth-promoting and -inhibition has been observed that suggests that administration of supraphysiological androgen levels mediates growth reduction in AR expressing PCa cells. Detection of senescence markers, three dimensional interphase fluorescence in situ hybridization (3D-iFISH), qRT-PCR, Western blotting, detection of GFP fusions, prostatectomy, ex vivo culturing. Here, we describe that supraphysiological levels of androgens induce cell cycle arrest and markers of cellular senescence in human PCa cells, which may in part explain the growth inhibitory role of androgens. The expression of the senescence associated beta galactosidase is observed by treatment with the natural androgen DHT or the less metabolized synthetic androgen R1881. The induction of senescence marker was detected in human PCa cell lines as well as in human primary PCa tissue derived from prostatectomy treated ex vivo. Using interphase FISH (iFISH) suggests that the androgen-induced cellular senescence is associated with localizing the genomic E2F1 locus to senescence associated heterochromatic foci. Analysis of different signaling pathways in LNCaP cells suggest that the p16-Rb-E2F1 pathway is essential for the induction of cellular senescence since treatment with siRNA directed against p16 reduces the level of androgen-induced cellular senescence. Based on the rapid induction of androgen-mediated cellular senescence we identified the Src-PI3K-Akt-signaling pathway and autophagy being in part involved in androgen regulation. Taken together, our data suggest that AR-agonists at

  16. Cold Spring Harbor symposia on quantitative biology: Volume 49, Recombination at the DNA level

    SciTech Connect

    Not Available

    1984-01-01

    This volume contains full papers prepared by the participants to the 1984 Cold Springs Harbor Symposia on Quantitative Biology. This year's theme is entitled Recombination at the DNA level. The volume consists of 93 articles grouped into subject areas entitled chromosome mechanics, yeast systems, mammalian homologous recombination, transposons, mu, plant transposons/T4 recombination, topoisomerase, resolvase and gyrase, Escherichia coli general recombination, RecA, repair, leukaryotic enzymes, integration and excision of bacteriophage, site-specific recombination, and recombination in vitro.

  17. Might carnitine status in animals indicate environmental/toxicological harm at the cellular level?

    SciTech Connect

    Garst, J.E.

    1995-12-01

    It is well known that R-(L)-carnitine (Cn) is essential for the energy-producing, mitochondrial beta-oxidation of long chain fatty acids. Cn can ameliorate the diverse effects of drugs, a chemicals and pollutants. Moreover, the toxicities of carbon monoxide, several heavy metals, and even the antibiotic cephaloridine seem mediated, in part, by actions affecting the Cn system. Data which could suggest that the Cn system is an integrator/regulator of the cellular response by the organism to it`s environment is described.

  18. Optical biosensors for probing at the cellular level: a review of recent progress and future prospects.

    PubMed

    Velasco-Garcia, M N

    2009-02-01

    New approaches are required to understand the complex processes taking place in the smallest unit of life. Recent years have seen an increasing activity in the use of optical devices and techniques for the investigation of the properties of single cells and also populations of cells including cell to cell communication. This article reviews relevant optical technologies to date as well as new advances in biosensor development, and goes on to explore reported applications in detection of biotargets and cellular signalling pathways inside individual living cells.

  19. Technosphere insulin: defining the role of Technosphere particles at the cellular level.

    PubMed

    Angelo, Robert; Rousseau, Kathleen; Grant, Marshall; Leone-Bay, Andrea; Richardson, Peter

    2009-05-01

    Technosphere Insulin (TI) is a novel inhalation powder for the treatment of diabetes mellitus. Technosphere Insulin delivers insulin with an ultra rapid pharmacokinetic profile that is distinctly different from all other insulin products but similar to natural insulin release. Such rapid absorption is often associated with penetration enhancers that disrupt cellular integrity. Technosphere Insulin was compared to a panel of known penetration enhancers in vitro using the Calu-3 lung cell line to investigate the effects of TI on insulin transport. Measures of tight junction integrity such as transepithelial electrical resistance, Lucifer yellow permeability, and F-actin staining patterns were all unaffected by TI. Cell viability and plasma membrane integrity were also not affected by TI. In contrast, cells treated with comparable (or lower) concentrations of penetration enhancers showed elevated Lucifer yellow permeability, disruption of the F-actin network, reduced cell viability, and compromised plasma membranes. These results demonstrate that TI is not cytotoxic in an in vitro human lung cell model and does not function as a penetration enhancer. Furthermore, TI does not appear to affect the transport of insulin across cellular barriers. 2009 Diabetes Technology Society.

  20. Lipidomics: a mass spectrometry based, systems level analysis of cellular lipids

    PubMed Central

    Ivanova, Pavlina T.; Milne, Stephen B.; Myers, David S.; Brown, H. Alex

    2009-01-01

    Lipidomics is a logical outcome of the history and traditions of lipid biochemistry and advances in mass spectrometry are at the heart of a renaissance in understanding the roles of lipids in cellular functions. Our desire to understand the complexity of lipids in biology has led to new techniques that allow us to identify over 1000 phospholipids in mammalian cell types and tissues. Improvements in chromatographic separation and mass spectrometry have positioned us to determine not only the lipid composition (i.e., parts list) of cells and tissues, but also address questions regarding lipid substrates and products that previously overwhelmed traditional analytical technologies. In the decade since lipidomics was conceived much of the efforts have been on new methodologies, development of computer programs to decipher the gigabytes of raw data, and struggling with the highly variable nature of biological systems where absolute quantities of a given metabolite may be less important than its relative change in concentration. It is clear that the technology is now sufficiently developed to address fundamental questions about the roles of lipids in cellular signaling and metabolic pathways. PMID:19744877

  1. 5-aminolevulinic acid for quantitative seek-and-treat of high-grade dysplasia in Barrett's esophagus cellular models.

    PubMed

    Yeh, Shu-Chi Allison; Ling, Celine S N; Andrews, David W; Patterson, Michael S; Diamond, Kevin R; Hayward, Joseph E; Armstrong, David; Fang, Qiyin

    2015-02-01

    High-grade dysplasia (HGD) in Barrett’s esophagus (BE) poses increased risk for developing esophageal adenocarcinoma. To date, early detection and treatment of HGD regions are still challenging due to the sampling error from tissue biopsy and relocation error during the treatment after histopathological analysis. In this study, CP-A (metaplasia) and CP-B (HGD) cell lines were used to investigate the “seek-and-treat” potential using 5-aminolevulinic acid-induced protoporphyrin IX (PpIX). The photodynamic therapy photosensitizer then provides both a phototoxic effect and additional image contrast for automatic detection and real-time laser treatment. Complementary to our studies on automatic classification, this work focused on characterizing subcellular irradiation and the potential phototoxicity on both metaplasia and HGD. The treatment results showed that the HGD cells are less viable than metaplastic cells due to more PpIX production at earlier times. Also, due to mitochondrial localization of PpIX, a better killing effect was achieved by involving mitochondria or whole cells compared with just nucleus irradiation in the detected region. With the additional toxicity given by PpIX and potential morphological/textural differences for pattern recognition, this cellular platform serves as a platform to further investigate real-time “seek-and-treat” strategies in three-dimensional models for improving early detection and treatment of BE. © 2015 Society of Photo-Optical Instrumentation Engineers (SPIE)

  2. 5-aminolevulinic acid for quantitative seek-and-treat of high-grade dysplasia in Barrett's esophagus cellular models

    NASA Astrophysics Data System (ADS)

    Yeh, Shu-Chi Allison; Ling, Celine S. N.; Andrews, David W.; Patterson, Michael S.; Diamond, Kevin R.; Hayward, Joseph E.; Armstrong, David; Fang, Qiyin

    2015-02-01

    High-grade dysplasia (HGD) in Barrett's esophagus (BE) poses increased risk for developing esophageal adenocarcinoma. To date, early detection and treatment of HGD regions are still challenging due to the sampling error from tissue biopsy and relocation error during the treatment after histopathological analysis. In this study, CP-A (metaplasia) and CP-B (HGD) cell lines were used to investigate the "seek-and-treat" potential using 5-aminolevulinic acid-induced protoporphyrin IX (PpIX). The photodynamic therapy photosensitizer then provides both a phototoxic effect and additional image contrast for automatic detection and real-time laser treatment. Complementary to our studies on automatic classification, this work focused on characterizing subcellular irradiation and the potential phototoxicity on both metaplasia and HGD. The treatment results showed that the HGD cells are less viable than metaplastic cells due to more PpIX production at earlier times. Also, due to mitochondrial localization of PpIX, a better killing effect was achieved by involving mitochondria or whole cells compared with just nucleus irradiation in the detected region. With the additional toxicity given by PpIX and potential morphological/textural differences for pattern recognition, this cellular platform serves as a platform to further investigate real-time "seek-and-treat" strategies in three-dimensional models for improving early detection and treatment of BE.

  3. Effects of Water Loss on New Mexico Spadefoot Toad (Spea multiplicata) Development, Spleen Cellularity, and Corticosterone Levels.

    PubMed

    Bagwill, April L; Lovern, Matthew B; Worthington, Thomas A; Smith, Loren M; McMurry, Scott T

    2016-10-01

    Amphibian metamorphosis is complex and larval morphology and physiology are completely restructured during this time. Amphibians that live in unpredictable environments are often exposed to stressors that can directly and indirectly alter physiological systems during development, with subsequent consequences (carryover effects) later in life. In this study, we investigated the effects of water level reduction on development rate, spleen size and cellularity, and examined the role of corticosterone levels in premetamorphic, metamorphic, and postmetamorphic New Mexico spadefoot toads (Spea multiplicata). Based on previous studies, we hypothesized that declining water level would increase tadpole developmental rate, but with the trade-off of increasing corticosterone to a level that would subsequently affect spleen size and cellularity, thus prolonging potential immunological suppression. Declining water levels increased developmental rate by 3 days; however, there were no significant body size effects. Corticosterone (CORT) was negatively correlated with total length, snout vent length, body weight, and spleen weight at metamorphosis, suggesting that size at metamorphosis and the immune system may be affected by excessive CORT levels. When compared to other studies, our results support the view that multiple factors may be acting as stressors in the field affecting amphibian responses, and simple pathways as tested in this study may not adequately represent field conditions. © 2016 Wiley Periodicals, Inc.

  4. Epidermal cellular proliferation and differentiation in sexually mature male Salmo salar with androgen levels depressed by oil.

    PubMed

    Burton, D; Burton, M P; Truscott, B; Idler, D R

    1985-07-22

    Sexually mature male Salmo salar exhibit epidermal thickening and an increase in goblet cell concentration during the spawning season. The ventral skin, which is likely to experience most abrasive contact during the spawning period, has the thickest epidermis and the greatest goblet cell concentration. Following exposure to crude oil there is inhibition of cellular proliferation and elongation associated with epidermal thickening, and also inhibition of mucigenesis. Data on the androgen levels in these fish, and data from earlier studies involving treatment with hormones, indicate that oil-related epidermal effects during the spawning period are most likely systemic in origin, probably arising from reduced plasmatic androgen levels.

  5. Quantitative assessment of developmental levels in overarm throwing using wearable inertial sensing technology.

    PubMed

    Grimpampi, Eleni; Masci, Ilaria; Pesce, Caterina; Vannozzi, Giuseppe

    2016-09-01

    Motor proficiency in childhood has been recently recognised as a public health determinant, having a potential impact on the physical activity level and possible sedentary behaviour of the child later in life. Among fundamental motor skills, ballistic skills assessment based on in-field quantitative observations is progressively needed in the motor development community. The aim of this study was to propose an in-field quantitative approach to identify different developmental levels in overarm throwing. Fifty-eight children aged 5-10 years performed an overarm throwing task while wearing three inertial sensors located at the wrist, trunk and pelvis level and were then categorised using a developmental sequence of overarm throwing. A set of biomechanical parameters were defined and analysed using multivariate statistics to evaluate whether they can be used as developmental indicators. Trunk and pelvis angular velocities and time durations before the ball release showed increasing/decreasing trends with increasing developmental level. Significant differences between developmental level pairs were observed for selected biomechanical parameters. The results support the suitability and feasibility of objective developmental measures in ecological learning contexts, suggesting their potential supportiveness to motor learning experiences in educational and youth sports training settings.

  6. Difference between Extra- and Intracellular T1 Values of Carboxylic Acids Affects the Quantitative Analysis of Cellular Kinetics by Hyperpolarized NMR.

    PubMed

    Karlsson, Magnus; Jensen, Pernille Rose; Ardenkjaer-Larsen, Jan Henrik; Lerche, Mathilde H

    2016-10-17

    Incomplete knowledge of the longitudinal relaxation time constant (T1 ) leads to incorrect assumptions in quantitative kinetic models of cellular systems, studied by hyperpolarized real-time NMR. Using an assay that measures the intracellular signal of small carboxylic acids in living cells, the intracellular T1 of the carboxylic acid moiety of acetate, keto-isocaproate, pyruvate, and butyrate was determined. The intracellular T1 is shown to be up to four-fold shorter than the extracellular T1 . Such a large difference in T1 values between the inside and the outside of the cell has significant influence on the quantification of intracellular metabolic activity. It is expected that the significantly shorter T1 value of the carboxylic moieties inside cells is a result of macromolecular crowding. An artificial cytosol has been prepared and applied to predict the T1 of other carboxylic acids. We demonstrate the value of this prediction tool.

  7. Analysis of Students' Aptitude to Provide Meaning to Images that Represent Cellular Components at the Molecular Level

    PubMed Central

    Dahmani, Hassen-Reda; Schneeberger, Patricia

    2009-01-01

    The number of experimentally derived structures of cellular components is rapidly expanding, and this phenomenon is accompanied by the development of a new semiotic system for teaching. The infographic approach is shifting from a schematic toward a more realistic representation of cellular components. By realistic we mean artist-prepared or computer graphic images that closely resemble experimentally derived structures and are characterized by a low level of styling and simplification. This change brings about a new challenge for teachers: designing course instructions that allow students to interpret these images in a meaningful way. To determine how students deal with this change, we designed several image-based, in-course assessments. The images were highly relevant for the cell biology course but did not resemble any of the images in the teaching documents. We asked students to label the cellular components, describe their function, or both. What we learned from these tests is that realistic images, with a higher apparent level of complexity, do not deter students from investigating their meaning. When given a choice, the students do not necessarily choose the most simplified representation, and they were sensitive to functional indications embedded in realistic images. PMID:19723817

  8. A quantitative analysis of L-glutamate-regulated Na+ dynamics in mouse cortical astrocytes: implications for cellular bioenergetics.

    PubMed

    Chatton, J Y; Marquet, P; Magistretti, P J

    2000-11-01

    The mode of Na+ entry and the dynamics of intracellular Na+ concentration ([Na+]i) changes consecutive to the application of the neurotransmitter glutamate were investigated in mouse cortical astrocytes in primary culture by video fluorescence microscopy. An elevation of [Na+]i was evoked by glutamate, whose amplitude and initial rate were concentration dependent. The glutamate-evoked Na+ increase was primarily due to Na+-glutamate cotransport, as inhibition of non-NMDA ionotropic receptors by 6-cyano-7-nitroquinoxiline-2,3-dione (CNQX) only weakly diminished the response and D-aspartate, a substrate of the glutamate transporter, produced [Na+]i elevations similar to those evoked by glutamate. Non-NMDA receptor activation could nevertheless be demonstrated by preventing receptor desensitization using cyclothiazide. Thus, in normal conditions non-NMDA receptors do not contribute significantly to the glutamate-evoked Na+ response. The rate of Na+ influx decreased during glutamate application, with kinetics that correlate well with the increase in [Na+]i and which depend on the extracellular concentration of glutamate. A tight coupling between Na+ entry and Na+/K+ ATPase activity was revealed by the massive [Na+]i increase evoked by glutamate when pump activity was inhibited by ouabain. During prolonged glutamate application, [Na+]i remains elevated at a new steady-state where Na+ influx through the transporter matches Na+ extrusion through the Na+/K+ ATPase. A mathematical model of the dynamics of [Na+]i homeostasis is presented which precisely defines the critical role of Na+ influx kinetics in the establishment of the elevated steady state and its consequences on the cellular bioenergetics. Indeed, extracellular glutamate concentrations of 10 microM already markedly increase the energetic demands of the astrocytes.

  9. Cellular level robotic surgery: Nanodissection of intermediate filaments in live keratinocytes.

    PubMed

    Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C; Seiffert-Sinha, Kristina; Sinha, Animesh A; Xi, Ning

    2015-01-01

    We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis.

  10. Cellular Level Robotic Surgery: Nanodissection of Intermediate Filaments in Live Keratinocytes

    PubMed Central

    Yang, Ruiguo; Song, Bo; Sun, Zhiyong; Lai, King Wai Chiu; Fung, Carmen Kar Man; Patterson, Kevin C.; Seiffert-Sinha, Kristina; Sinha, Animesh A.; Xi, Ning

    2014-01-01

    We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both, the case of biochemical modulation of the desmosome junctions or mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. PMID:25200612

  11. Hierarchical random cellular neural networks for system-level brain-like signal processing.

    PubMed

    Kozma, Robert; Puljic, Marko

    2013-09-01

    Sensory information processing and cognition in brains are modeled using dynamic systems theory. The brain's dynamic state is described by a trajectory evolving in a high-dimensional state space. We introduce a hierarchy of random cellular automata as the mathematical tools to describe the spatio-temporal dynamics of the cortex. The corresponding brain model is called neuropercolation which has distinct advantages compared to traditional models using differential equations, especially in describing spatio-temporal discontinuities in the form of phase transitions. Phase transitions demarcate singularities in brain operations at critical conditions, which are viewed as hallmarks of higher cognition and awareness experience. The introduced Monte-Carlo simulations obtained by parallel computing point to the importance of computer implementations using very large-scale integration (VLSI) and analog platforms.

  12. Understanding the Remodelling of Cell Walls during Brachypodium distachyon Grain Development through a Sub-Cellular Quantitative Proteomic Approach

    PubMed Central

    Francin-Allami, Mathilde; Lollier, Virginie; Pavlovic, Marija; San Clemente, Hélène; Rogniaux, Hélène; Jamet, Elisabeth; Guillon, Fabienne; Larré, Colette

    2016-01-01

    Brachypodium distachyon is a suitable plant model for studying temperate cereal crops, such as wheat, barley or rice, and helpful in the study of the grain cell wall. Indeed, the most abundant hemicelluloses that are in the B. distachyon cell wall of grain are (1-3)(1-4)-β-glucans and arabinoxylans, in a ratio similar to those of cereals such as barley or oat. Conversely, these cell walls contain few pectins and xyloglucans. Cell walls play an important role in grain physiology. The modifications of cell wall polysaccharides that occur during grain development and filling are key in the determination of the size and weight of the cereal grains. The mechanisms required for cell wall assembly and remodelling are poorly understood, especially in cereals. To provide a better understanding of these processes, we purified the cell wall at three developmental stages of the B. distachyon grain. The proteins were then extracted, and a quantitative and comparative LC-MS/MS analysis was performed to investigate the protein profile changes during grain development. Over 466 cell wall proteins (CWPs) were identified and classified according to their predicted functions. This work highlights the different proteome profiles that we could relate to the main phases of grain development and to the reorganization of cell wall polysaccharides that occurs during these different developmental stages. These results provide a good springboard to pursue functional validation to better understand the role of CWPs in the assembly and remodelling of the grain cell wall of cereals. PMID:28248231

  13. Quantitative Proteomics and Immunohistochemistry Reveal Insights into Cellular and Molecular Processes in the Infarct Border Zone One Month after Myocardial Infarction.

    PubMed

    Yang, Libang; Gregorich, Zachery R; Cai, Wenxuan; Zhang, Patrick; Young, Bernice; Gu, Yiwen; Zhang, Jianyi; Ge, Ying

    2017-04-07

    Postinfarction remodeling and expansion of the peri-infarct border zone (BZ) directly correlate with mortality following myocardial infarction (MI); however, the cellular and molecular mechanisms underlying remodeling processes in the BZ remain unclear. Herein, we utilized a label-free quantitative proteomics approach in combination with immunohistochemical analyses to gain a better understanding of processes contributing to postinfarction remodeling of the peri-infarct BZ in a swine model of MI with reperfusion. Our analysis uncovered a significant down-regulation of proteins involved in energy metabolism, indicating impaired myocardial energetics and possibly mitochondrial dysfunction, in the peri-scar BZ. An increase in endothelial and vascular smooth muscles cells, as well as up-regulation of proteins implicated in vascular endothelial growth factor (VEGF) signaling and marked changes in the expression of extracellular matrix and subendothelial basement membrane proteins, is indicative of active angiogenesis in the infarct BZ. A pronounced increase in macrophages in the peri-infarct BZ was also observed, and proteomic analysis uncovered evidence of persistent inflammation in this tissue. Additional evidence suggested an increase in cellular proliferation that, concomitant with increased nestin expression, indicates potential turnover of endogenous stem cells in the BZ. A marked up-regulation of pro-apoptotic proteins, as well as the down-regulation of proteins important for adaptation to mechanical, metabolic, and oxidative stress, likely contributes to increased apoptosis in the peri-infarct BZ. The cellular processes and molecular pathways identified herein may have clinical utility for therapeutic intervention aimed at limiting remodeling and expansion of the BZ myocardium and preventing the development of heart failure post-MI.

  14. The F7 gene and clotting factor VII levels: dissection of a human quantitative trait locus.

    PubMed

    Soria, Jose Manuel; Almasy, Laura; Souto, Juan Carlos; Sabater-Lleal, Maria; Fontcuberta, Jordi; Blangero, John

    2005-10-01

    Localization of human quantitative trait loci (QTLs) is now routine. However, identifying their functional DNA variants is still a formidable challenge. We present a complete dissection of a human QTL using novel statistical techniques to infer the most likely functional polymorphisms of a QTL that influence plasma levels of clotting factor VII (FVII), a risk factor for cardiovascular disease. Resequencing of 15 kb in and around the F7 gene identified 49 polymorphisms, which were then genotyped in 398 people. Using a Bayesian quantitative trait nucleotide (BQTN) method, we identified four to seven functional variants that completely account for this QTL. These variants include both rare coding variants and more common, potentially regulatory polymorphisms in intronic and promoter regions.

  15. Quantitative structure-ion intensity relationship strategy to the prediction of absolute levels without authentic standards.

    PubMed

    Wu, Liang; Wu, Yuzheng; Shen, Hanyuan; Gong, Ping; Cao, Lijuan; Wang, Guangji; Hao, Haiping

    2013-09-10

    The lack of authentic standards represents a major bottleneck in the quantitative analysis of complex samples. Here we propose a quantitative structure and ionization intensity relationship (QSIIR) approach to predict the absolute levels of compounds in complex matrixes. An absolute quantitative method for simultaneous quantification of 25 organic acids was firstly developed and validated. Napierian logarithm (LN) of the relative slope rate derived from the calibration curves was applied as an indicator of the relative ionization intensity factor (RIIF) and serves as the dependent variable for building a QSIIR model via a multiple linear regression (MLR) approach. Five independent variables representing for hydrogen bond acidity, HOMO energy, the number of hydrogen bond donating group, the ratio of organic phase, and the polar solvent accessible surface area were found as the dominant contributors to the RIIF of organic acids. This QSIIR model was validated to be accurate and robust, with the correlation coefficients (R(2)), R(2) adjusted, and R(2) prediction at 0.945, 0.925, and 0.89, respectively. The deviation of accuracy between the predicted and experimental value in analyzing a real complex sample was less than 20% in most cases (15/18). Furthermore, the high adaptability of this model was validated one year later in another LC/MS system. The QSIIR approach is expected to provide better understanding of quantitative structure and ionization efficiency relationship of analogous compounds, and also to be useful in predicting the absolute levels of analogous analytes in complex mixtures. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Direct aqueous measurement of 25-hydroxyvitamin D levels in a cellular environment by LC-MS/MS using the novel chemical derivatization reagent MDBP.

    PubMed

    Müller, Miriam J; Bruns, Heiko; Volmer, Dietrich A

    2017-01-30

    Vitamin D measurements in biological fluids by mass spectrometry are challenging at very low concentration levels. As a result, chemical derivatization is often employed to enhance the ionization properties of low abundant vitamin D compounds. Cookson-type reagents such as 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) or similar derivatives work well but require careful, water-free experimental conditions, as traces of water inactivate the reagent and inhibit or stop the derivatization reactions, thus making quantitative measurements in aqueous samples impossible. We describe a novel electrospray liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for determining 25-hydroxyvitamin D3 (25(OH)D3) directly in aqueous cellular systems using a new derivatization reagent, the ionic liquid 12-(maleimidyl)dodecyl-tri-n-butylphosphonium bromide (MDBP). The proof-of-concept for the MDBP assay was demonstrated by measuring the levels of 25(OH)D3 in four different human cell types, namely T cells, helper T cells, B cells, and macrophages. In addition to the ability to determine the levels of 25(OH)D3 directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D3 into the investigated cells to demonstrate the applicability of the new label. Furthermore, the MDBP derivatization technique may be equally useful in imaging mass spectrometry, where it could be used for response enhancements of spatially localized vitamin D metabolites on wet tissue surfaces, without destroying the integrity of the tissue surface. Graphical Abstract MDBP labelling of 25-hydroxyvitamin D in the extracellular space.

  17. A Local Increase in Red Blood Cell Aggregation Can Trigger Deep Vein Thrombosis: Evidence Based on Quantitative Cellular Ultrasound Imaging

    PubMed Central

    Yu, François T.H.; Armstrong, Jonathan K.; Tripette, Julien; Meiselman, Herbert J.

    2010-01-01

    Background Recurrent DVT risk factors include a first idiopathic DVT, strongly suggesting the existence of undiagnosed and/or unidentified prothrombotic abnormalities. Objectives To evaluate the impact of locally increased RBC aggregation on DVT pathogenesis in a rabbit model. Methods DVT presence, flow and aggregation were measured in-situ with ultrasound. Greatly enhanced aggregation was achieved by covalent linkage of Pluronic F98 to the RBC surface; coating with Pluronic F68, which very mildly enhances aggregation, was used as a coating control. On day 1, endothelial damage and a partial stenosis were surgically created on the left femoral vein while the right femoral vein was not manipulated. Results A thrombus was formed within 30 minutes in 6 of 7 left femoral veins of animals receiving a 30% volume blood exchange with F98-coated RBC, whereas a thrombus occurred in only 1 of 7 veins in F68-transfused controls. In vivo imaging using quantitative ultrasound confirmed increased aggregation in the thrombosed veins of the F98 group compared with the F68 group and the contralateral vessel. For each group, 5 animals were followed for 2 weeks before sacrifice. In F98-transfused animals, lysis of clots occurred and the presence of chronic thrombi totally occluding the vein in 3 of 5 animals was confirmed by histology. Conversely, in the F68 group, a single disorganized blood clot was observed in 1 of 5 animals. Conclusions A marked increase in RBC aggregation promotes thrombosis in rabbit femoral veins, confirming a pathophysiological role of locally altered hemorheology in the onset of DVT. PMID:21143377

  18. Quantitative Proteomics Reveals Dynamic Interactions of the Minichromosome Maintenance Complex (MCM) in the Cellular Response to Etoposide Induced DNA Damage.

    PubMed

    Drissi, Romain; Dubois, Marie-Line; Douziech, Mélanie; Boisvert, François-Michel

    2015-07-01

    The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2-7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Quantitative myocardial perfusion PET parametric imaging at the voxel-level

    NASA Astrophysics Data System (ADS)

    Mohy-ud-Din, Hassan; Lodge, Martin A.; Rahmim, Arman

    2015-08-01

    Quantitative myocardial perfusion (MP) PET has the potential to enhance detection of early stages of atherosclerosis or microvascular dysfunction, characterization of flow-limiting effects of coronary artery disease (CAD), and identification of balanced reduction of flow due to multivessel stenosis. We aim to enable quantitative MP-PET at the individual voxel level, which has the potential to allow enhanced visualization and quantification of myocardial blood flow (MBF) and flow reserve (MFR) as computed from uptake parametric images. This framework is especially challenging for the 82Rb radiotracer. The short half-life enables fast serial imaging and high patient throughput; yet, the acquired dynamic PET images suffer from high noise-levels introducing large variability in uptake parametric images and, therefore, in the estimates of MBF and MFR. Robust estimation requires substantial post-smoothing of noisy data, degrading valuable functional information of physiological and pathological importance. We present a feasible and robust approach to generate parametric images at the voxel-level that substantially reduces noise without significant loss of spatial resolution. The proposed methodology, denoted physiological clustering, makes use of the functional similarity of voxels to penalize deviation of voxel kinetics from physiological partners. The results were validated using extensive simulations (with transmural and non-transmural perfusion defects) and clinical studies. Compared to post-smoothing, physiological clustering depicted enhanced quantitative noise versus bias performance as well as superior recovery of perfusion defects (as quantified by CNR) with minimal increase in bias. Overall, parametric images obtained from the proposed methodology were robust in the presence of high-noise levels as manifested in the voxel time-activity-curves.

  20. CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels.

    PubMed

    Kartasalo, Kimmo; Pölönen, Risto-Pekka; Ojala, Marisa; Rasku, Jyrki; Lekkala, Jukka; Aalto-Setälä, Katriina; Kallio, Pasi

    2015-10-26

    Orientation and the degree of isotropy are important in many biological systems such as the sarcomeres of cardiomyocytes and other fibrillar structures of the cytoskeleton. Image based analysis of such structures is often limited to qualitative evaluation by human experts, hampering the throughput, repeatability and reliability of the analyses. Software tools are not readily available for this purpose and the existing methods typically rely at least partly on manual operation. We developed CytoSpectre, an automated tool based on spectral analysis, allowing the quantification of orientation and also size distributions of structures in microscopy images. CytoSpectre utilizes the Fourier transform to estimate the power spectrum of an image and based on the spectrum, computes parameter values describing, among others, the mean orientation, isotropy and size of target structures. The analysis can be further tuned to focus on targets of particular size at cellular or subcellular scales. The software can be operated via a graphical user interface without any programming expertise. We analyzed the performance of CytoSpectre by extensive simulations using artificial images, by benchmarking against FibrilTool and by comparisons with manual measurements performed for real images by a panel of human experts. The software was found to be tolerant against noise and blurring and superior to FibrilTool when analyzing realistic targets with degraded image quality. The analysis of real images indicated general good agreement between computational and manual results while also revealing notable expert-to-expert variation. Moreover, the experiment showed that CytoSpectre can handle images obtained of different cell types using different microscopy techniques. Finally, we studied the effect of mechanical stretching on cardiomyocytes to demonstrate the software in an actual experiment and observed changes in cellular orientation in response to stretching. CytoSpectre, a versatile, easy

  1. Profiling cellular bioenergetics, glutathione levels, and caspase activities in stomach biopsies of patients with upper gastrointestinal symptoms

    PubMed Central

    Alfazari, Ali S; Al-Dabbagh, Bayan; Al-Dhaheri, Wafa; Taha, Mazen S; Chebli, Ahmad A; Fontagnier, Eva M; Koutoubi, Zaher; Kochiyi, Jose; Karam, Sherif M; Souid, Abdul-Kader

    2015-01-01

    AIM: To measure biochemical parameters in stomach biopsies and test their suitability as diagnostic biomarkers for gastritis and precancerous lesions. METHODS: Biopsies were obtained from the stomachs of two groups of patients (n = 40) undergoing fiber-optic endoscopy due to upper gastrointestinal symptoms. In the first group (n = 17), only the corpus region was examined. Biopsies were processed for microscopic examination and measurement of mitochondrial O2 consumption (cellular respiration), cellular adenosine triphosphate (ATP), glutathione (GSH), and caspase activity. In the second group of patients (n = 23), both corpus and antral regions were studied. Some biopsies were processed for microscopic examination, while the others were used for measurements of cellular respiration and GSH level. RESULTS: Microscopic examinations of gastric corpus biopsies from 17 patients revealed normal mucosae in 8 patients, superficial gastritis in 7 patients, and chronic atrophic gastritis in 1 patient. In patients with normal histology, the rate (mean ± SD) of cellular respiration was 0.17 ± 0.02 μmol/L O2 min-1 mg-1, ATP content was 487 ± 493 pmol/mg, and GSH was 469 ± 98 pmol/mg. Caspase activity was detected in 3 out of 8 specimens. The values of ATP and caspase activity were highly variable. The presence of superficial gastritis had insignificant effects on the measured biomarkers. In the patient with atrophic gastritis, cellular respiration was high and ATP was relatively low, suggesting uncoupling oxidative phosphorylation. In the second cohort of patients, the examined biopsies showed either normal or superficial gastritis. The rate of cellular respiration (O2. μmol/L min-1 mg-1) was slightly higher in the corpus than the antrum (0.18 ± 0.05 vs 0.15 ± 0.04, P = 0.019). The value of GSH was about the same in both tissues (310 ± 135 vs 322 ± 155, P = 0.692). CONCLUSION: The corpus mucosa was metabolically more active than the antrum tissue. The data in this

  2. Relationship between chicken cellular immunity and endotoxin levels in dust from chicken housing environments

    PubMed Central

    Roque, Katharine; Shin, Kyung-Min; Jo, Ji-Hoon; Kim, Hyoung-Ah

    2015-01-01

    Hazardous biochemical agents in animal husbandry indoor environments are known to promote the occurrence of various illnesses among workers and animals. The relationship between endotoxin levels in dust collected from chicken farms and various immunological markers was investigated. Peripheral blood was obtained from 20 broiler chickens and 20 laying hens from four different chicken farms in Korea. Concentrations of total or respirable dust in the inside the chicken farm buildings were measured using a polyvinyl chloride membrane filter and mini volume sampler. Endotoxin levels in the dust were determined by the Limulus Amebocyte Lysate Kinetic method. Interferon-γ production by peripheral blood mononuclear cells stimulated with concanavalin A was significantly lower in broilers or layers from the farms with higher endotoxin concentrations than the chickens from the farms with lower endotoxin levels. An opposite pattern was observed for plasma cortisol concentrations with higher cortisol levels found in chickens from the farms with higher endotoxin levels. When peripheral lymphocytes were examined, the percentage of CD3-Ia+ B cells was lower in layers from farms with higher endotoxin levels than those from locations with lower endotoxin levels. Overall, these results suggest a probable negative association between dust endotoxin levels and cell-mediated immunity in chickens. PMID:25549222

  3. Relationship between chicken cellular immunity and endotoxin levels in dust from chicken housing environments.

    PubMed

    Roque, Katharine; Shin, Kyung-Min; Jo, Ji-Hoon; Kim, Hyoung-Ah; Heo, Yong

    2015-01-01

    Hazardous biochemical agents in animal husbandry indoor environments are known to promote the occurrence of various illnesses among workers and animals. The relationship between endotoxin levels in dust collected from chicken farms and various immunological markers was investigated. Peripheral blood was obtained from 20 broiler chickens and 20 laying hens from four different chicken farms in Korea. Concentrations of total or respirable dust in the inside the chicken farm buildings were measured using a polyvinyl chloride membrane filter and mini volume sampler. Endotoxin levels in the dust were determined by the Limulus Amebocyte Lysate Kinetic method. Interferon-γ production by peripheral blood mononuclear cells stimulated with concanavalin A was significantly lower in broilers or layers from the farms with higher endotoxin concentrations than the chickens from the farms with lower endotoxin levels. An opposite pattern was observed for plasma cortisol concentrations with higher cortisol levels found in chickens from the farms with higher endotoxin levels. When peripheral lymphocytes were examined, the percentage of CD3(-)Ia(+) B cells was lower in layers from farms with higher endotoxin levels than those from locations with lower endotoxin levels. Overall, these results suggest a probable negative association between dust endotoxin levels and cell-mediated immunity in chickens.

  4. Cellular-level near-wall unsteadiness of high-hematocrit erythrocyte flow using confocal μPIV

    NASA Astrophysics Data System (ADS)

    Patrick, Michael J.; Chen, Chia-Yuan; Frakes, David H.; Dur, Onur; Pekkan, Kerem

    2011-04-01

    In hemodynamics, the inherent intermittency of two-phase cellular-level flow has received little attention. Unsteadiness is reported and quantified for the first time in the literature using a combination of fluorescent dye labeling, time-resolved scanning confocal microscopy, and micro-particle image velocimetry (μPIV). The near-wall red blood cell (RBC) motion of physiologic high-hematocrit blood in a rectangular microchannel was investigated under pressure-driven flow. Intermittent flow was associated with (1) the stretching of RBCs as they passed through RBC clusters with twisting motions; (2) external flow through local obstacles; and (3) transitionary rouleaux formations. Velocity profiles are presented for these cases. Unsteady flow clustered in local regions. Extra-cellular fluid flow generated by individual RBCs was examined using submicron fluorescent microspheres. The capabilities of confocal μPIV post-processing were verified using synthetic raw PIV data for validation. Cellular interactions and oscillating velocity profiles are presented, and 3D data are made available for computational model validation.

  5. Naringin Levels in Citrus Tissues : II. Quantitative Distribution of Naringin in Citrus paradisi MacFad.

    PubMed

    Jourdan, P S; McIntosh, C A; Mansell, R L

    1985-04-01

    The quantitative distribution of the flavanone-7-neohesperidoside, naringin, in seeds, seedlings, young plants, branches, flowers, and fruit of Citrus paradisi Macfad., cv ;Duncan' was analyzed by radioimmunoassay. High levels of naringin were associated with very young tissue and lower levels were found in older tissues. Seed coats of ungerminated seeds and young shoots had high naringin concentrations whereas cotyledons and roots had very low concentrations. Light-grown seedlings contained nearly twice as much naringin as etiolated seedlings and, in young plants and branches, the naringin content was highest in developing leaves and stem tissue. In flowers, the ovary had the highest levels of naringin, accounting for nearly 11% of the fresh weight. There was a net increase in the total naringin content of fruits during growth. However, due to the large increase in fruit size, there was a concomitant decrease in the naringin concentration as the fruit matured.

  6. Investigation of heavy metal hyperaccumulation at the cellular level: development and characterization of Thlaspi caerulescens suspension cell lines.

    PubMed

    Klein, Melinda A; Sekimoto, Hitoshi; Milner, Matthew J; Kochian, Leon V

    2008-08-01

    The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens.

  7. Investigation of Heavy Metal Hyperaccumulation at the Cellular Level: Development and Characterization of Thlaspi caerulescens Suspension Cell Lines1[OA

    PubMed Central

    Klein, Melinda A.; Sekimoto, Hitoshi; Milner, Matthew J.; Kochian, Leon V.

    2008-01-01

    The ability of Thlaspi caerulescens, a zinc (Zn)/cadmium (Cd) hyperaccumulator, to accumulate extremely high foliar concentrations of toxic heavy metals requires coordination of uptake, transport, and sequestration to avoid damage to the photosynthetic machinery. The study of these metal hyperaccumulation processes at the cellular level in T. caerulescens has been hampered by the lack of a cellular system that mimics the whole plant, is easily transformable, and competent for longer term studies. Therefore, to better understand the contribution of the cellular physiology and molecular biology to Zn/Cd hyperaccumulation in the intact plant, T. caerulescens suspension cell lines were developed. Differences in cellular metal tolerance and accumulation between the cell lines of T. caerulescens and the related nonhyperaccumulator, Arabidopsis (Arabidopsis thaliana), were examined. A number of Zn/Cd transport-related differences between T. caerulescens and Arabidopsis cell lines were identified that also are seen in the whole plant. T. caerulescens suspension cell lines exhibited: (1) higher growth requirements for Zn; (2) much greater Zn and Cd tolerance; (3) enhanced expression of specific metal transport-related genes; and (4) significant differences in metal fluxes compared with Arabidopsis. One interesting feature exhibited by the T. caerulescens cell lines was that they accumulated less Zn and Cd than the Arabidopsis cell lines, most likely due to a greater metal efflux. This finding suggests that the T. caerulescens suspension cells represent cells of the Zn/Cd transport pathway between the root epidermis and leaf. We also show it is possible to stably transform T. caerulescens suspension cells, which will allow us to alter the expression of candidate hyperaccumulation genes and thus dissect the molecular and physiological processes underlying metal hyperaccumulation in T. caerulescens. PMID:18550685

  8. A quantitative Kirkpatrick Level 1 and 2 study of equipment specialist apprentice operations training

    NASA Astrophysics Data System (ADS)

    Hughes, Dirk D.

    The primary purpose of the quantitative experimental study is to compare employee-learning outcomes for a course of study that is offered in two formats: explicit and tacit instructor led and explicit e-learning operations training. A Kirkpatrick Level 2 course examination is used to establish a pretest knowledge baseline and to measure posttest learning outcomes for each instructional format. A secondary purpose is to compare responses of the two groups using a Kirkpatrick Level 1 customer satisfaction index survey. Several authors reported the United States electric utility industry would have an employee attrition issue during the 2010 through 2015 period. This is at the same time the industry will be experiencing an increased demand for electricity. There now is a demand for highly training powerplant operators. A review of literature yielded few studies comparing instructor led training and e-based training. Though the Electric Power Research Institute stated the two training modes would be acceptable instruction, the organization did not develop a quantifiable justified recommendation as to the training. Subjects participated in a basic operations course and decided to take either the instructor led or e-based training course. Results of the study concluded that both instructor led and e-based training provided significant learning to the participants. The Kirkpatrick Level 1 results indicated significantly better results for instructor led training. There was not a significant difference in the Kirkpatrick Level 2 results between the two training modalities. Recommendation for future research include conducting a quantitative studies including a Phillips Level 5 study and qualitative studies including a more detailed examination of the customer satisfaction survey (Kirkpatrick Level 1).

  9. Quantitative PCR assays to survey the bovine adenovirus levels in environmental samples.

    PubMed

    Wong, K; Xagoraraki, I

    2010-08-01

    Previous studies suggested bovine adenoviruses (BAdVs) could be used as cattle faecal indicators. The main aim of this study was to survey the levels of BAdV in environmental samples using quantitative polymerase chain reaction (qPCR). Two qPCR assays were developed to identify and quantify BAdVs in environmental samples. BAdVs were detected in all dairy manure, and in most cases, the sample concentrations were around 10(3)-10(4) copies ml(-1). Farm tile drainage samples were also detected, but the concentrations were about 1-3 log(10) lower than the BAdV concentrations in the manure samples. The genome equivalent copy (GEC) levels of BAdV and the phage forming unit (PFU) levels of somatic phage in manure samples were comparable. Four of twenty individual cattle faeces were positive with concentrations similar to that found in the manure samples. Sequencing results confirmed the presence of BAdV in the environmental samples, and phylogenetic analysis indicated that BAdV 2 and 4 were the most prevalent serotypes in all the manure samples tested. The qPCR assays developed in this study showed higher sensitivity in detecting BAdV 1 and 2 than the previous published nested assay. The high levels of BAdV in the environmental samples may suggest it could be used for bovine faecal indicator. The significant levels of BAdV in the drainage samples may indicate the potential of surface water pollution by the manure applied to farm fields. This is the first study that reports the quantitative level of BAdV in environmental samples. These results could be useful when it comes to determining whether BAdV could be utilized as a bovine faecal indicator.

  10. Could the Extended Phenotype Extend to the Cellular and Subcellular Levels in Insect-Induced Galls?

    PubMed Central

    Carneiro, Renê Gonçalves da Silva; Pacheco, Priscilla; Isaias, Rosy Mary dos Santos

    2015-01-01

    Neo-ontogenesis of plant galls involves redifferentiation of host plant tissues to express new phenotypes, when new cell properties are established via structural-functional remodeling. Herein, Psidium cattleianum leaves and Nothotrioza cattleiani galls are analyzed by developmental anatomy, cytometry and immunocytochemistry of cell walls. We address hypothesis-driven questions concerning the organogenesis of globoid galls in the association of P. cattleianum - N. cattleianum, and P. myrtoides - N. myrtoidis. These double co-generic systems represent good models for comparing final gall shapes and cell lineages functionalities under the perspective of convergent plant-dependent or divergent insect-induced characteristics. Gall induction, and growth and development are similar in both galls, but homologous cell lineages exhibit divergent degrees of cell hypertrophy and directions of elongation. Median cortical cells in P. cattleianum galls hypertrophy the most, while in P. myrtoides galls there is a centrifugal gradient of cell hypertrophy. Cortical cells in P. cattleianum galls tend to anisotropy, while P. myrtoidis galls have isotropically hypertrophied cells. Immunocytochemistry evidences the chemical identity and functional traits of cell lineages: epidermal cells walls have homogalacturonans (HGAs) and galactans, which confer rigidity to sites of enhanced cell division; oil gland cell walls have arabinogalactan proteins (AGPs) that help avoiding cell death; and parenchyma cell walls have HGAs, galactans and arabinans, which confer porosity. Variations in such chemical identities are related to specific sites of hypertrophy. Even though the double co-generic models have the same macroscopic phenotype, the globoid morphotype, current analyses indicate that the extended phenotype of N. cattleiani is substantiated by cellular and subcellular specificities. PMID:26053863

  11. Mechanistic understanding of cellular level of water in plant-based food material

    NASA Astrophysics Data System (ADS)

    Khan, Md. Imran H.; Kumar, C.; Karim, M. A.

    2017-06-01

    Understanding of water distribution in plant-based food material is crucial for developing an accurate heat and mass transfer drying model. Generally, in plant-based food tissue, water is distributed in three different spaces namely, intercellular water, intracellular water, and cell wall water. For hygroscopic material, these three types of water transport should be considered for actual understanding of heat and mass transfer during drying. However, there is limited study dedicated to the investigation of the moisture distribution in a different cellular environment in the plant-based food material. Therefore, the aim of the present study was to investigate the proportion of intercellular water, intracellular water, and cell wall water inside the plant-based food material. During this study, experiments were performed for two different plant-based food tissues namely, eggplant and potato tissue using 1H-NMR-T2 relaxometry. Various types of water component were calculated by using multicomponent fits of the T2 relaxation curves. The experimental result showed that in potato tissue 80-82% water exist in intracellular space; 10-13% water in intercellular space and only 4-6% water exist in the cell wall space. In eggplant tissue, 90-93% water in intracellular space, 4-6% water exists in intercellular space and the remaining percentage of water is recognized as cell wall water. The investigated results quantify different types of water in plant-based food tissue. The highest proportion of water exists in intracellular spaces. Therefore, it is necessary to include different transport mechanism for intracellular, intercellular and cell wall water during modelling of heat and mass transfer during drying.

  12. Could the Extended Phenotype Extend to the Cellular and Subcellular Levels in Insect-Induced Galls?

    PubMed

    Carneiro, Renê Gonçalves da Silva; Pacheco, Priscilla; Isaias, Rosy Mary dos Santos

    2015-01-01

    Neo-ontogenesis of plant galls involves redifferentiation of host plant tissues to express new phenotypes, when new cell properties are established via structural-functional remodeling. Herein, Psidium cattleianum leaves and Nothotrioza cattleiani galls are analyzed by developmental anatomy, cytometry and immunocytochemistry of cell walls. We address hypothesis-driven questions concerning the organogenesis of globoid galls in the association of P. cattleianum-N. cattleianum, and P. myrtoides-N. myrtoidis. These double co-generic systems represent good models for comparing final gall shapes and cell lineages functionalities under the perspective of convergent plant-dependent or divergent insect-induced characteristics. Gall induction, and growth and development are similar in both galls, but homologous cell lineages exhibit divergent degrees of cell hypertrophy and directions of elongation. Median cortical cells in P. cattleianum galls hypertrophy the most, while in P. myrtoides galls there is a centrifugal gradient of cell hypertrophy. Cortical cells in P. cattleianum galls tend to anisotropy, while P. myrtoidis galls have isotropically hypertrophied cells. Immunocytochemistry evidences the chemical identity and functional traits of cell lineages: epidermal cells walls have homogalacturonans (HGAs) and galactans, which confer rigidity to sites of enhanced cell division; oil gland cell walls have arabinogalactan proteins (AGPs) that help avoiding cell death; and parenchyma cell walls have HGAs, galactans and arabinans, which confer porosity. Variations in such chemical identities are related to specific sites of hypertrophy. Even though the double co-generic models have the same macroscopic phenotype, the globoid morphotype, current analyses indicate that the extended phenotype of N. cattleiani is substantiated by cellular and subcellular specificities.

  13. A Model of the joint motion of agents with a three-level hierarchy based on a cellular automaton

    NASA Astrophysics Data System (ADS)

    Kuznetsov, A. V.

    2017-02-01

    The collective interaction of agents for jointly overcoming (negotiating) obstacles is simulated. The simulation uses a cellular automaton. The automaton's cells are filled with agents and obstacles of various complexity. The agents' task is to negotiate the obstacles while moving to a prescribed target point. Each agent is assigned to one of three levels, which specifies a hierarchy of subordination between the agents. The complexity of an obstacle is determined by the amount of time needed to overcome it. The proposed model is based on the probabilities of going from one cell to another.

  14. Hypoxia and DNA-damaging agent bleomycin both increase the cellular level of the protein 4E-BP.

    PubMed

    Le Bouffant, Ronan; Cormier, Patrick; Mulner-Lorillon, Odile; Bellé, Robert

    2006-09-01

    The 4E-binding proteins (4E-BPs) regulate the cap-dependent eukaryotic initiation factor 4E (eIF4E). The level of 4E-BP protein is regulated during early development of sea urchin embryos. Fertilization leads to the rapid disappearance of the protein that reappears later in development. We show that two important cellular stresses, hypoxia and bleomycin prolonged checkpoint mobilization provoked the overexpression of the protein 4E-BP in developing sea urchin embryos. Hypoxia resulted after 1 h in a reversible gradual increase in the protein 4E-BP level. At 20 h, the protein 4E-BP had reached the level existing in the unfertilized eggs. Bleomycin used as a DNA-damaging agent for checkpoint activation, provoked cell cycle inhibition and after prolonged exposure (20 h), induced the expression of the protein 4E-BP. The effect of bleomycin on 4E-BP protein overexpression was dose-dependent between 0.4 and 1.2 mM. The role of the overexpression of the protein 4E-BP is discussed in relation with cellular stress responses.

  15. Ag Nanoparticles (Ag NM300K) in the Terrestrial Environment: Effects at Population and Cellular Level in Folsomia candida (Collembola)

    PubMed Central

    Mendes, Luís André; Maria, Vera L.; Scott-Fordsmand, Janeck J.; Amorim, Mónica J. B.

    2015-01-01

    The effects of nanomaterials have been primarily assessed based on standard ecotoxicity guidelines. However, by adapting alternative measures the information gained could be enhanced considerably, e.g., studies should focus on more mechanistic approaches. Here, the environmental risk posed by the presence of silver nanoparticles (Ag NM300K) in soil was investigated, anchoring population and cellular level effects, i.e., survival, reproduction (28 days) and oxidative stress markers (0, 2, 4, 6, 10 days). The standard species Folsomia candida was used. Measured markers included catalase (CAT), glutathione reductase (GR), glutathione S-transferase (GST), total glutathione (TG), metallothionein (MT) and lipid peroxidation (LPO). Results showed that AgNO3 was more toxic than AgNPs at the population level: reproduction EC20 and EC50 was ca. 2 and 4 times lower, respectively. At the cellular level Correspondence Analysis showed a clear separation between AgNO3 and AgNP throughout time. Results showed differences in the mechanisms, indicating a combined effect of released Ag+ (MT and GST) and of AgNPs (CAT, GR, TG, LPO). Hence, clear advantages from mechanistic approaches are shown, but also that time is of importance when measuring such responses. PMID:26473892

  16. Differential expression of growth factors at the cellular level in virus-infected brain

    PubMed Central

    Prosniak, Mikhail; Zborek, Anna; Scott, Gwen S.; Roy, Anirban; Phares, Timothy W.; Koprowski, Hilary; Hooper, D. Craig

    2003-01-01

    The contribution of host factors to rabies virus (RV) transcription/replication and axonal/transsynaptic spread is largely unknown. We previously identified several host genes that are up-regulated in the mouse brain during RV infection, including neuroleukin, which is involved in neuronal growth and survival, cell motility, and differentiation, and fibroblast growth factor homologous factor 4 (FHF4), which has been implicated in limb and nervous system development. In this study, we used real-time quantitative RT-PCR to assess the expression of mRNAs specific for neuroleukin, the two isoforms of FHF4 (FHF4-1a and -1b) encoded by the FHF4 gene, and N protein of RV in neurons and astrocytes isolated by laser capture microdissection from mouse brains infected with the laboratory-adapted RV strain CVS-N2c or with a street RV of silver-haired bat origin. Differences in the gene expression patterns suggest that the capacity of RV strains to infect nonneuronal cells and differentially modulate host gene expression may be important in virus replication and spread in the CNS. PMID:12736376

  17. A protocol for quantitative measurement of light intensity levels in food service operations.

    PubMed

    Kassa, Hailu; Keil, Charles; Fent, Kenneth W

    2004-09-01

    Food service workers conduct informal inspections of food service operations to ensure that food contamination does not occur. This activity requires proper lighting. Compliance with lighting standards is usually assessed by a qualitative, visual inspection method. Recent studies suggest, however, that qualitative inspections only minimally reduce risks of foodborne-disease outbreaks. To evaluate the efficacy of qualitative lighting level assessments, this study quantitatively measured lighting in walk-in coolers and at food preparation counters in 57 food service establishments. Measurements were conducted alongside regular sanitarian inspections. Of the 55 walk-in coolers evaluated, 12 had fluorescent and 43 had incandescent lighting. The geometic mean (GM) light level at the center of coolers with fluorescent lighting was 15.3 foot-candles (ft-c) (range: 6.4-85.5). Seventy-five percent of these coolers met the standard of 10 ft-c. The GM level at the center of coolers with incandescent lighting was 3.43 ft-c (range: 1.0-16.7). Seven percent of the incandescently lit coolers met the standard. Inadequate cooler illumination was indicated on only four sanitarian inspection reports. The GM level at 185 food preparation counters was 38.7 ft-c (range: 2.9-196.8). At 47 percent of the establishments, no counters met the 50 ft-c standard. Twenty-three percent of the establishments met the standard at all evaluated counters. Inadequate counter illumination was noted only once by a sanitarian. Qualitative assessments did not identify most of the lighting standard violations. Additional training and integration of quantitative assessments into inspections are warranted. Fluorescent lighting produced significantly higher light levels and pass rates in coolers and should become the lighting type of choice. Finally, gaps in the standards were identified and should be addressed.

  18. Quantitative measurement of hard exudates in patients with diabetes and their associations with serum lipid levels.

    PubMed

    Sasaki, Mariko; Kawasaki, Ryo; Noonan, Jonathan E; Wong, Tien Yin; Lamoureux, Ecosse; Wang, Jie Jin

    2013-08-15

    To describe a reproducible method of quantifying macular hard exudates (HEs) in diabetic maculopathy and determine the associations of HEs with systemic risk factors. Patients with diabetes were recruited from a tertiary eye hospital in Melbourne, Australia. Total macular area covered by HEs (total HE area) and the distance from the foveal center to the nearest HE were measured in a semi-automated manner. Associations between HE parameters and diabetic complication risk factors were examined using multiple linear regression models. Of 593 participants (mean age 60.5-years old), 97 (16.4%) had HEs in at least one eye, due to diabetic maculopathy. The intraclass correlation coefficients (ICC) for inter- and intra-observer agreements were 0.982 and 0.999, respectively. Total HE area was positively associated with qualitative HE severity scale determined by photographic graders. The median of total HE area was 0.089 mm(2) (interquartile range, 0.027-0.246). The median distance between foveal center and the nearest HE was 791.1 μm (431.9-1385.4). After adjusting for age, sex, duration of diabetes, glycated hemoglobin, mean arterial pressure, diabetic retinopathy level, and use of lipid-lowering medication, low density lipoprotein (LDL) cholesterol (P = 0.009), and triglyceride levels (P = 0.036) were positively associated with total HE area. Higher triglyceride levels were associated with central involvement (P = 0.023). Quantitative measurement of HEs in patients with diabetes is associated with lipid levels, and higher triglyceride levels are associated with a higher risk of central involvement. Quantitative information may be useful to monitor HE progression or treatment response in persons with diabetic maculopathy.

  19. Cellular responses and biodegradation of amoxicillin in Microcystis aeruginosa at different nitrogen levels.

    PubMed

    Liu, Ying; Wang, Feng; Chen, Xiao; Zhang, Jian; Gao, Baoyu

    2015-01-01

    The influence of nitrogen on the interactions between amoxicillin and Microcystis aeruginosa was investigated using a 7-day exposure test. Growth of M. aeruginosa was not significantly (p>0.05) affected by amoxicillin at the lowest nitrogen level of 0.05 mg L(-1), stimulated by 500 ng L(-1) of amoxicillin at a moderate nitrogen level of 0.5 mg L(-1) and enhanced by 200-500 ng L(-1) of amoxicillin at the highest nitrogen level of 5 mg L(-1). The generation of reactive oxygen species (ROS) and the synthesis of glutathione S-transferases (GST) and glutathione (GSH) were more sensitive to amoxicillin and were stimulated at all nitrogen levels. At the lowest nitrogen level of 0.05 mg L(-1), superoxide dismutase and peroxidase were not effective at eliminating amoxicillin-induced ROS, resulting in the highest malondialdehyde content in M. aeruginosa. The biodegradation of 18.5-30.5% of amoxicillin by M. aeruginosa was coupled to increasing GST activity and GSH content. Elevated nitrogen concentrations significantly enhanced (p<0.05) the stimulation effect of amoxicillin on the growth of M. aeruginosa, the antioxidant responses to amoxicillin and the biodegradation of amoxicillin in M. aeruginosa. The nitrogen-dependent hormesis effect of the coexisting amoxicillin contaminant on the M. aeruginosa bloom should be fully considered during the control of M. aeruginosa bloom.

  20. Evaluation of RF electromagnetic field exposure levels from cellular base stations in Korea.

    PubMed

    Kim, Byung Chan; Park, Seong-Ook

    2010-09-01

    This article presents the measurement results of human exposure to CDMA800 and CDMA1800 signals at locations in Korea where the general public has expressed concern. Measurements were performed at 50 locations across the country to compare the electromagnetic field levels with the general public exposure compliance limits. At each site, the distances between the nearest single or co-located base station and measurement positions were within a range of approximately 32-422 m. The measured exposure levels were very low compared with the international standard and the Korean human protection notice. The highest field level was 1.5 V/m, which corresponds to 0.15% of the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines for human exposure. 2010 Wiley-Liss, Inc.

  1. Quantitative hepatitis B core antibody levels in the natural history of hepatitis B virus infection.

    PubMed

    Song, L-W; Liu, P-G; Liu, C-J; Zhang, T-Y; Cheng, X-D; Wu, H-L; Yang, H-C; Hao, X-K; Yuan, Q; Zhang, J; Kao, J-H; Chen, D-S; Chen, P-J; Xia, N-S

    2015-02-01

    We previously demonstrated that pretreatment quantitative anti-hepatitis B core protein (qAnti-HBc) levels can predict the treatment response for both interferon and nucleoside analogue therapy, but the characteristics of qAnti-HBc during chronic hepatitis B virus (HBV) infection remain poorly understood. To understand this issue, the qAnti-HBc levels were evaluated in individuals with past HBV infection, occult HBV infection and chronic HBV infection in the immune tolerance phase, immune clearance phase, low-replicative phase and hepatitis B e antigen (HBeAg)-negative hepatitis phase. Individuals with hepatitis B surface antigen (n = 598, 3.74 ± 0.90 log10 IU/mL) had significantly higher (p < 0.001, approximately 1000-fold) serum qAnti-HBc levels than those who had occult HBV, and serum qAnti-HBc levels were significantly higher in the occult HBV group than in the past HBV infection group (p < 0.001). qAnti-HBc levels were positively correlated with alanine aminotransferase levels (R = 0.663, p < 0.001), and subjects with an abnormal alanine aminotransferase level had a higher qAnti-HBc level (p < 0.001). Serum qAnti-HBc level varied in different phases of HBV infection, as determined by host immune status. Serum qAnti-HBc level is strongly associated with hepatitis activity in subjects with chronic HBV infection. Copyright © 2014 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  2. Quantitative analysis of Paratethys sea level change during the Messinian Salinity Crisis

    NASA Astrophysics Data System (ADS)

    de la Vara, Alba; Meijer, Paul; van Baak, Christiaan; Marzocchi, Alice; Grothe, Arjen

    2016-04-01

    At the time of the Messinian Salinity Crisis in the Mediterranean Sea (i.e., the Pontian stage of the Paratethys), the Paratethys sea level dropped also. Evidence found in the sedimentary record of the Black Sea and the Caspian Sea has been interpreted to indicate that a sea level fall occurred between 5.6 and 5.5 Ma. Estimates for the magnitude of the fall range between tens of meters to more than 1500 m. The purpose of this study is to provide quantitative insight into the sensitivity of the water level of the Black Sea and the Caspian Sea to the hydrologic budget, for the case that the Paratethys is disconnected from the Mediterranean. Using a Late Miocene bathymetry based on a palaeographic map by Popov et al. (2004) we quantify the fall in sea level, the mean salinity, and the time to reach equilibrium for a wide range of negative hydrologic budgets. By combining our results with (i) estimates derived from a recent global Late Miocene climate simulation and (ii) reconstructed basin salinities, we are able to rule out a drop in sea level of the order of 1000 m in the Caspian Sea during this time period. In the Black Sea, however, such a large sea level fall cannot be fully discarded.

  3. Quantitative prediction of cellular metabolism with constraint-based models: the COBRA Toolbox v2.0

    PubMed Central

    Schellenberger, Jan; Que, Richard; Fleming, Ronan M. T.; Thiele, Ines; Orth, Jeffrey D.; Feist, Adam M.; Zielinski, Daniel C.; Bordbar, Aarash; Lewis, Nathan E.; Rahmanian, Sorena; Kang, Joseph; Hyduke, Daniel R.; Palsson, Bernhard Ø.

    2012-01-01

    Over the past decade, a growing community of researchers has emerged around the use of COnstraint-Based Reconstruction and Analysis (COBRA) methods to simulate, analyze and predict a variety of metabolic phenotypes using genome-scale models. The COBRA Toolbox, a MATLAB package for implementing COBRA methods, was presented earlier. Here we present a significant update of this in silico ToolBox. Version 2.0 of the COBRA Toolbox expands the scope of computations by including in silico analysis methods developed since its original release. New functions include: (1) network gap filling, (2) 13C analysis, (3) metabolic engineering, (4) omics-guided analysis, and (5) visualization. As with the first version, the COBRA Toolbox reads and writes Systems Biology Markup Language formatted models. In version 2.0, we improved performance, usability, and the level of documentation. A suite of test scripts can now be used to learn the core functionality of the Toolbox and validate results. This Toolbox lowers the barrier of entry to use powerful COBRA methods. PMID:21886097

  4. Quantitation of tyrosine hydroxylase, protein levels: Spot immunolabeling with an affinity-purified antibody

    SciTech Connect

    Haycock, J.W. )

    1989-09-01

    Tyrosine hydroxylase was purified from bovine adrenal chromaffin cells and rat pheochromocytoma using a rapid (less than 2 days) procedure performed at room temperature. Rabbits were immunized with purified enzyme that was denatured with sodium dodecylsulfate, and antibodies to tyrosine hydroxylase were affinity-purified from immune sera. A Western blot procedure using the affinity-purified antibodies and {sup 125}I-protein A demonstrated a selective labeling of a single Mr approximately 62,000 band in samples from a number of different tissues. The relative lack of background {sup 125}I-protein A binding permitted the development of a quantitative spot immunolabeling procedure for tyrosine hydroxylase protein. The sensitivity of the assay is 1-2 ng of enzyme. Essentially identical standard curves were obtained with tyrosine hydroxylase purified from rat pheochromocytoma, rat corpus striatum, and bovine adrenal medulla. An extract of PC 12 cells (clonal rat pheochromocytoma cells) was calibrated against purified rat pheochromocytoma tyrosine hydroxylase and used as an external standard against which levels of tyrosine hydroxylase in PC12 cells and other tissue were quantified. With this procedure, qualitative assessment of tyrosine hydroxylase protein levels can be obtained in a few hours and quantitative assessment can be obtained in less than a day.

  5. Hormesis effects of amoxicillin on growth and cellular biosynthesis of Microcystis aeruginosa at different nitrogen levels.

    PubMed

    Liu, Ying; Chen, Xiao; Zhang, Jian; Gao, Baoyu

    2015-04-01

    Coexisting antibiotic contaminants have potential to regulate cyanobacterial bloom, and the regulation is likely affected by nitrogen supply. A typical cyanobaterium Microcystis aeruginosa was cultured with 0.05-50 mg L(-1) of nitrogen and exposed to 100-600 ng L(-1) of amoxicillin for 7 days. Algal growth was not significantly (p > 0.05) affected by amoxicillin at the lowest nitrogen level of 0.05 mg L(-1), stimulated by 600 ng L(-1) of amoxicillin at a moderate nitrogen level of 0.5 mg L(-1) and enhanced by 100-600 ng L(-1) of amoxicillin at higher nitrogen levels of 5-50 mg L(-1). Amoxicillin affected chlorophyll-a, psbA gene, and rbcL gene in a similar manner as algal growth, suggesting a regulation of algal growth via the photosynthesis system. At each nitrogen level, synthesis of protein and polysaccharides as well as production and release of microcystins (MCs) increased in response to environmental stress caused by amoxicillin. Expression of ntcA and mcyB showed a positive correlation with the total content of MCs under exposure to amoxicillin at nitrogen levels of 0.05-50 mg L(-1). Nitrogen and amoxicillin significantly (p < 0.05) interact with each other on the regulation of algal growth, synthesis of chlorophyll-a, production and release of MCs, and expression of ntcA and mcyB. The nitrogen-dependent stimulation effect of coexisting amoxicillin contaminant on M. aeruginosa bloom should be fully considered during the combined pollution control of M. aeruginosa and amoxicillin.

  6. Quantitative gradient of subsidies reveals a threshold in community-level trophic cascades.

    PubMed

    Klemmer, Amanda J; Richardson, John S

    2013-09-01

    Evidence varies on how subsidies affect trophic cascades within recipient food webs. This could be due to complex nonlinearities being masked by single-level manipulations (presence/absence) of subsidies in past studies. We predicted that trophic cascade strength would increase nonlinearly across a gradient of subsidies. We set out to reveal these complex, nonlinear relationships through manipulating a quantitative gradient of detrital subsidies to lake benthic food webs along with the presence/absence of trout. Contrary to our prediction, we found that trophic cascades only occurred at low subsidy levels, disappearing as subsidies increased. This threshold in trophic cascade strength may be due to an increase in intermediate predators in the absence of top predators, as well as changes in the proportion of armored vs. un-armored primary consumers. Future studies on the effect of subsidies on trophic cascade strength need to incorporate naturally occurring gradients to reveal the complex direct and indirect interactions within food webs.

  7. Cellular-level mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) by oversampling.

    PubMed

    Nazari, Milad; Muddiman, David C

    2015-03-01

    Mass spectrometry imaging (MSI) allows for the direct and simultaneous analysis of the spatial distribution of molecular species from sample surfaces such as tissue sections. One of the goals of MSI is monitoring the distribution of compounds at the cellular resolution in order to gain insights about the biology that occurs at this spatial level. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) imaging of cervical tissue sections was performed using a spot-to-spot distance of 10 μm by utilizing the method of oversampling, where the target plate is moved by a distance that is less than the desorption radius of the laser. In addition to high spatial resolution, high mass accuracy (±1 ppm) and high mass resolving power (140,000 at m/z = 200) were achieved by coupling the IR-MALDESI imaging source to a hybrid quadrupole Orbitrap mass spectrometer. Ion maps of cholesterol in tissues were generated from voxels containing <1 cell, on average. Additionally, the challenges of imaging at the cellular level in terms of loss of sensitivity and longer analysis time are discussed.

  8. An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels.

    PubMed

    Lang, Michael J; Strunk, Bethany S; Azad, Nadia; Petersen, Jason L; Weisman, Lois S

    2017-04-01

    Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2 Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

  9. Mechano-regulation of the beating heart at the cellular level--mechanosensitive channels in normal and diseased heart.

    PubMed

    Friedrich, Oliver; Wagner, Soeren; Battle, Andrew R; Schürmann, Sebastian; Martinac, Boris

    2012-01-01

    The heart as a contractile hollow organ finely tunes mechanical parameters such as stroke volume, stroke pressure and cardiac output according to filling volumes, filling pressures via intrinsic and neuronal routes. At the cellular level, cardiomyocytes in beating hearts are exposed to large mechanical stress during successive heart beats. Although the mechanisms of excitation-contraction coupling are well established in mammalian heart cells, the putative contribution of mechanosensitive channels to Ca²⁺ homeostasis, Ca²⁺ signaling and force generation has been primarily investigated in relation to heart disease states. For instance, transient receptor potential channels (TRPs) are up-regulated in animal models of congestive heart failure or hypertension models and seem to play a vital role in pathological Ca²⁺ overload to cardiomyocytes, thus aggravating the pathology of disease at the cellular level. Apart from that, the contribution of mechanosensitive channels (MsC) in the normal beating heart to the downstream force activation cascade has not been addressed. We present an overview of the current literature and concepts of mechanosensitive channel involvement in failing hearts and cardiomyopathies and novel data showing a likely contribution of Ca²⁺ influx via mechanosensitive channels in beating normal cardiomyocytes during systolic shortening.

  10. Semiautomated hybrid algorithm for estimation of three-dimensional liver surface in CT using dynamic cellular automata and level-sets.

    PubMed

    Dakua, Sarada Prasad; Abinahed, Julien; Al-Ansari, Abdulla

    2015-04-01

    Liver segmentation continues to remain a major challenge, largely due to its intense complexity with surrounding anatomical structures (stomach, kidney, and heart), high noise level and lack of contrast in pathological computed tomography (CT) data. We present an approach to reconstructing the liver surface in low contrast CT. The main contributions are: (1) a stochastic resonance-based methodology in discrete cosine transform domain is developed to enhance the contrast of pathological liver images, (2) a new formulation is proposed to prevent the object boundary, resulting from the cellular automata method, from leaking into the surrounding areas of similar intensity, and (3) a level-set method is suggested to generate intermediate segmentation contours from two segmented slices distantly located in a subject sequence. We have tested the algorithm on real datasets obtained from two sources, Hamad General Hospital and medical image computing and computer-assisted interventions grand challenge workshop. Various parameters in the algorithm, such as [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], [Formula: see text], and [Formula: see text], play imperative roles, thus their values are precisely selected. Both qualitative and quantitative evaluation performed on liver data show promising segmentation accuracy when compared with ground truth data reflecting the potential of the proposed method.

  11. Erk5 contributes to maintaining the balance of cellular nucleotide levels and erythropoiesis

    PubMed Central

    Angulo-Ibáñez, Maria; Rovira-Clavé, Xavier; Granados-Jaén, Alba; Downs, Bradley; Kim, Yeong C; Wang, San Ming; Reina, Manuel; Espel, Enric

    2015-01-01

    An adequate supply of nucleotides is essential for accurate DNA replication, and inappropriate deoxyribonucleotide triphosphate (dNTP) concentrations can lead to replication stress, a common source of DNA damage, genomic instability and tumourigenesis. Here, we provide evidence that Erk5 is necessary for correct nucleotide supply during erythroid development. Mice with Erk5 knockout in the haematopoietic lineage showed impaired erythroid development in bone marrow, accompanied by altered dNTP levels and increased DNA mutagenesis in erythroid progenitors as detected by exome sequencing. Moreover, Erk5-depleted leukemic Jurkat cells presented a marked sensitivity to thymidine-induced S phase stalling, as evidenced by increased H2AX phosphorylation and apoptosis. The increase in thymidine sensitivity correlated with a higher dTTP/dCTP ratio. These results indicate that Erk5 is necessary to maintain the balance of nucleotide levels, thus preventing dNTP misincorporation and DNA damage in proliferative erythroid progenitors and leukemic Jurkat T cells. PMID:26697837

  12. Thermal conductivity of biological cells at cellular level and correlation with disease state

    NASA Astrophysics Data System (ADS)

    Park, Byoung Kyoo; Woo, Yunho; Jeong, Dayeong; Park, Jaesung; Choi, Tae-Youl; Simmons, Denise Perry; Ha, Jeonghong; Kim, Dongsik

    2016-06-01

    This paper reports the thermal conductivity k of matched pair cell lines: two pairs of a normal and a cancer cell, one pair of a primary and metastatic cell. The 3ω method with a nanoscale thermal sensor was used to measure k at the single-cell level. To observe the difference in k between normal and cancer cells, the measurements were conducted for Hs 578Bst/Hs 578 T (human breast cells) and TE 353.Sk/TE 354.T (human skin cells). Then k of WM-115/WM-266-4, a primary and metastatic pair of human skin cell, was measured to find the effect of disease progression on k. The measured k data for normal and disease cell samples show statistically meaningful differences. In all cases, k decreased as the disease progressed. This work shows that thermal-analysis schemes, such as the 3ω method, have a potential to detect diseases at the cell level.

  13. Diacylglycerol levels modulate the cellular distribution of the nicotinic acetylcholine receptor.

    PubMed

    Kamerbeek, Constanza B; Mateos, Melina V; Vallés, Ana S; Pediconi, María F; Barrantes, Francisco J; Borroni, Virginia

    2016-05-01

    Diacylglycerol (DAG), a second messenger involved in different cell signaling cascades, activates protein kinase C (PKC) and D (PKD), among other kinases. The present work analyzes the effects resulting from the alteration of DAG levels on neuronal and muscle nicotinic acetylcholine receptor (AChR) distribution. We employ CHO-K1/A5 cells, expressing adult muscle-type AChR in a stable manner, and hippocampal neurons, which endogenously express various subtypes of neuronal AChR. CHO-K1/A5 cells treated with dioctanoylglycerol (DOG) for different periods showed augmented AChR cell surface levels at short incubation times (30min-4h) whereas at longer times (18h) the AChR was shifted to intracellular compartments. Similarly, in cultured hippocampal neurons surface AChR levels increased as a result of DOG incubation for 4h. Inhibition of endogenous DAG catabolism produced changes in AChR distribution similar to those induced by DOG treatment. Specific enzyme inhibitors and Western blot assays revealed that DAGs exert their effect on AChR distribution through the modulation of the activity of classical PKC (cPKC), novel PKC (nPKC) and PKD activity.

  14. A new rhodamine derived fluorescent sensor: Detection of Hg2+ at cellular level

    NASA Astrophysics Data System (ADS)

    Kuchlyan, Jagannath; Basak, Shyam; Dutta, Debabrata; Das, Amit Kumar; Mal, Dipakranjan; Sarkar, Nilmoni

    2017-04-01

    Mercury ion is of great threat to the human kind due to its high toxicity in live systems. Consequently, its detection at nanolevel is of current interest. The rhodamine derivative is one of the rarest examples of fluorescent chemosensors for Hg2+, wherein a phthalaldehydic acid moiety which shows antibacterial activity, is responsible for specific binding. By using fluorescence correlation spectroscopic (FCS) study of the probe, it has been possible to detect Hg2+ in solution at a level as low as ∼5 pM. This sensing, applicable to fluorescence lifetime imaging of live cells, provides a novel microscopic technique.

  15. Aquatic ecotoxicology: from the ecosystem to the cellular and molecular levels.

    PubMed

    Boudou, A; Ribeyre, F

    1997-02-01

    This review of aquatic ecotoxicology is presented in three parts. First, we discuss the fundamental concepts and stress the importance of its ecological basis and the complexity and diversity of the field of investigation, which result from actions and interactions between the physicochemical characteristics of the biotopes, the structural and functional properties of the living organisms, and the contamination modalities. Ecotoxicological mechanisms, regardless of the level of biological complexity, primarily depend on the bioavailability of the toxic products. Numerous processes control the chemical fate of contaminants in the water column and/or sediment compartments; accessibility to the biological barriers that separate the organisms from their surrounding medium depends directly on bioavailability. Second, we review the principal methodologies of the field, from in situ studies at the ecosystem/ecocomplex level to bioassays or single species tests. Third, we focus on mercury, selected as a reference contaminant, in order to illustrate the main ecotoxicological concepts, the complementarity between field and laboratory studies, and the indispensable multidisciplinarity of the approaches.

  16. Aquatic ecotoxicology: from the ecosystem to the cellular and molecular levels.

    PubMed Central

    Boudou, A; Ribeyre, F

    1997-01-01

    This review of aquatic ecotoxicology is presented in three parts. First, we discuss the fundamental concepts and stress the importance of its ecological basis and the complexity and diversity of the field of investigation, which result from actions and interactions between the physicochemical characteristics of the biotopes, the structural and functional properties of the living organisms, and the contamination modalities. Ecotoxicological mechanisms, regardless of the level of biological complexity, primarily depend on the bioavailability of the toxic products. Numerous processes control the chemical fate of contaminants in the water column and/or sediment compartments; accessibility to the biological barriers that separate the organisms from their surrounding medium depends directly on bioavailability. Second, we review the principal methodologies of the field, from in situ studies at the ecosystem/ecocomplex level to bioassays or single species tests. Third, we focus on mercury, selected as a reference contaminant, in order to illustrate the main ecotoxicological concepts, the complementarity between field and laboratory studies, and the indispensable multidisciplinarity of the approaches. PMID:9114275

  17. Systems engineering meets quantitative systems pharmacology: from low-level targets to engaging the host defenses.

    PubMed

    Androulakis, Ioannis P

    2015-01-01

    Quantitative systems pharmacology aims at systematizing, in a model-based manner, the integration of systems biology and pharmacology in an effort to rationalize the process of assessing the ability of a drug to enhance well-being by off-setting the effects of a disease. Systems engineering, on the other hand, has enabled us to develop principles and methodologies for designing and operating engineered networks of structures exploring the integration of the underlying governing (design) laws. Although the computational tools which have resulted in major advances in the design, analysis, and operation of complex engineered structures have had tremendous success in the analysis of systems pharmacology models, it is argued in this opinion paper, that exploring the underlying conceptual foundation of complex systems engineering will enable us to move toward integrated models at the host level to explore, and possibly, induce synergies between low-level drug targets and higher level, systemic, defense mechanisms. This is an approach which would require refocusing of the key activities; however, it is likely the more promising approach as we enter the new era of personalized and precision medicine. We finally argue for the development of an allostatic approach to quantitative systems pharmacology and the development of an integrated framework for considering drugs in their broader context, beyond their local site of action. WIREs Syst Biol Med 2015, 7:101-112. doi: 10.1002/wsbm.1294 For further resources related to this article, please visit the WIREs website. The author has declared no conflicts of interest for this article. © 2015 Wiley Periodicals, Inc.

  18. The expression level of the transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT) determines cellular survival after radiation treatment.

    PubMed

    Mandl, Markus; Lieberum, Maria- Katharina; Dunst, Juergen; Depping, Reinhard

    2015-11-16

    Tumour hypoxia promotes radioresistance and is associated with poor prognosis. The transcription factor Aryl hydrocarbon receptor nuclear translocator (ARNT), also designated as Hypoxia-inducible factor (HIF)-1β, is part of the HIF pathway which mediates cellular adaptations to oxygen deprivation and facilitates tumour progression. The subunits HIF-1α and ARNT are key players within this pathway. HIF-1α is regulated in an oxygen-dependent manner whereas ARNT is considered to be constitutively expressed. However, there is mounting evidence that certain tumour cells are capable to elevate ARNT in hypoxia which suggests a survival benefit. Therefore the objective of this study was to elucidate effects of an altered ARNT expression level on the cellular response to radiation. Different human cell lines (Hep3B, MCF-7, 786-Owt, 786-Ovhl, RCC4wt and RCC4vhl) originating from various tumour entities (Hepatocellular carcinoma, breast cancer and renal cell carcinoma respectively) were X-irradiated using a conventional linear accelerator. Knockdown of ARNT expression was achieved by transient siRNA transfection. Complementary experiments were performed by forced ARNT overexpression using appropriate plasmids. Presence/absence of ARNT protein was confirmed by Western blot analysis. Clonogenic survival assays were performed in order to determine cellular survival post irradiation. Statistical comparison of two groups was achieved by the unpaired t-test. The results of this study indicate that ARNT depletion renders tumour cells susceptible to radiation whereas overexpression of this transcription factor confers radioresistance. These findings provide evidence to consider ARNT as a drug target and as a predictive marker in clinical applications concerning the response to radiation.

  19. Localizing Organomercury Uptake And Accumulation in Zebrafish Larvae at the Tissue And Cellular Level

    SciTech Connect

    Korbas, M.; Blechinger, S.R.; Krone, P.H.; Pickering, I.J.; George, G.N.

    2009-05-20

    Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.

  20. Quantitative analysis of associations between DNA hypermethylation, hypomethylation, and DNMT RNA levels in ovarian tumors

    PubMed Central

    Ehrlich, Melanie; Woods, Christian B.; Yu, Mimi C.; Dubeau, Louis; Yang, Fan; Campan, Mihaela; Weisenberger, Daniel J.; Long, Tiffany I.; Youn, Byungwoo; Fiala, Emerich S.; Laird, Peter W.

    2005-01-01

    How hypermethylation and hypomethylation of different parts of the genome in cancer are related to each other and to DNA methyltransferase (DNMT) gene expression is ill defined. We used ovarian epithelial tumors of different malignant potential to look for associations between 5’ gene region or promoter hypermethylation, satellite or global DNA hypomethylation, and RNA levels for ten DNMT isoforms. In the quantitative MethyLight assay, 6 of the 55 examined gene loci (LTB4R, MTHFR, CDH13, PGR, CDH1, and IGSF4) were significantly hypermethylated relative to the degree of malignancy (after adjustment for multiple comparisons; P<0.001). Importantly, hypermethylation of these genes was associated with degree of malignancy independently of the association of satellite or global DNA hypomethylation with degree of malignancy. Cancer-related increases in methylation of only two studied genes, LTB4R and MTHFR, which were appreciably methylated even in control tissues, were associated with DNMT1 RNA levels. Cancer-linked satellite DNA hypomethylation was independent of RNA levels for all DNMT3B isoforms, despite the ICF syndrome-linked DNMT3B deficiency causing juxtacentromeric satellite DNA hypomethylation. Our results suggest that there is not a simple association of gene hypermethylation in cancer with altered DNMT RNA levels, and that this hypermethylation is neither the result nor cause of satellite and global DNA hypomethylation. PMID:16532039

  1. Gene Level Meta-Analysis of Quantitative Traits by Functional Linear Models.

    PubMed

    Fan, Ruzong; Wang, Yifan; Boehnke, Michael; Chen, Wei; Li, Yun; Ren, Haobo; Lobach, Iryna; Xiong, Momiao

    2015-08-01

    Meta-analysis of genetic data must account for differences among studies including study designs, markers genotyped, and covariates. The effects of genetic variants may differ from population to population, i.e., heterogeneity. Thus, meta-analysis of combining data of multiple studies is difficult. Novel statistical methods for meta-analysis are needed. In this article, functional linear models are developed for meta-analyses that connect genetic data to quantitative traits, adjusting for covariates. The models can be used to analyze rare variants, common variants, or a combination of the two. Both likelihood-ratio test (LRT) and F-distributed statistics are introduced to test association between quantitative traits and multiple variants in one genetic region. Extensive simulations are performed to evaluate empirical type I error rates and power performance of the proposed tests. The proposed LRT and F-distributed statistics control the type I error very well and have higher power than the existing methods of the meta-analysis sequence kernel association test (MetaSKAT). We analyze four blood lipid levels in data from a meta-analysis of eight European studies. The proposed methods detect more significant associations than MetaSKAT and the P-values of the proposed LRT and F-distributed statistics are usually much smaller than those of MetaSKAT. The functional linear models and related test statistics can be useful in whole-genome and whole-exome association studies.

  2. Gene Level Meta-Analysis of Quantitative Traits by Functional Linear Models

    PubMed Central

    Fan, Ruzong; Wang, Yifan; Boehnke, Michael; Chen, Wei; Li, Yun; Ren, Haobo; Lobach, Iryna; Xiong, Momiao

    2015-01-01

    Meta-analysis of genetic data must account for differences among studies including study designs, markers genotyped, and covariates. The effects of genetic variants may differ from population to population, i.e., heterogeneity. Thus, meta-analysis of combining data of multiple studies is difficult. Novel statistical methods for meta-analysis are needed. In this article, functional linear models are developed for meta-analyses that connect genetic data to quantitative traits, adjusting for covariates. The models can be used to analyze rare variants, common variants, or a combination of the two. Both likelihood-ratio test (LRT) and F-distributed statistics are introduced to test association between quantitative traits and multiple variants in one genetic region. Extensive simulations are performed to evaluate empirical type I error rates and power performance of the proposed tests. The proposed LRT and F-distributed statistics control the type I error very well and have higher power than the existing methods of the meta-analysis sequence kernel association test (MetaSKAT). We analyze four blood lipid levels in data from a meta-analysis of eight European studies. The proposed methods detect more significant associations than MetaSKAT and the P-values of the proposed LRT and F-distributed statistics are usually much smaller than those of MetaSKAT. The functional linear models and related test statistics can be useful in whole-genome and whole-exome association studies. PMID:26058849

  3. Influence of toxoplasmosis on acetylcholinesterase activity, nitric oxide levels and cellular lesion on the brain of mice.

    PubMed

    Tonin, Alexandre A; Da Silva, Aleksandro S; Thomé, Gustavo R; Sangoi, Manuela B; Oliveira, Lizielle S; Flores, Mariana M; Schetinger, Maria Rosa C; Fighera, Rafael A; Moresco, Rafael N; Camillo, Giovana; Vogel, Fernanda S F; Lopes, Sonia T A

    2014-08-01

    The objective of this study was to investigate the activity of acetylcholinesterase (AChE), nitrite/nitrate (NOx) levels, as well as the biomarkers of cellular damage in the brain of mice experimentally infected with Toxoplasma gondii. Sixty mice were divided into two experiments: in experiment I the mice were infected with T. gondii/RH strain, while in experiment II they were infected with T. gondii, strains VEG and ME-49. Our evaluations were carried out on brain homogenized samples, assessing the AChE and glutathione reductase (GR) activities, and NOx, TBARS and AOPP levels in all the infected animals, compared with the control group. In both experiments, I and II, it was observed an increase in the activity of AChE and GR, as well as in the levels of NOx in the brain of infected mice with T. gondii. TBARS levels were increased in mice infected with the three different strains, RH, ME-49, and VEG. AOPP concentration was increased only in mice infected with the RH strain. Animals infected with the strains VEG and ME-49 showed histological lesions, associated with the presence of the parasite in the brain. Therefore, the infection by T. gondii is able to interfere in cholinesterase activity and NO levels, in association with oxidative stress and histological lesion. Copyright © 2014 Elsevier GmbH. All rights reserved.

  4. Quantitation of cellular deoxynucleoside triphosphates

    PubMed Central

    Ferraro, Paola; Franzolin, Elisa; Pontarin, Giovanna; Reichard, Peter; Bianchi, Vera

    2010-01-01

    Eukaryotic cells contain a delicate balance of minute amounts of the four deoxyribonucleoside triphosphates (dNTPs), sufficient only for a few minutes of DNA replication. Both a deficiency and a surplus of a single dNTP may result in increased mutation rates, faulty DNA repair or mitochondrial DNA depletion. dNTPs are usually quantified by an enzymatic assay in which incorporation of radioactive dATP (or radioactive dTTP in the assay for dATP) into specific synthetic oligonucleotides by a DNA polymerase is proportional to the concentration of the unknown dNTP. We find that the commonly used Klenow DNA polymerase may substitute the corresponding ribonucleotide for the unknown dNTP leading in some instances to a large overestimation of dNTPs. We now describe assay conditions for each dNTP that avoid ribonucleotide incorporation. For the dTTP and dATP assays it suffices to minimize the concentrations of the Klenow enzyme and of labeled dATP (or dTTP); for dCTP and dGTP we had to replace the Klenow enzyme with either the Taq DNA polymerase or Thermo Sequenase. We suggest that in some earlier reports ribonucleotide incorporation may have caused too high values for dGTP and dCTP. PMID:20008099

  5. Quantitative evaluation of berberine subcellular distribution and cellular accumulation in non-small cell lung cancer cells by UPLC-MS/MS.

    PubMed

    Yuan, Zhong-Wen; Leung, Elaine Lai-Han; Fan, Xing-Xing; Zhou, Hua; Ma, Wen-Zhe; Liu, Liang; Xie, Ying

    2015-11-01

    Berberine, an isoquinoline alkaloid, has been demonstrated to be a safe anti-cancer agent with multiple effects on mitochondria. Intracellular concentration and distribution around the targeting sites are determinants of efficacy, but subcellular distribution of berberine has not been fully elucidated yet, which relies on the sensitive and robustness assay. In this study, a sensitive and robust UPLC-MS/MS method has been developed and validated with optimized extraction solvents and detection conditions. Key factors such as the purity and integrity of isolated organelle fractions, and the effects of isolation procedures on the subcellular concentration of berberine were systemically evaluated. With the developed assay, we found that the intracellular accumulations of berberine in two gefitinib resistant NSCLC cell lines H1650 and H1975 were 2-3 folds higher than that of normal epithelial cells BEAS-2B. Moreover, significantly different subcellular distribution profiles in NSCLC cancer cells from that of BEAS-2B cells with a striking increase in content in most organelles may contribute to its selective cytotoxicity to cancer cells. Furthermore, a predominant accumulation of berberine was observed for the first time in microsomal fraction for all three cell lines. Therefore, this method could be used for quantitative evaluation of subcellular distribution and cellular accumulation of berberine and for further evaluation of the concentration-effects relationship.

  6. Cellular Response to Ciprofloxacin in Low-Level Quinolone-Resistant Escherichia coli

    PubMed Central

    Machuca, Jesús; Recacha, Esther; Briales, Alejandra; Díaz-de-Alba, Paula; Blazquez, Jesús; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

    2017-01-01

    Bactericidal activity of quinolones has been related to a combination of DNA fragmentation, reactive oxygen species (ROS) production and programmed cell death (PCD) systems. The underlying molecular systems responsible for reducing bactericidal effect during antimicrobial therapy in low-level quinolone resistance (LLQR) phenotypes need to be clarified. To do this and also define possible new antimicrobial targets, the transcriptome profile of isogenic Escherichia coli harboring quinolone resistance mechanisms in the presence of a clinical relevant concentration of ciprofloxacin was evaluated. A marked differential response to ciprofloxacin of either up- or downregulation was observed in LLQR strains. Multiple genes implicated in ROS modulation (related to the TCA cycle, aerobic respiration and detoxification systems) were upregulated (sdhC up to 63.5-fold) in mutants with LLQR. SOS system components were downregulated (recA up to 30.7-fold). yihE, a protective kinase coding for PCD, was also upregulated (up to 5.2-fold). SdhC inhibition sensitized LLQR phenotypes (up to ΔLog = 2.3 after 24 h). At clinically relevant concentrations of ciprofloxacin, gene expression patterns in critical systems to bacterial survival and mutant development were significantly modified in LLQR phenotypes. Chemical inhibition of SdhC (succinate dehydrogenase) validated modulation of ROS as an interesting target for bacterial sensitization. PMID:28769919

  7. Cellular Response to Ciprofloxacin in Low-Level Quinolone-Resistant Escherichia coli.

    PubMed

    Machuca, Jesús; Recacha, Esther; Briales, Alejandra; Díaz-de-Alba, Paula; Blazquez, Jesús; Pascual, Álvaro; Rodríguez-Martínez, José-Manuel

    2017-01-01

    Bactericidal activity of quinolones has been related to a combination of DNA fragmentation, reactive oxygen species (ROS) production and programmed cell death (PCD) systems. The underlying molecular systems responsible for reducing bactericidal effect during antimicrobial therapy in low-level quinolone resistance (LLQR) phenotypes need to be clarified. To do this and also define possible new antimicrobial targets, the transcriptome profile of isogenic Escherichia coli harboring quinolone resistance mechanisms in the presence of a clinical relevant concentration of ciprofloxacin was evaluated. A marked differential response to ciprofloxacin of either up- or downregulation was observed in LLQR strains. Multiple genes implicated in ROS modulation (related to the TCA cycle, aerobic respiration and detoxification systems) were upregulated (sdhC up to 63.5-fold) in mutants with LLQR. SOS system components were downregulated (recA up to 30.7-fold). yihE, a protective kinase coding for PCD, was also upregulated (up to 5.2-fold). SdhC inhibition sensitized LLQR phenotypes (up to ΔLog = 2.3 after 24 h). At clinically relevant concentrations of ciprofloxacin, gene expression patterns in critical systems to bacterial survival and mutant development were significantly modified in LLQR phenotypes. Chemical inhibition of SdhC (succinate dehydrogenase) validated modulation of ROS as an interesting target for bacterial sensitization.

  8. Toxic effects of perfluorinated compounds at human cellular level and on a model vertebrate.

    PubMed

    Rainieri, Sandra; Conlledo, Nadia; Langerholc, Tomaž; Madorran, Eneko; Sala, Martin; Barranco, Alejandro

    2017-06-01

    This work aims at deepening the understanding of the mode of action of some of the most prominent perfluorinated compounds (PFCs) by detecting in a realistic way their effects. To this end, after adjusting the exposure media taking into account the biological model employed and the physico-chemical properties of PFCs, we evaluated the toxic effects of PFOA, PFOS and PFNA in a human macrophage cell line (TLT cells) and in zebrafish embryos. We performed such evaluation on individual compounds and mixtures. Acute toxicity was greater for PFOS in zebrafish; however, it was greater for PFNA in TLT cells. PFNA was also the compound producing the greatest levels of oxidative stress, both in zebrafish and TLT cells. Additionally, in both biological systems, it showed a much stronger effect on mixtures in comparison to the others PFCs tested in this work. Mixture studies in zebrafish showed that acute toxicity depended on the concentration and that the mixture was far more toxic than the individual compounds. This study highlights the importance of studying PFCs in realistic conditions on various biological models. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Effect of subclinical levels of T-2 toxin on the bovine cellular immune system.

    PubMed Central

    Mann, D D; Buening, G M; Osweiler, G D; Hook, B S

    1984-01-01

    The effect of subclinical levels of mycotoxin T-2 on the cells of the bovine immune system was investigated in two in vivo experiments. In experiment 1, five calves were orally dosed with 0.3 mg/kg/day of T-2 toxin for 56 days and five calves were pair fed controls. The neutrophil function as measured by nitroblue tetrazolium reduction was reduced in the mycotoxin treated calves. The cutaneous reaction to intradermally injected phytohemagglutinin was reduced in the T-2 toxin treated calves. B-cell (SIg+) numbers increased slightly, but T-cell (PNA+) numbers were not affected during the experimental period. In the second experiment, six calves were given 0.5 mg/kg/day T-2 toxin orally for 28 days and six calves were pair fed controls. B-cell numbers and the response of a B-cell enriched fraction to phytohemagglutinin increased after toxin administration. T-cell numbers and the response of a T-cell enriched fraction and the whole mononuclear cell population to phytohemagglutinin was reduced only on day 19 posttoxin administration. The in vitro (T-2 toxin) exposure of the mononuclear cell population, B-cell enriched, or T-cell enriched fraction reduced their lymphoblastic response to mitogens. A 50% reduction was induced by as little as 1.4 ng/mL of T-2 toxin. PMID:6332662

  10. Quantitative PCR estimates Angiostrongylus cantonensis (rat lungworm) infection levels in semi-slugs (Parmarion martensi)

    PubMed Central

    Jarvi, Susan I.; Farias, Margaret E.M.; Howe, Kay; Jacquier, Steven; Hollingsworth, Robert; Pitt, William

    2013-01-01

    The life cycle of the nematode Angiostrongylus cantonensis involves rats as the definitive host and slugs and snails as intermediate hosts. Humans can become infected upon ingestion of intermediate or paratenic (passive carrier) hosts containing stage L3 A. cantonensis larvae. Here, we report a quantitative PCR (qPCR) assay that provides a reliable, relative measure of parasite load in intermediate hosts. Quantification of the levels of infection of intermediate hosts is critical for determining A. cantonensis intensity on the Island of Hawaii. The identification of high intensity infection ‘hotspots’ will allow for more effective targeted rat and slug control measures. qPCR appears more efficient and sensitive than microscopy and provides a new tool for quantification of larvae from intermediate hosts, and potentially from other sources as well. PMID:22902292

  11. Hyperaccumulation of cadmium and zinc in Thlaspi caerulescens and Arabidopsis halleri at the leaf cellular level.

    PubMed

    Cosio, Claudia; Martinoia, Enrico; Keller, Catherine

    2004-02-01

    Vacuolar compartmentalization or cell wall binding in leaves could play a major role in hyperaccumulation of heavy metals. However, little is known about the physiology of intracellular cadmium (Cd) sequestration in plants. We investigated the role of the leaf cells in allocating metal in hyperaccumulating plants by measuring short-term (109)Cd and (65)Zn uptake in mesophyll protoplasts of Thlaspi caerulescens "Ganges" and Arabidopsis halleri, both hyperaccumulators of zinc (Zn) and Cd, and T. caerulescens "Prayon," accumulating Cd at a lower degree. The effects of low temperature, several divalent cations, and pre-exposure of the plants to metals were investigated. There was no significant difference between the Michaelis-Menten kinetic constants of the three plants. It indicates that differences in metal uptake cannot be explained by different constitutive transport capacities at the leaf protoplast level and that plasma and vacuole membranes of mesophyll cells are not responsible for the differences observed in heavy metal allocation. This suggests the existence of regulation mechanisms before the plasma membrane of leaf mesophyll protoplasts. However, pre-exposure of the plants to Cd induced an increase in Cd accumulation in protoplasts of "Ganges," whereas it decreased Cd accumulation in A. halleri protoplasts, indicating that Cd-permeable transport proteins are differentially regulated. The experiment with competitors has shown that probably more than one single transport system is carrying Cd in parallel into the cell and that in T. caerulescens "Prayon," Cd could be transported by a Zn and Ca pathway, whereas in "Ganges," Cd could be transported mainly by other pathways.

  12. Sperm quality evaluation in Solea senegalensis during the reproductive season at cellular level.

    PubMed

    Beirão, J; Soares, F; Herráez, M P; Dinis, M T; Cabrita, E

    2009-12-01

    Sperm quality seems to be one of the reasons for the reproduction constraints faced by Senegalese sole (Solea senegalensis) aquaculturists. Previous studies in this species indicated that the sperm quality of individuals kept in culture varies throughout the year and that different sperm subpopulations can be identified in ejaculates according to the motility pattern of spermatozoa. Aiming to better understand factors affecting sole sperm quality in captivity, sperm of 11 males was assessed during the reproductive season using different parameters: motility characteristics using CASA analysis; cell plasma membrane resistance to seawater hyperosmolarity; DNA fragmentation with single-cell gel electrophoresis; and early apoptosis, labeled with Annexin-V FITC. Computer-assisted sperm analyses motility data were treated using multivariate analysis to identify the presence of different spermatozoa subpopulations according to their motility pattern. Four distinct sperm subpopulations were obtained: Subpop1, which includes fast linear spermatozoa; Subpop2, made up of fast nonlinear spermatozoa; Subpop3, which includes slow linear spermatozoa; and Subpop4, which contains slow nonlinear spermatozoa. The sperm subpopulation structure varied with time after activation and with male. Low cell resistance to the seawater hyperosmotic conditions was noticed. The Annexin-V assay allowed the identification of an apoptotic population ranging from 6% to 20%. A high percentage of cells (64.1%) showed a DNA fragmentation level below 30%, but these values varied significantly between males. DNA fragmentation appears to be related to cell membrane resistance to hyperosmotic conditions faced by the cells when in contact with seawater. This condition seems to modulate the composition of the motile sperm population and performance after activation. This phenomenon could be related to the spermatozoa maturation process.

  13. Australia’s first national level quantitative environmental justice assessment of industrial air pollution

    NASA Astrophysics Data System (ADS)

    Chakraborty, Jayajit; Green, Donna

    2014-04-01

    This study presents the first national level quantitative environmental justice assessment of industrial air pollution in Australia. Specifically, our analysis links the spatial distribution of sites and emissions associated with industrial pollution sources derived from the National Pollution Inventory, to Indigenous status and social disadvantage characteristics of communities derived from Australian Bureau of Statistics indicators. Our results reveal a clear national pattern of environmental injustice based on the locations of industrial pollution sources, as well as volume, and toxicity of air pollution released at these locations. Communities with the highest number of polluting sites, emission volume, and toxicity-weighted air emissions indicate significantly greater proportions of Indigenous population and higher levels of socio-economic disadvantage. The quantities and toxicities of industrial air pollution are particularly higher in communities with the lowest levels of educational attainment and occupational status. These findings emphasize the need for more detailed analysis in specific regions and communities where socially disadvantaged groups are disproportionately impacted by industrial air pollution. Our empirical findings also underscore the growing necessity to incorporate environmental justice considerations in environmental planning and policy-making in Australia.

  14. Interrater reliability of quantitative ultrasound using force feedback among examiners with varied levels of experience

    PubMed Central

    Ismail, Catheeja; Monfaredi, Reza; Hernandez, Haniel J.; Pennington, Donte; Woletz, Paula; McIntosh, Valerie; Adams, Bernadette; Blackman, Marc R.

    2016-01-01

    Background. Quantitative ultrasound measures are influenced by multiple external factors including examiner scanning force. Force feedback may foster the acquisition of reliable morphometry measures under a variety of scanning conditions. The purpose of this study was to determine the reliability of force-feedback image acquisition and morphometry over a range of examiner-generated forces using a muscle tissue-mimicking ultrasound phantom. Methods. Sixty material thickness measures were acquired from a muscle tissue mimicking phantom using B-mode ultrasound scanning by six examiners with varied experience levels (i.e., experienced, intermediate, and novice). Estimates of interrater reliability and measurement error with force feedback scanning were determined for the examiners. In addition, criterion-based reliability was determined using material deformation values across a range of examiner scanning forces (1–10 Newtons) via automated and manually acquired image capture methods using force feedback. Results. All examiners demonstrated acceptable interrater reliability (intraclass correlation coefficient, ICC = .98, p < .001) for material thickness measures obtained using force feedback. Individual examiners exhibited acceptable reliability with the criterion-based reference measures (ICC > .90, p < .001), independent of their level of experience. The measurement error among all examiners was 1.5%–2.9% across all applied stress conditions. Conclusion. Manual image capture with force feedback may aid the reliability of morphometry measures across a range of examiner scanning forces, and allow for consistent performance among examiners with differing levels of experience. PMID:27366647

  15. Cotton Defense Induction Patterns Under Spatially, Temporally and Quantitatively Varying Herbivory Levels.

    PubMed

    Eisenring, Michael; Meissle, Michael; Hagenbucher, Steffen; Naranjo, Steven E; Wettstein, Felix; Romeis, Jörg

    2017-01-01

    In its defense against herbivores, cotton (Gossypium sp.) relies in part on the production of a set of inducible, non-volatile terpenoids. Under uniform damage levels, in planta allocation of induced cotton terpenoids has been found to be highest in youngest leaves, supporting assumptions of the optimal defense theory (ODT) which predicts that plants allocate defense compounds to tissues depending on their value and the likelihood of herbivore attack. However, our knowledge is limited on how varying, and thus more realistic, damage levels might affect cotton defense organization. We hypothesized that the allocation of terpenoids and densities of terpenoid-storing glands in leaves aligns with assumptions of the ODT, even when plants are subjected to temporally, spatially and quantitatively varying caterpillar (Heliothis virescens) damage. As expected, cotton plants allocated most of their defenses to their youngest leaves regardless of damage location. However, defense induction in older leaves varied with damage location. For at least 14 days after damage treatments ended, plants reallocated defense resources from previously young leaves to newly developed leaves. Furthermore, we observed a positive hyperbolic relationship between leaf damage area and both terpenoid concentrations and gland densities, indicating that cotton plants can fine-tune defense allocation. Although it appears that factors like vascular constraints and chemical properties of individual defense compounds can affect defense levels, our results overall demonstrate that induced defense organization of cotton subjected to varying damage treatments is in alignment with key assumptions of the ODT.

  16. Cotton Defense Induction Patterns Under Spatially, Temporally and Quantitatively Varying Herbivory Levels

    PubMed Central

    Eisenring, Michael; Meissle, Michael; Hagenbucher, Steffen; Naranjo, Steven E.; Wettstein, Felix; Romeis, Jörg

    2017-01-01

    In its defense against herbivores, cotton (Gossypium sp.) relies in part on the production of a set of inducible, non-volatile terpenoids. Under uniform damage levels, in planta allocation of induced cotton terpenoids has been found to be highest in youngest leaves, supporting assumptions of the optimal defense theory (ODT) which predicts that plants allocate defense compounds to tissues depending on their value and the likelihood of herbivore attack. However, our knowledge is limited on how varying, and thus more realistic, damage levels might affect cotton defense organization. We hypothesized that the allocation of terpenoids and densities of terpenoid-storing glands in leaves aligns with assumptions of the ODT, even when plants are subjected to temporally, spatially and quantitatively varying caterpillar (Heliothis virescens) damage. As expected, cotton plants allocated most of their defenses to their youngest leaves regardless of damage location. However, defense induction in older leaves varied with damage location. For at least 14 days after damage treatments ended, plants reallocated defense resources from previously young leaves to newly developed leaves. Furthermore, we observed a positive hyperbolic relationship between leaf damage area and both terpenoid concentrations and gland densities, indicating that cotton plants can fine-tune defense allocation. Although it appears that factors like vascular constraints and chemical properties of individual defense compounds can affect defense levels, our results overall demonstrate that induced defense organization of cotton subjected to varying damage treatments is in alignment with key assumptions of the ODT. PMID:28270830

  17. Epifluorescence-based Quantitative Microvasculature Remodeling Using Geodesic Level-Sets and Shape-based Evolution

    PubMed Central

    Bunyak, F.; Palaniappan, K.; Glinskii, O.; Glinskii, V.; Glinsky, V.; Huxley, V.

    2009-01-01

    Accurate vessel segmentation is the first step in analysis of microvascular networks for reliable feature extraction and quantitative characterization. Segmentation of epifluorescent imagery of microvasculature presents a unique set of challenges and opportunities compared to traditional angiogram-based vessel imagery. This paper presents a novel system that combines methods from mathematical morphology, differential geometry, and active contours to reliably detect and segment microvasculature under varying background fluorescence conditions. The system consists of three main modules: vessel enhancement, shape-based initialization, and level-set based segmentation. Vessel enhancement deals with image noise and uneven background fluorescence using anisotropic diffusion and mathematical morphology techniques. Shape-based initialization uses features from the second-order derivatives of the enhanced vessel image and produces a coarse ridge (vessel) mask. Geodesic level-set based active contours refine the coarse ridge map and fix possible discontinuities or leakage of the level set contours that may arise from complex topology or high background fluorescence. The proposed system is tested on epifluorescence-based high resolution images of porcine dura mater microvasculature. Preliminary experiments show promising results. PMID:19163371

  18. Biological response at the cellular level within the periodontal ligament on application of orthodontic force – An update

    PubMed Central

    Meeran, Nazeer Ahmed

    2012-01-01

    Orthodontic force elicits a biological response in the tissues surrounding the teeth, resulting in remodeling of the periodontal ligament and the alveolar bone. The force-induced tissue strain result in reorganization of both cellular and extracellular matrix, besides producing changes in the local vascularity. This in turn leads to the synthesis and release of various neurotransmitters, arachidonic acid, growth factors, metabolites, cytokines, colony-stimulating factors, and enzymes like cathepsin K, matrix metalloproteinases, and aspartate aminotransferase. Despite the availability of many studies in the orthodontic and related scientific literature, a concise integration of all data is still lacking. Such a consolidation of the rapidly accumulating scientific information should help in understanding the biological processes that underlie the phenomenon of tooth movement in response to mechanical loading. Therefore, the aim of this review was to describe the biological processes taking place at the molecular level on application of orthodontic force and to provide an update of the current literature. PMID:24987618

  19. An advanced approach for the generation of complex cellular material representative volume elements using distance fields and level sets

    NASA Astrophysics Data System (ADS)

    Sonon, B.; François, B.; Massart, T. J.

    2015-08-01

    A general and widely tunable method for the generation of representative volume elements for cellular materials based on distance and level set functions is presented. The approach is based on random tessellations constructed from random inclusion packings. A general methodology to obtain arbitrary-shaped tessellations to produce disordered foams is presented and illustrated. These tessellations can degenerate either in classical Voronoï tessellations potentially additively weighted depending on properties of the initial inclusion packing used, or in Laguerre tessellations through a simple modification of the formulation. A versatile approach to control the particular morphology of the obtained foam is introduced. Specific local features such as concave triangular Plateau borders and non-constant thickness heterogeneous coatings can be built from the tessellation in a straightforward way and are tuned by a small set of parameters with a clear morphological interpretation.

  20. "PERFEXT": a direct method for quantitative assessment of cytokine production in vivo at the local level.

    PubMed

    Villavedra, M; Carol, H; Hjulström, M; Holmgren, J; Czerkinsky, C

    1997-05-01

    A method termed "PERFEXT", based on sequential perfusion and detergent extraction of lymphoid and non-lymphoid organs, has been developed for the quantitative measurement of cytokines produced at a local level in a given tissue. In vivo treatment of mice with Staphylococcus enterotoxin B (SEB) or lipopolysaccharide (LPS) served as the model systems. Interleukin-2 (IL2) and interferon-gamma (IFN gamma) levels were monitored by ELISA analysis of extracted samples. After local footpad (FP) injection with SEB, spleen and serum IL2 levels peaked at 2-4 h, while IL2 levels peaked at around 4-8 h in both FP and popliteal lymph nodes. SEB injection resulted in increased IFN gamma levels both in the FP and the draining lymph node. The detection of cytokines in the intestine allows for the application of the method at mucosal sites as well, provided enzyme inhibitors are present during the extraction procedure. After FP injection with LPS, IFN gamma production was significantly increased in the draining lymph node and was detectable in the FP, whereas IL2 was undetectable in any organ examined. IL2 and IFN gamma could also be detected at the site of elicitation of a delayed-type hypersensitivity reaction following local FP challenge. Local cytokine production correlated with the swelling response, whereas cytokine production in the spleen did not. IL2 peaked early, followed by a late increase in IFN gamma production, corresponding to the maximum swelling. This simple method should prove useful for analysing the production of other cytokines in vivo in distinct anatomical compartments.

  1. Metal-induced stress in bivalves living along a gradient of Cd contamination: relating sub-cellular metal distribution to population-level responses.

    PubMed

    Perceval, Olivier; Couillard, Yves; Pinel-Alloul, Bernadette; Giguère, Anik; Campbell, Peter G C

    2004-09-20

    The use of biomarkers to assess the impacts of contaminants on aquatic ecosystems has noticeably increased over the past few years. Few of these studies, however, have contributed to the prediction of ecologically significant effects (i.e., at the population or community levels). The present field study was designed to evaluate the potential of metallothionein (MT) and sub-cellular metal partitioning measurements for predicting toxic effects at higher levels of the biological organization in freshwater bivalves (Pyganodon grandis) chronically exposed to Cd. For that purpose, we quantitatively sampled P. grandis populations in the littoral zone of nine lakes on the Precambrian Canadian Shield during two consecutive summers (1998 and 1999); lakes were characterized by contrasting Cd levels but similar trophic status. We tested relationships between the population status of P. grandis (i.e., growth parameters, density, biomass, secondary production, turnover ratio and cumulative fecundity) and (i) ambient Cd concentrations, (ii) sub-organismal responses (MT concentrations in the gill cytosol of individuals and Cd concentrations in three metal-ligand pools identified as M-HMW, the high molecular weight pool, M-MT, the metallothionein-like pool and M-LMW, the low molecular weight pool) and (iii) ecological confounding factors (food resources, presence of host fishes for the obligatory parasitic larval stage of P. grandis). Our results show that littoral density, live weight, dry viscera biomass, production and cumulative fecundity decreased with increasing concentrations of the free-cadmium ion in the environment (Pearson's r ranging from -0.63 to -0.78). On the other hand, theoretical maximum shell lengths (L( infinity )) in our populations were related to both the dissolved Ca concentration and food quality (sestonic C and N concentrations). Overall, Cd concentrations in the gill cytosolic HMW pool of the individual molluscs were the biomarker response that was most

  2. Quantitative Analysis of Aquaporin Expression Levels during the Development and Maturation of the Inner Ear.

    PubMed

    Miyoshi, Takushi; Yamaguchi, Taro; Ogita, Kiyokazu; Tanaka, Yasuko; Ishibashi, Ken-Ichi; Ito, Hiroaki; Kobayashi, Taisuke; Nakagawa, Takayuki; Ito, Juichi; Omori, Koichi; Yamamoto, Norio

    2017-04-01

    Aquaporins (AQPs) are a family of small membrane proteins that transport water molecules across the plasma membrane along the osmotic gradient. Mammals express 13 subtypes of AQPs, including the recently reported "subcellular AQPs", AQP11 and 12. Each organ expresses specific subsets of AQP subtypes, and in the inner ear, AQPs are essential for the establishment and maintenance of two distinct fluids, endolymph and perilymph. To evaluate the contribution of AQPs during the establishment of inner ear function, we used quantitative reverse transcription polymerase chain reaction to quantify the expression levels of all known AQPs during the entire development and maturation of the inner ear. Using systematic and longitudinal quantification, we found that AQP11 was majorly and constantly expressed in the inner ear, and that the expression levels of several AQPs follow characteristic longitudinal patterns: increasing (Aqp0, 1, and 9), decreasing (Aqp6, 8, and 12), and peak of expression on E18 (Aqp2, 5, and 7). In particular, the expression level of Aqp9 increased by 70-fold during P3-P21. We also performed in situ hybridization of Aqp11, and determined the unique localization of Aqp11 in the outer hair cells. Immunohistochemistry of AQP9 revealed its localization in the supporting cells inside the organ of Corti, and in the root cells. The emergence of AQP9 expression in these cells was during P3-P21, which was coincident with the marked increase of its expression level. Combining these quantification and localization data, we discuss the possible contributions of these AQPs to inner ear function.

  3. Exposure of humans to ambient levels of ozone for 6. 6 hours causes cellular and biochemical changes in the lung

    SciTech Connect

    Devlin, R.B.; McDonnell, W.F.; Mann, R.; Becker, S.; House, D.E.

    1991-01-01

    An acute (2h) exposure of humans to 0.4 ppm ozone initiates biochemical changes in the lung resulting in the production of components which mediate inflammation and acute lung damage as well as components which have the potential to lead to long term effects such as fibrosis. However, many people are exposed to lower levels of ozone than this, but for periods of several hours. Therefore, it is important to determine if a prolonged exposure to low levels of ozone is also capable of causing cellular and biochemical changes in the lung. Non-smoking males were randomly exposed to filtered air and either 0.10 ppm ozone or 0.08 ppm ozone for 6.6 h with moderate exercise (40 1/min). Bronchalveolar lavage (BAL) was performed 18 h after each exposure, and cells and fluid analyzed. The BAL fluid of volunteers exposed to 0.10 ppm ozone had significant increases in neutrophils (PMNs), protein, PGE2, fibronectin, interleukin-6 (IL-6), and lactate dehydrogenase (LDH) compared with BAL fluid from the same volunteers exposed to filtered air. The study concludes that exposure of humans to low levels of ozone is sufficient to initiate an inflammatory reaction in the lung.

  4. Galectin-3 level and the severity of cardiac diastolic dysfunction using cellular and animal models and clinical indices.

    PubMed

    Wu, Cho-Kai; Su, Mao-Yuan; Lee, Jen-Kuang; Chiang, Fu-Tien; Hwang, Juey-Jen; Lin, Jiunn-Lee; Chen, Jin-Jer; Liu, Fu-Tong; Tsai, Chia-Ti

    2015-11-19

    Heart failure with preserved ejection fraction (HFPEF) is characterized by myocardial interstitial fibrosis. A total of 146 patients with HFPEF, were recruited. HFPEF severity was determined using Doppler imaging (E/Em) and also cardiac magnetic resonance imaging (CMRI). Canine modeling of HFPEF was induced by aortic banding. Hemodynamic and echocardiographic data were obtained before and after pressure loading and myocardial Galectin-3 was determined. Mechanical stretch of cultured cardiomyocytes served as the cellular model of HFPEF. Patients with severe HFPEF had significantly higher plasma Galectin-3 levels. Significant correlation between plasma Galectin-3 and E/Em in advanced HFPEF patients was noted. After 2 weeks of pressure overload in canine models, the protein expression of Galectin-3 from LV myocardial tissue was significantly increased (p < 0.01) compared with controls. Galectin-3 expression paralleled the severity of LV diastolic dysfunction by evaluation of CMRI (r = -0.58, p = 0.003) and tissue fibrosis (r = 0.59, p = 0.002). After adjusting for confounders for diastolic dysfunction, Galectin-3 levels were still associated with diastolic parameters both in humans (p < 0.001) and canine model (p = 0.041). Mechanical stretch increased Galectin-3 secretion in cultured cardiomyocytes. Both plasma and myocardial Galectin-3 levels correlated with severity of cardiac diastolic dysfunction.

  5. Galectin-3 level and the severity of cardiac diastolic dysfunction using cellular and animal models and clinical indices

    PubMed Central

    Wu, Cho-Kai; Su, Mao-Yuan; Lee, Jen-Kuang; Chiang, Fu-Tien; Hwang, Juey-Jen; Lin, Jiunn-Lee; Chen, Jin-Jer; Liu, Fu-Tong; Tsai, Chia-Ti

    2015-01-01

    Heart failure with preserved ejection fraction (HFPEF) is characterized by myocardial interstitial fibrosis. A total of 146 patients with HFPEF, were recruited. HFPEF severity was determined using Doppler imaging (E/Em) and also cardiac magnetic resonance imaging (CMRI). Canine modeling of HFPEF was induced by aortic banding. Hemodynamic and echocardiographic data were obtained before and after pressure loading and myocardial Galectin-3 was determined. Mechanical stretch of cultured cardiomyocytes served as the cellular model of HFPEF. Patients with severe HFPEF had significantly higher plasma Galectin-3 levels. Significant correlation between plasma Galectin-3 and E/Em in advanced HFPEF patients was noted. After 2 weeks of pressure overload in canine models, the protein expression of Galectin-3 from LV myocardial tissue was significantly increased (p < 0.01) compared with controls. Galectin-3 expression paralleled the severity of LV diastolic dysfunction by evaluation of CMRI (r = −0.58, p = 0.003) and tissue fibrosis (r = 0.59, p = 0.002). After adjusting for confounders for diastolic dysfunction, Galectin-3 levels were still associated with diastolic parameters both in humans (p < 0.001) and canine model (p = 0.041). Mechanical stretch increased Galectin-3 secretion in cultured cardiomyocytes. Both plasma and myocardial Galectin-3 levels correlated with severity of cardiac diastolic dysfunction. PMID:26582585

  6. Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress.

    PubMed

    Jamsheer K, Muhammed; Laxmi, Ashverya

    2015-01-01

    Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

  7. Hydrophilic interaction liquid chromatography-tandem mass spectrometry quantitative method for the cellular analysis of varying structures of gemini surfactants designed as nanomaterial drug carriers.

    PubMed

    Donkuru, McDonald; Michel, Deborah; Awad, Hanan; Katselis, George; El-Aneed, Anas

    2016-05-13

    Diquaternary gemini surfactants have successfully been used to form lipid-based nanoparticles that are able to compact, protect, and deliver genetic materials into cells. However, what happens to the gemini surfactants after they have released their therapeutic cargo is unknown. Such knowledge is critical to assess the quality, safety, and efficacy of gemini surfactant nanoparticles. We have developed a simple and rapid liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative determination of various structures of gemini surfactants in cells. Hydrophilic interaction liquid chromatography (HILIC) was employed allowing for a short simple isocratic run of only 4min. The lower limit of detection (LLOD) was 3ng/mL. The method was valid to 18 structures of gemini surfactants belonging to two different structural families. A full method validation was performed for two lead compounds according to USFDA guidelines. The HILIC-MS/MS method was compatible with the physicochemical properties of gemini surfactants that bear a permanent positive charge with both hydrophilic and hydrophobic elements within their molecular structure. In addition, an effective liquid-liquid extraction method (98% recovery) was employed surpassing previously used extraction methods. The analysis of nanoparticle-treated cells showed an initial rise in the analyte intracellular concentration followed by a maximum and a somewhat more gradual decrease of the intracellular concentration. The observed intracellular depletion of the gemini surfactants may be attributable to their bio-transformation into metabolites and exocytosis from the host cells. Obtained cellular data showed a pattern that grants additional investigations, evaluating metabolite formation and assessing the subcellular distribution of tested compounds. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Quantitative analyses of the hepatic proteome of methylmercury-exposed Atlantic cod (Gadus morhua) suggest oxidative stress-mediated effects on cellular energy metabolism.

    PubMed

    Yadetie, Fekadu; Bjørneklett, Silje; Garberg, Hilde Kristin; Oveland, Eystein; Berven, Frode; Goksøyr, Anders; Karlsen, Odd André

    2016-08-05

    Methylmecury (MeHg) is a widely distributed environmental pollutant with considerable risk to both human health and wildlife. To gain better insight into the underlying mechanisms of MeHg-mediated toxicity, we have used label-free quantitative mass spectrometry to analyze the liver proteome of Atlantic cod (Gadus morhua) exposed in vivo to MeHg (0, 0.5, 2 mg/kg body weight) for 2 weeks. Out of a toltal of 1143 proteins quantified, 125 proteins were differentially regulated between MeHg-treated samples and controls. Using various bioinformatics tools, we performed gene ontology, pathway and network enrichment analysis, which indicated that proteins and pathways mainly related to energy metabolism, antioxidant defense, cytoskeleton remodeling, and protein synthesis were regulated in the hepatic proteome after MeHg exposure. Comparison with previous gene expression data strengthened these results, and further supported that MeHg predominantly affects many energy metabolism pathways, presumably through its strong induction of oxidative stress. Some enzymes known to have functionally important oxidation-sensitive cysteine residues in other animals are among the differentially regulated proteins, suggesting their modulations by MeHg-induced oxidative stress. Integrated analysis of the proteomics dataset combined with previous gene expression dataset showed a more pronounced effect of MeHg on amino acid, glucose and fatty acid metabolic pathways, and suggested possible interactions of the cellular energy metabolism and antioxidant defense pathways. MeHg disrupts mainly redox homeostasis and energy generating metabolic pathways in cod liver. The energy pathways appear to be modulated through MeHg-induced oxidative stress, possibly mediated by oxidation sensitive enzymes.

  9. Iron-Restricted Diet Affects Brain Ferritin Levels, Dopamine Metabolism and Cellular Prion Protein in a Region-Specific Manner.

    PubMed

    Pino, Jessica M V; da Luz, Marcio H M; Antunes, Hanna K M; Giampá, Sara Q de Campos; Martins, Vilma R; Lee, Kil S

    2017-01-01

    Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrP(C)) and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrP(C) and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR) or with normal diet (CTL) for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA) was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrP(C) levels were increased only in the striatum of IR group, where ferritin level was also increased. PrP(C) is known to play roles in iron uptake. Thus, the increase of PrP(C) in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological

  10. Iron-Restricted Diet Affects Brain Ferritin Levels, Dopamine Metabolism and Cellular Prion Protein in a Region-Specific Manner

    PubMed Central

    Pino, Jessica M. V.; da Luz, Marcio H. M.; Antunes, Hanna K. M.; Giampá, Sara Q. de Campos; Martins, Vilma R.; Lee, Kil S.

    2017-01-01

    Iron is an essential micronutrient for several physiological functions, including the regulation of dopaminergic neurotransmission. On the other hand, both iron, and dopamine can affect the folding and aggregation of proteins related with neurodegenerative diseases, such as cellular prion protein (PrPC) and α-synuclein, suggesting that deregulation of iron homeostasis and the consequential disturbance of dopamine metabolism can be a risk factor for conformational diseases. These proteins, in turn, are known to participate in the regulation of iron and dopamine metabolism. In this study, we evaluated the effects of dietary iron restriction on brain ferritin levels, dopamine metabolism, and the expression levels of PrPC and α-synuclein. To achieve this goal, C57BL/6 mice were fed with iron restricted diet (IR) or with normal diet (CTL) for 1 month. IR reduced iron and ferritin levels in liver. Ferritin reduction was also observed in the hippocampus. However, in the striatum of IR group, ferritin level was increased, suggesting that under iron-deficient condition, each brain area might acquire distinct capacity to store iron. Increased lipid peroxidation was observed only in hippocampus of IR group, where ferritin level was reduced. IR also generated discrete results regarding dopamine metabolism of distinct brain regions: in striatum, the level of dopamine metabolites (DOPAC and HVA) was reduced; in prefrontal cortex, only HVA was increased along with the enhanced MAO-A activity; in hippocampus, no alterations were observed. PrPC levels were increased only in the striatum of IR group, where ferritin level was also increased. PrPC is known to play roles in iron uptake. Thus, the increase of PrPC in striatum of IR group might be related to the increased ferritin level. α-synuclein was not altered in any regions. Abnormal accumulation of ferritin, increased MAO-A activity or lipid peroxidation are molecular features observed in several neurological disorders. Our

  11. Field-assisted paper spray mass spectrometry for the quantitative evaluation of imatinib levels in plasma.

    PubMed

    D'Aronco, Sara; Calandra, Eleonora; Crotti, Sara; Toffoli, Giuseppe; Marangon, Elena; Posocco, Bianca; Traldi, Pietro; Agostini, Marco

    Drug levels in patients' bloodstreams vary among individuals and consequently therapeutic drug monitoring (TDM) is fundamental to controlling the effective therapeutic range. For TDM purposes, different analytical approaches have been used, mainly based on immunoassay, liquid chromatography- ultraviolet, liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. More recently a matrix-assisted laser desorption/ionisation method has been proposed for the determination of irinotecan levels in the plasma of subjects under therapy and this method has been cross- validated by comparison with data achieved by LC-MS/MS. However, to reach an effective point-of-care monitoring of plasma drug concentrations, a TDM platform technology for fast, accurate, low-cost assays is required. In this frame, recently the use of paper spray mass spectrometry, which is becoming a popular and widely employed MS method, has been proposed. In this paper we report the results obtained by the development of a paper spray-based method for quantitative analysis in plasma samples of imatinib, a new generation of anticancer drug. Preliminary experiments showed that poor sensitivity, reproducibility and linear response were obtained by the "classical" paper spray set-up. In order to achieve better results, it was thought of interest to operate in presence of a higher and more homogeneous electrical field. For this aim, a stainless steel needle connected with the high voltage power supply was mounted below the paper triangle. Furthermore, in order to obtain valid quantitative data, we analysed the role of the different equilibria participating to the phenomena occurring in paper spray experiments, depending either on instrumental parameters or on the chemical nature of analyte and solvents. A calibration curve was obtained by spiking plasma samples containing different amounts of imatinib (1) with known amounts of deuterated imatinib (1d3) as

  12. Quantitation of mRNA levels of steroid 5alpha-reductase isozymes: a novel method that combines quantitative RT-PCR and capillary electrophoresis.

    PubMed

    Torres, Jesús M; Ortega, Esperanza

    2004-01-01

    A novel, accurate, rapid and modestly labor-intensive method has been developed to quantitate specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR). This strategy combines the high degree of specificity of competitive PCR with the sensitivity of laser-induced fluorescence capillary electrophoresis (LIF-CE). The specific target mRNA and a mimic DNA fragment, used as an internal standard (IS), were co-amplified in a single reaction in which the same primers are used. The amount of mRNA was then quantitated by extrapolation from the standard curve generated with the internal standard. PCR primers were designed to amplify both a 185 bp fragment of the target cDNA for steroid 5alpha-reductase 1 (5alpha-R1) and a 192 bp fragment of the target cDNA for steroid 5alpha-reductase type 2 (5alpha-R2). The 5' forward primers were end-labeled with 6-carboxy-fluorescein (6-FAM). Two synthetic internal standard DNAs of 300 bp were synthesized from the sequence of plasmid pEGFP-C1. The ratio of fluorescence intensity between amplified products of the target cDNA (185 or 192 bp fragments) and the competitive DNA (300 bp fragment) was determined quantitatively after separation by capillary electrophoresis and fluorescence analysis. The accurate quantitation of low-abundance mRNAs by the present method allows low-level gene expression to be characterized.

  13. Quantitative impact of changes in marrow cellularity, skeletal size, and bone mineral density on active marrow dosimetry based upon a reference model.

    PubMed

    Geyer, Amy M; Schwarz, Bryan C; Hobbs, Robert F; Sgouros, George; Bolch, Wesley E

    2017-01-01

    The hematopoietically active tissues of skeletal bone marrow are a prime target for computational dosimetry given potential risks of leukemia and, at higher dose levels, acute marrow toxicity. The complex three-dimensional geometry of trabecular spongiosa, however, complicates schema for dose assessment in such a way that only a few reference skeletal models have been developed to date, and which are based upon microimaging of a limited number of cadaveric bone spongiosa cores. The question then arises as to what degree of accuracy is achievable from reference skeletal dose models when applied to individual patients or specific exposed populations? Patient variability in marrow dosimetry were quantified for three skeletal sites - the ribs, lumbar vertebrae, and cranium - for the beta-emitters (45) Ca, (153) Sm, and (90) Y, and the alpha-particle emitters (223) Ra, (219) Rn, and (215) Po, the latter two being the immediate progeny of the former. For each radionuclide and bone site, three patient parameters were altered from their values in the reference model: (1) bone size as a surrogate for patient stature, (2) marrow cellularity as a surrogate for age- or disease-related changes in marrow adiposity, and (3) the trabecular bone volume fraction as a surrogate for bone mineral density. Marrow dose variability is expressed as percent differences in the radionuclide S value given by the reference model and the patient-parameterized model. The impact of radionuclide biokinetics on marrow dosimetry was not considered. Variations in overall bone size play a very minor role in active marrow dose variability. Marrow cellularity is a significant factor in dose variability for active marrow self-irradiation, but it plays no role for radionuclides localized to the trabecular bone matrix. Variations in trabecular bone volume fractions impact the active marrow dose variability for short-range particle emitters (45) Ca, (223) Ra, (219) Rn, and (215) Po in the vertebrae and ribs

  14. Engineered mutations in fibrillin-1 leading to Marfan syndrome act at the protein, cellular and organismal levels.

    PubMed

    Zeyer, Karina A; Reinhardt, Dieter P

    2015-01-01

    Fibrillins are the major components of microfibrils in the extracellular matrix of elastic and non-elastic tissues. They are multi-domain proteins, containing primarily calcium binding epidermal growth factor-like (cbEGF) domains and 8-cysteine/transforming growth factor-beta binding protein-like (TB) domains. Mutations in the fibrillin-1 gene give rise to Marfan syndrome, a connective tissue disorder with clinical complications in the cardiovascular, skeletal, ocular and other organ systems. Here, we review the consequences of engineered Marfan syndrome mutations in fibrillin-1 at the protein, cellular and organismal levels. Representative point mutations associated with Marfan syndrome in affected individuals have been introduced and analyzed in recombinant fibrillin-1 fragments. Those mutations affect fibrillin-1 on a structural and functional level. Mutations which impair folding of cbEGF domains can affect protein trafficking. Protein folding disrupted by some mutations can lead to defective secretion in mutant fibrillin-1 fragments, whereas fragments with other Marfan mutations are secreted normally. Many Marfan mutations render fibrillin-1 more susceptible to proteolysis. There is also evidence that some mutations affect heparin binding. Few mutations have been further analyzed in mouse models. An extensively studied mouse model of Marfan syndrome expresses mouse fibrillin-1 with a missense mutation (p.C1039G). The mice display similar characteristics to human patients with Marfan syndrome. Overall, the analyses of engineered mutations leading to Marfan syndrome provide important insights into the pathogenic molecular mechanisms exerted by mutated fibrillin-1.

  15. Effects of hypotonic stress and ouabain on the apparent diffusion coefficient of water at cellular and tissue levels in Aplysia.

    PubMed

    Jelescu, Ileana Ozana; Ciobanu, Luisa; Geffroy, Françoise; Marquet, Pierre; Le Bihan, Denis

    2014-03-01

    There is evidence that physiological or pathological cell swelling is associated with a decrease of the apparent diffusion coefficient (ADC) of water in tissues, as measured with MRI. However the mechanism remains unclear. Magnetic resonance microscopy, performed on small tissue samples, has the potential to distinguish effects occurring at cellular and tissue levels. A three-dimensional diffusion prepared fast imaging with steady-state free precession sequence for MR microscopy was implemented on a 17.2 T imaging system and used to investigate the effect of two biological challenges known to cause cell swelling, exposure to a hypotonic solution or to ouabain, on Aplysia nervous tissue. The ADC was measured inside isolated neuronal soma and in the region of cell bodies of the buccal ganglia. Both challenges resulted in an ADC increase inside isolated neuronal soma (+31 ± 24% and +30 ± 11%, respectively) and an ADC decrease at tissue level in the buccal ganglia (-12 ± 5% and -18 ± 8%, respectively). A scenario involving a layer of water molecules bound to the inflating cell membrane surface is proposed to reconcile this apparent discrepancy.

  16. Electromagnetic energy as a bridge between atomic and cellular levels in the genetics approach to cancer treatment.

    PubMed

    Tofani, Santi

    2015-01-01

    Literature on magnetic fields (MF) and gene expression, as well as on DNA damage, supports the hypothesis that electromagnetic energy may act at atomic level influencing genetic stability. According to quantum physics, MF act on the interconversion of singlet and triplet spin states, and therefore on genetic instability, activating oxidative processes connected to biological free radicals formation, particularly ROS. In the above frame, the results of in vitro and in vivo laboratory trials have been analyzed. The use of a static MF amplitude modulated by 50 Hz MF, with a time average total intensity of 5.5 mT, has been shown to influence tumor cell functions such as cell proliferation, apoptosis, p53 expression, inhibition of tumor growth and prolongation of survival in animals, evidence that MF can be more effective than chemotherapy (cyclophosphamide) in inhibiting metastatic spread and growth, having synergistic activity with chemotherapy (Cis-platin), and no observable side effects or toxicity in animals or in humans. The beneficial biological/clinical effects observed, without any adverse effects, have been confirmed by various authors and augur well for the potentiality of this new approach to treat genetically based diseases like cancer. Further studies are needed to develop a quantum physics approach to biology, allowing a stable bridge to be built between atomic and cellular levels, therefore developing quantum biology.

  17. Interactions between 7-hydroxymethotrexate and methotrexate at the cellular level in the Ehrlich ascites tumor in vitro.

    PubMed

    Fabre, G; Matherly, L H; Fabre, I; Cano, J P; Goldman, I D

    1984-03-01

    Studies were undertaken to characterize the cellular pharmacology of 7-hydroxymethotrexate (7-OH-MTX) in Ehrlich ascites tumor cells, compare it to that of methotrexate (MTX), and define the interactions between the parent compound and its catabolite. Transport of 7-OH-MTX is mediated by the MTX-tetrahydrofolate cofactor carrier, with a Km of 9 microM in comparison to the MTX Km of 5 microM. Both compounds mutually inhibit their influx and steady-state levels of free drug accumulated. While influx of 7-OH-MTX is slower than influx of MTX, 7-OH-MTX efflux is likewise slower, so that the steady-state level of 7-OH-MTX achieved is comparable to that of MTX. Influx of 7-OH-MTX is inhibited by extracellular 5-formyltetrahydrofolate and trans-stimulated in cells preloaded with this tetrahydrofolate cofactor. The energetics of 7-OH-MTX transport is similar to that of MTX in the influx and net transport are stimulated by sodium azide, while net transport is reduced by glucose. As observed for MTX, 7-OH-MTX transport is sensitive to the anionic composition of the extracellular compartment and was shown to be inhibited by organic and inorganic phosphates. 7-OH-MTX does not, alone, inhibit [3H]deoxyuridine incorporation into DNA at concentrations of up to 50 microM. However, the catabolite reduces MTX inhibition of deoxyuridine metabolism, presumably due to the reduction in the free level of intracellular MTX achieved. These findings support the possibility that when 7-OH-MTX accumulates to high levels relative to MTX in clinical regimens, it may modulate the pharmacological effects of MTX.

  18. The Contribution of RNA Decay Quantitative Trait Loci to Inter-Individual Variation in Steady-State Gene Expression Levels

    PubMed Central

    Pai, Athma A.; Cain, Carolyn E.; Mizrahi-Man, Orna; De Leon, Sherryl; Lewellen, Noah; Veyrieras, Jean-Baptiste; Degner, Jacob F.; Gaffney, Daniel J.; Pickrell, Joseph K.; Stephens, Matthew; Pritchard, Jonathan K.; Gilad, Yoav

    2012-01-01

    Recent gene expression QTL (eQTL) mapping studies have provided considerable insight into the genetic basis for inter-individual regulatory variation. However, a limitation of all eQTL studies to date, which have used measurements of steady-state gene expression levels, is the inability to directly distinguish between variation in transcription and decay rates. To address this gap, we performed a genome-wide study of variation in gene-specific mRNA decay rates across individuals. Using a time-course study design, we estimated mRNA decay rates for over 16,000 genes in 70 Yoruban HapMap lymphoblastoid cell lines (LCLs), for which extensive genotyping data are available. Considering mRNA decay rates across genes, we found that: (i) as expected, highly expressed genes are generally associated with lower mRNA decay rates, (ii) genes with rapid mRNA decay rates are enriched with putative binding sites for miRNA and RNA binding proteins, and (iii) genes with similar functional roles tend to exhibit correlated rates of mRNA decay. Focusing on variation in mRNA decay across individuals, we estimate that steady-state expression levels are significantly correlated with variation in decay rates in 10% of genes. Somewhat counter-intuitively, for about half of these genes, higher expression is associated with faster decay rates, possibly due to a coupling of mRNA decay with transcriptional processes in genes involved in rapid cellular responses. Finally, we used these data to map genetic variation that is specifically associated with variation in mRNA decay rates across individuals. We found 195 such loci, which we named RNA decay quantitative trait loci (“rdQTLs”). All the observed rdQTLs are located near the regulated genes and therefore are assumed to act in cis. By analyzing our data within the context of known steady-state eQTLs, we estimate that a substantial fraction of eQTLs are associated with inter-individual variation in mRNA decay rates. PMID:23071454

  19. Techniques for rapid quantitative assessment of activity levels in small-group tutorials.

    PubMed

    Prinz, J F; Yip, H Y; Tipoe, G L; Lucas, P W; Lenstrup, M

    1998-07-01

    Two techniques for the rapid quantitative analysis of student participation in small-group teaching were investigated. In the first approach an observer, who also acted as a 'critical friend', recorded the length of individual contributions using a computer keyboard as a simple timing device. In the second approach, small-group sessions were recorded with a portable stereophonic audiotape recorder. The teacher was recorded on one channel, all students on the other. A computer program produced automated analysis of these small group interactions by computing relative amount of speech on each channel. Simple analysis produced automatically by the programs revealed the overall style of the tutorial--variably 'mini-lectures' by teachers with very little participation by the student body, rapid 'question and answer' sessions with about equal teacher/student body involvement or 'mini-presentations' by students with the teacher offering sparse comments in the manner of a facilitator. By presenting results in a graphic format, teachers can be given rapid objective feedback on their teaching style. Coupled with short verbal/non-verbal quizzes at the end of tutorials and information from other assessments, the value of using levels of participation as a measure of the efficiency of such small-group sessions can itself be assessed.

  20. Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.

    PubMed Central

    Haddad, I Y; Pataki, G; Hu, P; Galliani, C; Beckman, J S; Matalon, S

    1994-01-01

    Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. Images PMID:7989597

  1. Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

    PubMed

    Nagayasu, Yuko; Morita, Shin-Ya; Hayashi, Hideki; Miura, Yutaka; Yokoyama, Kazuki; Michikawa, Makoto; Ito, Jin-Ichi

    2014-05-14

    We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress

  2. Quantitative Determination of Antibody to Gonococcal Pili. CHANGES IN ANTIBODY LEVELS WITH GONOCOCCAL INFECTION

    PubMed Central

    Buchanan, Thomas M.; Swanson, John; Holmes, King K.; Kraus, Stephen J.; Gotschlich, Emil C.

    1973-01-01

    Gonococcal pili, pure by the criteria of electron microscopic examination and polyacrylamide gel electrophoresis in sodium dodecyl sulfate, have been prepared by repeated cycles of precipitation with 0.1 M MgCl2, followed by dissolution in 0.01 M Tris pH 8, 0.01 M NaN3. Using a fluorescein-conjugated antibody prepared to pili from a single strain, pilar antigen(s) was found to be present in each of 18 strains of gonococci tested, and absent from strains of pilated meningococci, nonpathogenic Neisseria sp., and Escherichia coli. Purified pili, labeled with 125I were used in an antigen binding assay to quantitatively measure antibody to pili in rabbit sera and in 561 human sera. The range of antibody activity for 133 persons unlikely to have experienced gonorrhea was 0.1-1.6 μg/ml with a geometric mean of 0.5 μg/ml. This geometric mean antibody activity was significantly lower than the geometric mean for asymptomatically infected males (1.0 μg/ml, P < 0.002), males with symptomatic gonococcal anterior urethritis (1.6 μg/ml, P < 0.001), or asymptomatically infected females (4.2 μg/ml, P < 0.001). Antibody appeared in elevated levels (> 1.6 μg/ml) 2-3 wk after infection and returned toward control levels 1 or more months after treatment. Antibody levels higher than 1.6 μg/ml were found in 26 (50%) of 52 males with gonococcal anterior urethritis, in 10 (33%) of 30 males with asymptomatic urethral infection and in 50 (89%) of 56 asymptomatically infected females. In a high-risk group of 103 females for whom culture results and antibody to pili were compared, 58 (57%) had elevated antibody levels to pili and 86% of the infected females were within this seropositive group. The antigen binding assay may provide a means to detect asymptomatic gonococcal infection in women. Images PMID:4201268

  3. Analysis of Students' Aptitude to Provide Meaning to Images that Represent Cellular Components at the Molecular Level

    ERIC Educational Resources Information Center

    Dahmani, Hassen-Reda; Schneeberger, Patricia; Kramer, IJsbrand M.

    2009-01-01

    The number of experimentally derived structures of cellular components is rapidly expanding, and this phenomenon is accompanied by the development of a new semiotic system for teaching. The infographic approach is shifting from a schematic toward a more realistic representation of cellular components. By realistic we mean artist-prepared or…

  4. Analysis of Students' Aptitude to Provide Meaning to Images that Represent Cellular Components at the Molecular Level

    ERIC Educational Resources Information Center

    Dahmani, Hassen-Reda; Schneeberger, Patricia; Kramer, IJsbrand M.

    2009-01-01

    The number of experimentally derived structures of cellular components is rapidly expanding, and this phenomenon is accompanied by the development of a new semiotic system for teaching. The infographic approach is shifting from a schematic toward a more realistic representation of cellular components. By realistic we mean artist-prepared or…

  5. Reduction of Cellular Expression Levels Is a Common Feature of Functionally Affected Pendrin (SLC26A4) Protein Variants

    PubMed Central

    de Moraes, Vanessa C S; Bernardinelli, Emanuele; Zocal, Nathalia; Fernandez, Jhonathan A; Nofziger, Charity; Castilho, Arthur M; Sartorato, Edi L; Paulmichl, Markus; Dossena, Silvia

    2016-01-01

    Sequence alterations in the pendrin gene (SLC26A4) leading to functionally affected protein variants are frequently involved in the pathogenesis of syndromic and nonsyndromic deafness. Considering the high number of SLC26A4 sequence alterations reported to date, discriminating between functionally affected and unaffected pendrin protein variants is essential in contributing to determine the genetic cause of deafness in a given patient. In addition, identifying molecular features common to the functionally affected protein variants can be extremely useful to design future molecule-directed therapeutic approaches. Here we show the functional and molecular characterization of six previously uncharacterized pendrin protein variants found in a cohort of 58 Brazilian deaf patients. Two variants (p.T193I and p.L445W) were undetectable in the plasma membrane, completely retained in the endoplasmic reticulum and showed no transport function; four (p.P142L, p.G149R, p.C282Y and p.Q413R) showed reduced function and significant, although heterogeneous, expression levels in the plasma membrane. Importantly, total expression levels of all of the functionally affected protein variants were significantly reduced with respect to the wild-type and a fully functional variant (p.R776C), regardless of their subcellular localization. Interestingly, reduction of expression may also reduce the transport activity of variants with an intrinsic gain of function (p.Q413R). As reduction of overall cellular abundance was identified as a common molecular feature of pendrin variants with affected function, the identification of strategies to prevent reduction in expression levels may represent a crucial step of potential future therapeutic interventions aimed at restoring the transport activity of dysfunctional pendrin variants. PMID:26752218

  6. Exposure of humans to ambient levels of ozone for 6. 6 hours causes cellular and biochemical changes in the lung

    SciTech Connect

    Devlin, R.B.; McDonnell, W.F.; Mann, R.; Becker, S.; House, D.E.; Schreinemachers, D.; Koren, H.S. )

    1991-01-01

    An acute (2 h) exposure of humans to 0.4 ppm ozone initiates biochemical changes in the lung that result in the production of components mediating inflammation and acute lung damage as well as components having the potential to lead to long-term effects such as fibrosis. However, many people are exposed to lower levels of ozone than this, but for periods of several hours. Therefore, it is important to determine if a prolonged exposure to low levels of ozone is also capable of causing cellular and biochemical changes in the lung. Nonsmoking males were randomly exposed to filtered air and either 0.10 ppm ozone or 0.08 ppm ozone for 6.6 h with moderate exercise (40 liters/min). Bronchoalveolar lavage (BAL) was performed 18 h after each exposure, and cells and fluid were analyzed. The BAL fluid of volunteers exposed to 0.10 ppm ozone had significant increases in neutrophils (PMNs), protein, prostaglandin E2 (PGE2), fibronectin, interleukin-6 (IL-6), and lactate dehydrogenase (LDH) compared with BAL fluid from the same volunteers exposed to filtered air. In addition, there was a decrease in the ability of alveolar macrophages to phagocytize yeast via the complement receptor. Exposure to 0.08 ppm ozone resulted in significant increases in PMNs, PGE2, LDH, IL-6, alpha 1-antitrypsin, and decreased phagocytosis via the complement receptor. However, BAL fluid protein and fibronectin were no longer significantly elevated. We conclude that exposure of humans to as low a level as 0.08 ppm for 6.6 h is sufficient to initiate an inflammatory reaction in the lung.

  7. Modulation of cellular S1P levels with a novel, potent and specific inhibitor of sphingosine kinase-1.

    PubMed

    Schnute, Mark E; McReynolds, Matthew D; Kasten, Tom; Yates, Matthew; Jerome, Gina; Rains, John W; Hall, Troii; Chrencik, Jill; Kraus, Michelle; Cronin, Ciaran N; Saabye, Matthew; Highkin, Maureen K; Broadus, Richard; Ogawa, Shinji; Cukyne, Kristin; Zawadzke, Laura E; Peterkin, Vincent; Iyanar, Kaliapan; Scholten, Jeffrey A; Wendling, Jay; Fujiwara, Hideji; Nemirovskiy, Olga; Wittwer, Arthur J; Nagiec, Marek M

    2012-05-15

    SphK (sphingosine kinase) is the major source of the bioactive lipid and GPCR (G-protein-coupled receptor) agonist S1P (sphingosine 1-phosphate). S1P promotes cell growth, survival and migration, and is a key regulator of lymphocyte trafficking. Inhibition of S1P signalling has been proposed as a strategy for treatment of inflammatory diseases and cancer. In the present paper we describe the discovery and characterization of PF-543, a novel cell-permeant inhibitor of SphK1. PF-543 inhibits SphK1 with a K(i) of 3.6 nM, is sphingosine-competitive and is more than 100-fold selective for SphK1 over the SphK2 isoform. In 1483 head and neck carcinoma cells, which are characterized by high levels of SphK1 expression and an unusually high rate of S1P production, PF-543 decreased the level of endogenous S1P 10-fold with a proportional increase in the level of sphingosine. In contrast with past reports that show that the growth of many cancer cell lines is SphK1-dependent, specific inhibition of SphK1 had no effect on the proliferation and survival of 1483 cells, despite a dramatic change in the cellular S1P/sphingosine ratio. PF-543 was effective as a potent inhibitor of S1P formation in whole blood, indicating that the SphK1 isoform of sphingosine kinase is the major source of S1P in human blood. PF-543 is the most potent inhibitor of SphK1 described to date and it will be useful for dissecting specific roles of SphK1-driven S1P signalling.

  8. Chemical genetic screen in fission yeast reveals roles for vacuolar acidification, mitochondrial fission, and cellular GMP levels in lifespan extension.

    PubMed

    Stephan, Jessica; Franke, Jacqueline; Ehrenhofer-Murray, Ann E

    2013-08-01

    The discovery that genetic mutations in several cellular pathways can increase lifespan has lent support to the notion that pharmacological inhibition of aging pathways can be used to extend lifespan and to slow the onset of age-related diseases. However, so far, only few compounds with such activities have been described. Here, we have conducted a chemical genetic screen for compounds that cause the extension of chronological lifespan of Schizosaccharomyces pombe. We have characterized eight natural products with such activities, which has allowed us to uncover so far unknown anti-aging pathways in S. pombe. The ionophores monensin and nigericin extended lifespan by affecting vacuolar acidification, and this effect depended on the presence of the vacuolar ATPase (V-ATPase) subunits Vma1 and Vma3. Furthermore, prostaglandin J₂ displayed anti-aging properties due to the inhibition of mitochondrial fission, and its effect on longevity required the mitochondrial fission protein Dnm1 as well as the G-protein-coupled glucose receptor Git3. Also, two compounds that inhibit guanosine monophosphate (GMP) synthesis, mycophenolic acid (MPA) and acivicin, caused lifespan extension, indicating that an imbalance in guanine nucleotide levels impinges upon longevity. We furthermore have identified diindolylmethane (DIM), tschimganine, and the compound mixture mangosteen as inhibiting aging. Taken together, these results reveal unanticipated anti-aging activities for several phytochemicals and open up opportunities for the development of novel anti-aging therapies. © 2013 John Wiley & Sons Ltd and the Anatomical Society.

  9. Clinical concerns of immunogenicity produced at cellular levels by biopharmaceuticals following their parenteral administration into human body.

    PubMed

    Tamilvanan, Shunmugaperumal; Raja, Natarajan Livingston; Sa, Biswanath; Basu, Sanat Kumar

    2010-08-01

    Similar to the low molecular weight traditional drugs, biopharmaceuticals are capable of producing not only therapeutic effects but also side effects provided if the dose of these compounds exceeds certain concentration and/or if the exposure duration of these compounds at subtoxic doses is being lengthened. In addition, a major drawback of biopharmaceuticals is the risk of antibody formation. Following the administration of biopharmaceuticals into human body, the formation of antidrug-antibody (ADA) or neutralizing antibody and other general immune system effects (including allergy, anaphylaxis, or serum sickness) are of clinical concern regarding therapeutic efficacy and patient safety. For example, drug-induced neutralizing antibodies to erythropoietin (EPO) result in pure red cell aplasia, whereas drug-induced acquired anti-factor VIII antibodies worsen the pathology associated with hemophilia. Since most of the already developed or under development biopharmaceuticals are to some extent immunogenic, the regulatory agencies insist to conduct potential ADA formation during the drug development process itself. This review encompasses a short overview on the clinical concerns of immunogenicity produced at cellular levels by growth hormone, interferon-alpha, EPO, factor VIII, and factor IX following their parenteral administration into human body. Clinical concerns related to immunogenicity produced by the biosimilar versions of these drugs are also presented wherever possible.

  10. Modulation of p-eIF2α cellular levels and stress granule assembly/disassembly by trehalose

    PubMed Central

    Dimasi, Pasquale; Quintiero, Annamaria; Shelkovnikova, Tatyana A.; Buchman, Vladimir L.

    2017-01-01

    Stress granules (SGs) are an important component of cellular stress response. Compromised assembly of SGs as well as their premature or delayed disassembly affect physiology and survival of cells under stress or during recovery from stress. Consequently, abnormal turnover of SGs has been implicated in the development of various pathologies, including neurodegeneration. We found that pretreatment of cells with a natural disaccharide trehalose, a known autophagy enhancer, delays SG assembly and facilitates their premature post-stress disassembly. Mechanistically, the effect of trehalose on SGs is mediated via the p-eIF2α rather than autophagosome pathway. Trehalose increases pre-stress levels of p-eIF2α and its phosphatase subunits and promotes post-stress translational recovery. Upon prolonged treatment, trehalose impairs basal translation affecting production of transiently expressed proteins. Early translational recovery and SG disassembly induced by trehalose pretreatment can sensitise cells to stress and impair survival. Our study has important implications for the use of trehalose in studies of autophagic clearance of misfolded proteins and for targeting SGs as a possible therapeutic approach in neurodegenerative and other diseases. PMID:28276506

  11. High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level

    PubMed Central

    Gong, Hui; Xu, Dongli; Yuan, Jing; Li, Xiangning; Guo, Congdi; Peng, Jie; Li, Yuxin; Schwarz, Lindsay A.; Li, Anan; Hu, Bihe; Xiong, Benyi; Sun, Qingtao; Zhang, Yalun; Liu, Jiepeng; Zhong, Qiuyuan; Xu, Tonghui; Zeng, Shaoqun; Luo, Qingming

    2016-01-01

    The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei. PMID:27374071

  12. Serum levels of metalloproteinases and their inhibitors during infection with pathogens having integrin receptor-mediated cellular entry.

    PubMed

    Krajinović, Lidija Cvetko; Soprek, Silvija; Korva, Miša; Dželalija, Boris; Rode, Oktavija Đaković; Skerk, Višnja; Avšič-Županc, Tatjana; Markotić, Alemka

    2012-09-01

    Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes with numerous roles in the normal immune response to infection. However, excess MMP activity following infection may lead to immunopathological processes that cause tissue damage. Their activity in normal tissues is subject to tight control, which is regulated by its specific endogenous tissue inhibitors (TIMPs). It is known that MMPs bind to cell surface proteins (e.g. integrins) and that such interactions can have modulatory effects on MMP functionality. The objective of this study was to determine whether there are differences in MMP and TIMP production during the acute phase of infection with different pathogens that use β-integrins as their receptors for cell entry. We measured the total amounts of soluble MMP-2, MMP-9, TIMP-1, and TIMP-2 in the sera from patients infected with Dobrava virus (DOBV), Coxiella burnetii, or uropathogenic Escherichia coli. Statistical analyses were used to correlate MMP/TIMP serum levels with different clinical laboratory parameters. The results showed that both of the bacterial infections generally manifested the stronger effect on MMP production, while in contrast, viral infection introduced stronger changes to metalloproteinase inhibitors. MMPs and TIMPs were significantly correlated with some of the clinical laboratory parameters in both bacterial infections, but no correlations were found for DOBV infection. These findings suggest diverse mechanisms by which MMP activity could be implicated in the pathology of these 2 bacterial infections versus the viral DOBV infection, despite the type of their cellular entry receptors.

  13. Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils

    PubMed Central

    Bakken, Lars R.; Bergaust, Linda; Liu, Binbin; Frostegård, Åsa

    2012-01-01

    Denitrifying prokaryotes use NOx as terminal electron acceptors in response to oxygen depletion. The process emits a mixture of NO, N2O and N2, depending on the relative activity of the enzymes catalysing the stepwise reduction of NO3− to N2O and finally to N2. Cultured denitrifying prokaryotes show characteristic transient accumulation of NO2−, NO and N2O during transition from oxic to anoxic respiration, when tested under standardized conditions, but this character appears unrelated to phylogeny. Thus, although the denitrifying community of soils may differ in their propensity to emit N2O, it may be difficult to predict such characteristics by analysis of the community composition. A common feature of strains tested in our laboratory is that the relative amounts of N2O produced (N2O/(N2+N2O) product ratio) is correlated with acidity, apparently owing to interference with the assembly of the enzyme N2O reductase. The same phenomenon was demonstrated for soils and microbial communities extracted from soils. Liming could be a way to reduce N2O emissions, but needs verification by field experiments. More sophisticated ways to reduce emissions may emerge in the future as we learn more about the regulation of denitrification at the cellular level. PMID:22451108

  14. Elevated levels of alpha-synuclein blunt cellular signal transduction downstream of Gq protein-coupled receptors.

    PubMed

    Volta, Mattia; Lavdas, Alexandros A; Obergasteiger, Julia; Überbacher, Christa; Picard, Anne; Pramstaller, Peter P; Hicks, Andrew A; Corti, Corrado

    2017-01-01

    Alpha-synuclein is central to Parkinson's disease pathogenesis and pathology, however its precise functions are still unclear. It has been shown to bind both PLCβ1 and MAPKs, but how this property influences the downstream signaling of Gq protein-coupled receptors has not been elucidated. Here we show that recombinant expression of alpha-synuclein in human neuroblastoma cells enhances cellular levels of PLCβ1 but blunts its signaling pathway, preventing the agonist-dependent rise of cytoplasmic Ca(2+). In addition, overexpressing alpha-synuclein abolishes the activation of ERK1/2 upon agonist stimulation, indicating an upstream action in the signal transduction pathway. This data demonstrates that alpha-synuclein, when recombinantly expressed, interferes with the normal signaling of Gq-protein coupled receptors, which are then dysfunctional. Since many neurotransmitter systems utilize these receptor signaling pathways to mediate different abilities affected in Parkinson's disease, we argue this novel perspective might be helpful in designing treatment strategies for some of the non-motor symptoms in Parkinson's disease and synucleinopathies.

  15. Platelet-Rich Plasma Increases the Levels of Catabolic Molecules and Cellular Dedifferentiation in the Meniscus of a Rabbit Model.

    PubMed

    Lee, Hye-Rim; Shon, Oog-Jin; Park, Se-Il; Kim, Han-Jun; Kim, Sukyoung; Ahn, Myun-Whan; Do, Sun Hee

    2016-01-16

    Despite the susceptibility to frequent intrinsic and extrinsic injuries, especially in the inner zone, the meniscus does not heal spontaneously owing to its poor vascularity. In this study, the effect of platelet-rich plasma (PRP), containing various growth factors, on meniscal mechanisms was examined under normal and post-traumatic inflammatory conditions. Isolated primary meniscal cells of New Zealand white (NZW) rabbits were incubated for 3, 10, 14 and 21 days with PRP(-), 10% PRP (PRP(+)), IL(+) or IL(+)PRP(+). The meniscal cells were collected and examined using reverse-transcription polymerase chain reaction (RT-PCR). Culture media were examined by immunoblot analyses for matrix metalloproteinases (MMP) catabolic molecules. PRP containing growth factors improved the cellular viability of meniscal cells in a concentration-dependent manner at Days 1, 4 and 7. However, based on RT-PCR, meniscal cells demonstrated dedifferentiation, along with an increase in type I collagen in the PRP(+) and in IL(+)PRP(+). In PRP(+), the aggrecan expression levels were lower than in the PRP(-) until Day 21. The protein levels of MMP-1 and MMP-3 were higher in each PRP group, i.e., PRP(+) and IL(+)PRP(+), at each culture time. A reproducible 2-mm circular defect on the meniscus of NZW rabbit was used to implant fibrin glue (control) or PRP in vivo. After eight weeks, the lesions in the control and PRP groups were occupied with fibrous tissue, but not with meniscal cells. This study shows that PRP treatment of the meniscus results in an increase of catabolic molecules, especially those related to IL-1α-induced inflammation, and that PRP treatment for an in vivo meniscus injury accelerates fibrosis, instead of meniscal cartilage.

  16. Increased Cellular NAD+ Level through NQO1 Enzymatic Action Has Protective Effects on Bleomycin-Induced Lung Fibrosis in Mice

    PubMed Central

    Oh, Gi-Su; Lee, Su-Bin; Karna, Anjani; Kim, Hyung-Jin; Shen, AiHua; Pandit, Arpana; Lee, SeungHoon

    2016-01-01

    Background Idiopathic pulmonary fibrosis is a common interstitial lung disease; it is a chronic, progressive, and fatal lung disease of unknown etiology. Over the last two decades, knowledge about the underlying mechanisms of pulmonary fibrosis has improved markedly and facilitated the identification of potential targets for novel therapies. However, despite the large number of antifibrotic drugs being described in experimental pre-clinical studies, the translation of these findings into clinical practices has not been accomplished yet. NADH:quinone oxidoreductase 1 (NQO1) is a homodimeric enzyme that catalyzes the oxidation of NADH to NAD+ by various quinones and thereby elevates the intracellular NAD+ levels. In this study, we examined the effect of increase in cellular NAD+ levels on bleomycin-induced lung fibrosis in mice. Methods C57BL/6 mice were treated with intratracheal instillation of bleomycin. The mice were orally administered with β-lapachone from 3 days before exposure to bleomycin to 1-3 weeks after exposure to bleomycin. Bronchoalveolar lavage fluid (BALF) was collected for analyzing the infiltration of immune cells. In vitro, A549 cells were treated with transforming growth factor β1 (TGF-β1) and β-lapachone to analyze the extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT). Results β-Lapachone strongly attenuated bleomycin-induced lung inflammation and fibrosis, characterized by histological staining, infiltrated immune cells in BALF, inflammatory cytokines, fibrotic score, and TGF-β1, α-smooth muscle actin accumulation. In addition, β-lapachone showed a protective role in TGF-β1–induced ECM expression and EMT in A549 cells. Conclusion Our results suggest that β-lapachone can protect against bleomycin-induced lung inflammation and fibrosis in mice and TGF-β1–induced EMT in vitro, by elevating the NAD+/NADH ratio through NQO1 activation. PMID:27790277

  17. Cellular and network-level adaptations to in utero methadone exposure along the ventral respiratory column in the neonate rat.

    PubMed

    Gourévitch, Boris; Cai, Jun; Mellen, Nicholas

    2016-03-20

    Neonatal abstinence syndrome (NAS) occurs in babies chronically exposed to opioids during pregnancy. NAS shares features with opioid withdrawal symptoms seen in adults, including autonomic dysregulation. Here, the effect of low-dose in utero methadone (MTD) exposure on respiration-modulated networks along the ventral respiratory column (VRC) in ventrolateral medulla was investigated in the neonate Sprague-Dawley rat. MTD was administered via drinking water (3mg/kg/day in drinking water of the mother E7-E21). Lower expression levels of myelin-associated proteins phosphorylated axonal neurofilament subunit H (pNFH), 2',3'-Cyclicnucleotide 3'-phosphodiesterase (CNPase) and myelin basic protein (MBP), in MTD-exposed pups compared to controls at P3, P6 and P10 indicated MTD transport across the placenta. We investigated whether in utero MTD exposure led to network-level excitability changes consistent with tolerance, and also probed for changes in endogenous opioid modulation of respiratory networks. To this end, high-speed (45.5Hz) optical recordings of respiratory network activity in control and MTD-exposed neonate (P0-P2) pups before and during administration of the μ-opioid receptor antagonist naloxone (NAL; 10μM) were carried out. Spike rate was estimated from optical traces via deconvolution, and coupling between all neuron pairs in recorded networks was quantified using the normalized transfer entropy (NTE). Recordings of local networks along the VRC, together with recordings of respiratory output from ventral root C1 did not reveal changes in respiratory activity at the system level, but cellular and network changes in MTD-exposed pups were consistent with the development of opioid tolerance. MTD-exposed pups were found to have i. higher neuronal firing rates; ii. higher covariance between neuronal activity and motor output; iii. more bidirectionally and unidirectionally coupled neurons, and fewer uncoupled neurons; iv. stronger coupling and shorter

  18. Elevated serum levels of T3 without metabolic effect in nutritionally deficient rats, attributable to reduced cellular uptake of T3

    SciTech Connect

    Okamura, K.; Taurog, A.; DiStefano, J.J.

    1981-08-01

    Rats receiving a nutritionally deficient diet displayed markedly elevated serum free T3 levels but showed no increase in oxygen consumption. This was associated with greatly reduced ratios of hepatic cellular and nuclear /sub 125/I-T3 to serum /sub 125/I-T3. Kinetic data supported the conclusion that cellular uptake of T3 was decreased in the nutritionally deficient rats. The lack of metabolic effect, despite the elevated serum T3 levels, is attributable to reduced availability of serum T3 to tissue nuclear receptor sites.

  19. Holocene relative sea-level change in Hiroshima Bay, Japan: A semi-quantitative reconstruction based on ostracodes

    USGS Publications Warehouse

    Yasuhara, Moriaki; Seto, Koji

    2006-01-01

    Holocene relative sea-level changes in Hiroshima Bay were reconstructed from fossil ostracodes from a core, using a semi-quantitative method. In Hiroshima Bay, relative sea level rose rapidly (about 25 m) between ca. 9000 cal yr BP and ca. 5800 cal yr BP, after which it gradually fell (about 5 m) to its present level. The peak in relative sea level occurred at ca. 5800 cal yr BP. The sea-level curve for Hiroshima Bay is similar to curves for tectonically stable areas of Japan (e.g., Osaka Bay). ?? by the Palaeontological Society of Japan.

  20. Determination of Cellular Phosphatidylinositol-3-phosphate (PI3P) Levels Using a Fluorescently Labelled Selective PI3P Binding Domain (PX)

    PubMed Central

    Munson, Michael J.; Ganley, Ian G.

    2017-01-01

    The lipid Phosphatidylinositol-3-phosphate [PtdIns3P or PI(3)P] plays many membrane trafficking roles and is primarily produced by the Class III PI3K, VPS34. Determining the level of cellular PI(3)P however can be complex. Extraction of cellular lipids by methanol/chloroform can struggle to separate and identify distinct phospholipid species. Alternately mass spectrometry may be utilised but this requires significant set up of specialised equipment and time to utilise. Use of a PI(3)P-binding-specific recombinant protein domain is a quick method for ascertaining cellular PI(3)P levels and can also allow visualisation of sub-cellular localisation. The PX domain of p40phox (herein referred to as PX) is very specific for PI(3)P over other phospholipid species (Kanai et al., 2001). However, expressing PX directly in cells can be problematic, as it will act in a dominant negative manner to bind and sequester PI(3)P with greater affinity than endogenous proteins, thus disturbing cellular pathways and the normal balance of PI(3)P levels. Using fluorescently labelled PX following cell fixation is therefore more suitable, as it is able to highlight PI(3)P rich structures without risk of perturbing the system. PMID:28127574

  1. Individuals with autism have higher 8-Iso-PGF2α levels than controls, but no correlation with quantitative assay of Paraoxonase 1 serum levels.

    PubMed

    Pop, Bianca; Niculae, Alexandru-Ștefan; Pop, Tudor Lucian; Răchișan, Andreea Liana

    2017-08-14

    Autism spectrum disorder (ASD) represents a very large set of neurodevelopmental issues with diverse clinical outcomes. Various hypotheses have been put forth for the etiology of autism spectrum disorder, including issues pertaining to oxidative stress. In this study, we conducted measurements of serum 8-Iso-Prostaglanding F2 α (8-iso-PGF2α, which is the results of non-enzimatically mediated polyunsaturated fatty acid oxidation) in a population of individuals with autism and a control group of age and sex matched controls. A quantitative assay of Paraoxonase 1 (PON1) was conducted. Data regarding comorbidities, structural MRI scans, medication, intelligence quotient (IQ) and Childhood Autism Rating Scale scores (CARS) were also included in our study. Our results show that patients diagnosed with autism have higher levels of 8-iso-PGF2α than their neurotypical counterparts. Levels of this particular metabolite, however, do not correlate with quantitative serum levels of Paraoxonase 1, which has been shown to be altered in individuals with autism. Neither 8-iso-PGF2α nor quantitative levels of PON1 provide any meaningful correlation with clinical or neuroimaging data in this study group. Future research should focus on providing data regarding PON 1 phenotype, in addition to standard quantitative measurements, in relation to 8-iso-PGF2α as well as other clinical and structural brain findings.

  2. Quantitation of Phenol Levels in Oil of Wintergreen Using Gas Chromatography-Mass Spectrometry with Selected Ion Monitoring

    ERIC Educational Resources Information Center

    Sobel, Robert M.; Ballantine, David S.; Ryzhov, Victor

    2005-01-01

    Industrial application of gas chromatography-mass spectrometry (GC-MS) analysis is a powerful technique that could be used to elucidate components of a complex mixture while offering the benefits of high-precision quantitative analysis. The natural wintergreen oil is examined for its phenol concentration to determine the level of refining…

  3. Quantitation of Phenol Levels in Oil of Wintergreen Using Gas Chromatography-Mass Spectrometry with Selected Ion Monitoring

    ERIC Educational Resources Information Center

    Sobel, Robert M.; Ballantine, David S.; Ryzhov, Victor

    2005-01-01

    Industrial application of gas chromatography-mass spectrometry (GC-MS) analysis is a powerful technique that could be used to elucidate components of a complex mixture while offering the benefits of high-precision quantitative analysis. The natural wintergreen oil is examined for its phenol concentration to determine the level of refining…

  4. The Role of Extracellular Binding Proteins in the Cellular Uptake of Drugs: Impact on Quantitative In Vitro-to-In Vivo Extrapolations of Toxicity and Efficacy in Physiologically Based Pharmacokinetic-Pharmacodynamic Research.

    PubMed

    Poulin, Patrick; Burczynski, Frank J; Haddad, Sami

    2016-02-01

    A critical component in the development of physiologically based pharmacokinetic-pharmacodynamic (PBPK/PD) models for estimating target organ dosimetry in pharmacology and toxicology studies is the understanding of the uptake kinetics and accumulation of drugs and chemicals at the cellular level. Therefore, predicting free drug concentrations in intracellular fluid will contribute to our understanding of concentrations at the site of action in cells in PBPK/PD research. Some investigators believe that uptake of drugs in cells is solely driven by the unbound fraction; conversely, others argue that the protein-bound fraction contributes a significant portion of the total amount delivered to cells. Accordingly, the current literature suggests the existence of a so-called albumin-mediated uptake mechanism(s) for the protein-bound fraction (i.e., extracellular protein-facilitated uptake mechanisms) at least in hepatocytes and cardiac myocytes; however, such mechanism(s) and cells from other organs deserve further exploration. Therefore, the main objective of this present study was to discuss further the implication of potential protein-facilitated uptake mechanism(s) on drug distribution in cells under in vivo conditions. The interplay between the protein-facilitated uptake mechanism(s) and the effects of a pH gradient, metabolism, transport, and permeation limitation potentially occurring in cells was also discussed, as this should violate the basic assumption on similar free drug concentration in cells and plasma. This was made because the published equations used to calculate drug concentrations in cells in a PBPK/PD model did not consider potential protein-facilitated uptake mechanism(s). Consequently, we corrected some published equations for calculating the free drug concentrations in cells compared with plasma in PBPK/PD modeling studies, and we proposed a refined strategy for potentially performing more accurate quantitative in vitro-to-in vivo extrapolations

  5. Hyperoxia-Induced Protein Alterations in Renal Rat Tissue: A Quantitative Proteomic Approach to Identify Hyperoxia-Induced Effects in Cellular Signaling Pathways

    PubMed Central

    Hinkelbein, Jochen; Böhm, Lennert; Spelten, Oliver; Sander, David; Soltész, Stefan; Braunecker, Stefan

    2015-01-01

    Introduction. In renal tissue as well as in other organs, supranormal oxygen pressure may lead to deleterious consequences on a cellular level. Additionally, hyperoxia-induced effect in cells and related free radicals may potentially contribute to renal failure. The aim of this study was to analyze time-dependent alterations of rat kidney protein expression after short-term normobaric hyperoxia using proteomics and bioinformatic approaches. Material and Methods. N = 36 Wistar rats were randomized into six different groups: three groups with normobaric hyperoxia (exposure to 100% oxygen for 3 h) and three groups with normobaric normoxia (NN; room air). After hyperoxia exposure, kidneys were removed immediately, after 3 days and after 7 days. Kidney lysates were analyzed by two-dimensional gel electrophoresis followed by peptide mass fingerprinting using tandem mass spectrometry. Statistical analysis was performed with DeCyder 2D software (p < 0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis and PathwayStudio). Results. Expression of 14 proteins was significantly altered (p < 0.01): eight proteins (MEP1A_RAT, RSSA_RAT, F16P1_RAT, STML2_RAT, BPNT1_RAT, LGMN_RAT, ATPA_RAT, and VDAC1_RAT) were downregulated and six proteins (MTUS1_RAT, F16P1_RAT, ACTG_RAT, ACTB_RAT, 2ABA_RAT, and RAB1A_RAT) were upregulated. Bioinformatic analyses revealed an association of regulated proteins with inflammation. Conclusions. Significant alterations in renal protein expression could be demonstrated for up to 7 days even after short-term hyperoxia. The identified proteins indicate an association with inflammation signaling cascades. MEP1A and VDAC1 could be promising candidates to identify hyperoxic injury in kidney cells. PMID:26106253

  6. Quantitative Study of Emotional Intelligence and Communication Levels in Information Technology Professionals

    ERIC Educational Resources Information Center

    Hendon, Michalina

    2016-01-01

    This quantitative non-experimental correlational research analyzes the relationship between emotional intelligence and communication due to the lack of this research on information technology professionals in the U.S. One hundred and eleven (111) participants completed a survey that measures both the emotional intelligence and communication…

  7. Quantitative Study of Emotional Intelligence and Communication Levels in Information Technology Professionals

    ERIC Educational Resources Information Center

    Hendon, Michalina

    2016-01-01

    This quantitative non-experimental correlational research analyzes the relationship between emotional intelligence and communication due to the lack of this research on information technology professionals in the U.S. One hundred and eleven (111) participants completed a survey that measures both the emotional intelligence and communication…

  8. Quantitative relationships between huntingtin levels, polyglutamine length, inclusion body formation, and neuronal death provide novel insight into Huntington’s disease molecular pathogenesis

    PubMed Central

    Miller, Jason; Arrasate, Montserrat; Shaby, Benjamin A.; Mitra, Siddhartha; Masliah, Eliezer; Finkbeiner, Steven

    2010-01-01

    An expanded polyglutamine (polyQ) stretch in the protein huntingtin (htt) induces self-aggregation into inclusion bodies (IBs) and causes Huntington’s disease (HD). Defining precise relationships between early observable variables and neuronal death at the molecular and cellular levels should improve our understanding of HD pathogenesis. Here, we utilized an automated microscope that can track thousands of neurons individually over their entire lifetime to quantify interconnected relationships between early variables, such as htt levels, polyQ length, and IB formation, and neuronal death in a primary striatal model of HD. The resulting model revealed that: mutant htt increases the risk of death by tonically interfering with homeostatic coping mechanisms rather than producing accumulated damage to the neuron; htt toxicity is saturable; the rate limiting steps for inclusion body formation and death can be traced to different conformational changes in monomeric htt; and IB formation reduces the impact of a neuron’s starting levels of htt on its risk of death. Finally, the model that emerges from our quantitative measurements places critical limits on the potential mechanisms by which mutant htt might induce neurodegeneration, which should help direct future research. PMID:20685997

  9. Prospective Elementary Science Teachers' Understanding of Photosynthesis and Cellular Respiration in the Context of Multiple Biological Levels as Nested Systems

    ERIC Educational Resources Information Center

    Akçay, Süleyman

    2017-01-01

    In this study, Turkish prospective elementary science teachers' understanding of photosynthesis and cellular respiration has been analysed within the contexts of ecosystem knowledge, organism knowledge and interconnection knowledge (IK). In the analysis, concept maps developed by 74 prospective teachers were used. The study was carried out with…

  10. Cellular Up-regulation of Nedd4 Family Interacting Protein 1 (Ndfip1) using Low Levels of Bioactive Cobalt Complexes*

    PubMed Central

    Schieber, Christine; Howitt, Jason; Putz, Ulrich; White, Jonathan M.; Parish, Clare L.; Donnelly, Paul S.; Tan, Seong-Seng

    2011-01-01

    The delivery of metal ions using cell membrane-permeable metal complexes represents a method for activating cellular pathways. Here, we report the synthesis and characterization of new [CoIII(salen)(acac)] complexes capable of up-regulating the ubiquitin ligase adaptor protein Ndfip1. Ndfip1 is a neuroprotective protein that is up-regulated in the brain after injury and functions in combination with Nedd4 ligases to ubiquitinate harmful proteins for removal. We previously showed that Ndfip1 can be increased in human neurons using CoCl2 that is toxic at high concentration. Here we demonstrate a similar effect can be achieved by low concentrations of synthetic CoIII complexes that are non-toxic and designed to be activated following cellular entry. Activation is achieved by intracellular reduction of CoIII to CoII leading to release of CoII ions for Ndfip1 up-regulation. The cellular benefit of Ndfip1 up-regulation by CoIII complexes includes demonstrable protection against cell death in SH-SY5Y cells during stress. In vivo, focal delivery of CoIII complexes into the adult mouse brain was observed to up-regulate Ndfip1 in neurons. These results demonstrate that a cellular response pathway can be advantageously manipulated by chemical modification of metal complexes, and represents a significant step of harnessing low concentration metal complexes for therapeutic benefit. PMID:21187286

  11. Prospective Elementary Science Teachers' Understanding of Photosynthesis and Cellular Respiration in the Context of Multiple Biological Levels as Nested Systems

    ERIC Educational Resources Information Center

    Akçay, Süleyman

    2017-01-01

    In this study, Turkish prospective elementary science teachers' understanding of photosynthesis and cellular respiration has been analysed within the contexts of ecosystem knowledge, organism knowledge and interconnection knowledge (IK). In the analysis, concept maps developed by 74 prospective teachers were used. The study was carried out with…

  12. Quantitative Genetic Analysis of Biomass and Wood Chemistry of Populus under Different Nitrogen Levels

    SciTech Connect

    Novaes, E.; Osorio, L.; Drost, D. R.; Miles, B. L.; Boaventura-Novaes, C. R. D.; Benedict, C.; Dervinis, C.; Yu, Q.; Sykes, R.; Davis, M.; Martin, T. A.; Peter, G. F.; Kirst, M.

    2009-01-01

    The genetic control of carbon allocation and partitioning in woody perennial plants is poorly understood despite its importance for carbon sequestration, biofuels and other wood-based industries. It is also unclear how environmental cues, such as nitrogen availability, impact the genes that regulate growth, biomass allocation and wood composition in trees. We phenotyped 396 clonally replicated genotypes of an interspecific pseudo-backcross pedigree of Populus for wood composition and biomass traits in above- and below-ground organs. The loci that regulate growth, carbon allocation and partitioning under two nitrogen conditions were identified, defining the contribution of environmental cues to their genetic control. Sixty-three quantitative trait loci were identified for the 20 traits analyzed. The majority of quantitative trait loci are specific to one of the two nitrogen treatments, demonstrating significant nitrogen-dependent genetic control. A highly significant genetic correlation was observed between plant growth and lignin/cellulose composition, and quantitative trait loci co-localization identified the genomic position of potential pleiotropic regulators. Pleiotropic loci linking higher growth rates to wood with less lignin are excellent targets to engineer tree germplasm improved for pulp, paper and cellulosic ethanol production. The causative genes are being identified with a genetical genomics approach.

  13. Deletion of Pim Kinases Elevates the Cellular Levels of Reactive Oxygen Species and Sensitizes to K-Ras-Induced Cell Killing

    PubMed Central

    Song, Jin H.; An, Ningfei; Chatterjee, Shilpak; Kistner-Griffin, Emily; Mahajan, Sandeep; Mehrotra, Shikhar; Kraft, Andrew S.

    2014-01-01

    The Pim protein kinases contribute to transformation by enhancing the activity of oncogenic Myc and Ras, which drives significant metabolic changes during tumorigenesis. In this report, we demonstrate that mouse embryo fibroblasts (MEFs) lacking all three isoforms of Pim protein kinases, triple knockout (TKO), cannot tolerate the expression of activated K-Ras (K-RasG12V) and undergo cell death. Transduction of K-RasG12V into these cells markedly increased the level of cellular reactive oxygen species (ROS). The addition of N-acetyl cysteine attenuates ROS production and reversed the cytotoxic effects of K-RasG12V in the TKO MEFs. The altered cellular redox state caused by the loss of Pim occurred as a result of lower levels of metabolic intermediates in the glycolytic and pentose phosphate pathways as well as abnormal mitochondrial oxidative phosphorylation. TKO MEFs exhibit reduced levels of superoxide dismutase (Sod), glutathione peroxidase 4 (Gpx4) and peroxiredoxin 3 (Prdx3) that render them susceptible to killing by K-RasG12V-mediated ROS production. In contrast, the transduction of c-Myc into TKO cells can overcome the lack of Pim protein kinases by regulating cellular metabolism and Sod2. In the absence of the Pim kinases, c-Myc transduction permitted K-RasG12V-induced cell growth by decreasing Ras-induced cellular ROS levels. These results demonstrate that the Pim protein kinases play an important role in regulating cellular redox, metabolism and K-Ras-stimulated cell growth. PMID:25241892

  14. Temporal dynamics of changes in reactive oxygen species (ROS) levels and cellular morphology are coordinated during complementary chromatic acclimation in Fremyella diplosiphon.

    PubMed

    Singh, Shailendra P; Miller, Haley L; Montgomery, Beronda L

    2013-10-14

    Fremyella diplosiphon alters the phycobiliprotein composition of its light-harvesting complexes, i.e., phycobilisomes, and its cellular morphology in response to changes in the prevalent wavelengths of light in the external environment in a phenomenon known as complementary chromatic acclimation (CCA). The organism primarily responds to red light (RL) and green light (GL) during CCA to maximize light absorption for supporting optimal photosynthetic efficiency. Recently, we found that RL-characteristic spherical cell morphology is associated with higher levels of reactive oxygen species (ROS) compared to growth under GL where lower ROS levels and rectangular cell shape are observed. The RL-dependent association of increased ROS levels with cellular morphology was demonstrated by treating cells with a ROS-scavenging antioxidant which resulted in the observation of GL-characteristic rectangular morphology under RL. To gain additional insights into the involvement of ROS in impacting cellular morphology changes during CCA, we conducted experiments to study the temporal dynamics of changes in ROS levels and cellular morphology during transition to growth under RL or GL. Alterations in ROS levels and cell morphology were found to be correlated with each other at early stages of acclimation of low white light-grown cells to growth under high RL or cells transitioned between growth in RL and GL. These results provide further general evidence that significant RL-dependent increases in ROS levels are temporally correlated with changes in morphology toward spherical. Future studies will explore the light-dependent mechanisms by which ROS levels may be regulated and the direct impacts of ROS on the observed morphology changes.

  15. Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles.

    PubMed

    Gonda, Kohsuke; Miyashita, Minoru; Watanabe, Mika; Takahashi, Yayoi; Goda, Hideki; Okada, Hisatake; Nakano, Yasushi; Tada, Hiroshi; Amari, Masakazu; Ohuchi, Noriaki

    2012-09-28

    The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal

  16. Phg1/TM9 Proteins Control Intracellular Killing of Bacteria by Determining Cellular Levels of the Kil1 Sulfotransferase in Dictyostelium

    PubMed Central

    Le Coadic, Marion; Froquet, Romain; Lima, Wanessa C.; Dias, Marco; Marchetti, Anna; Cosson, Pierre

    2013-01-01

    Dictyostelium discoideum has largely been used to study phagocytosis and intracellular killing of bacteria. Previous studies have shown that Phg1A, Kil1 and Kil2 proteins are necessary for efficient intracellular killing of Klebsiella bacteria. Here we show that in phg1a KO cells, cellular levels of lysosomal glycosidases and lysozyme are decreased, and lysosomal pH is increased. Surprisingly, overexpression of Kil1 restores efficient killing in phg1a KO cells without correcting these lysosomal anomalies. Conversely, kil1 KO cells are defective for killing, but their enzymatic content and lysosomal pH are indistinguishable from WT cells. The killing defect of phg1a KO cells can be accounted for by the observation that in these cells the stability and the cellular amount of Kil1 are markedly reduced. Since Kil1 is the only sulfotransferase characterized in Dictyostelium, an (unidentified) sulfated factor, defective in both phg1a and kil1 KO cells, may play a key role in intracellular killing of Klebsiella bacteria. In addition, Phg1B plays a redundant role with Phg1A in controlling cellular amounts of Kil1 and intracellular killing. Finally, cellular levels of Kil1 are unaffected in kil2 KO cells, and Kil1 overexpression does not correct the killing defect of kil2 KO cells, suggesting that Kil2 plays a distinct role in intracellular killing. PMID:23301051

  17. Investigation of Elemental Mass Spectrometry in Pharmacology for Peptide Quantitation at Femtomolar Levels.

    PubMed

    Cordeau, Emmanuelle; Arnaudguilhem, Carine; Bouyssiere, Brice; Hagège, Agnès; Martinez, Jean; Subra, Gilles; Cantel, Sonia; Enjalbal, Christine

    2016-01-01

    In the search of new robust and environmental-friendly analytical methods able to answer quantitative issues in pharmacology, we explore liquid chromatography (LC) associated with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biological matrices. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold standard to measure organic compound concentrations in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacological assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labelling of the peptide of interest. Selenium, that is scarcely present in biological media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chemistry and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacology is challenging due to the very high salt content and organic material complexity of the samples that produces polyatomic aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatographic separation was found compulsory. Noteworthy, we aimed to develop a straightforward quantitative protocol that can be performed in any laboratory equipped with a standard macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 femtomoles of injected Se) was achieved, the samples issued from the pharmacological assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and

  18. Investigation of Elemental Mass Spectrometry in Pharmacology for Peptide Quantitation at Femtomolar Levels

    PubMed Central

    Cordeau, Emmanuelle; Arnaudguilhem, Carine; Bouyssiere, Brice; Hagège, Agnès; Martinez, Jean; Subra, Gilles; Cantel, Sonia

    2016-01-01

    In the search of new robust and environmental-friendly analytical methods able to answer quantitative issues in pharmacology, we explore liquid chromatography (LC) associated with elemental mass spectrometry (ICP-MS) to monitor peptides in such complex biological matrices. The novelty is to use mass spectrometry to replace radiolabelling and radioactivity measurements, which represent up-to now the gold standard to measure organic compound concentrations in life science. As a proof of concept, we choose the vasopressin (AVP)/V1A receptor system for model pharmacological assays. The capacity of ICP-MS to provide highly sensitive quantitation of metallic and hetero elements, whatever the sample medium, prompted us to investigate this technique in combination with appropriate labelling of the peptide of interest. Selenium, that is scarcely present in biological media, was selected as a good compromise between ICP-MS response, covalent tagging ability using conventional sulfur chemistry and peptide detection specificity. Applying selenium monitoring by elemental mass spectrometry in pharmacology is challenging due to the very high salt content and organic material complexity of the samples that produces polyatomic aggregates and thus potentially mass interferences with selenium detection. Hyphenation with a chromatographic separation was found compulsory. Noteworthy, we aimed to develop a straightforward quantitative protocol that can be performed in any laboratory equipped with a standard macrobore LC-ICP-MS system, in order to avoid time-consuming sample treatment or special implementation of instrumental set-up, while allowing efficient suppression of all mass interferences to reach the targeted sensitivity. Significantly, a quantification limit of 57 ng Se L-1 (72 femtomoles of injected Se) was achieved, the samples issued from the pharmacological assays being directly introduced into the LC-ICP-MS system. The established method was successfully validated and

  19. Paper diagnostic device for quantitative electrochemical detection of ricin at picomolar levels.

    PubMed

    Cunningham, Josephine C; Scida, Karen; Kogan, Molly R; Wang, Bo; Ellington, Andrew D; Crooks, Richard M

    2015-01-01

    We report a paper-based assay platform for detection of ricin a chain. The paper platform is assembled by simple origami paper folding. The sensor is based on quantitative, electrochemical detection of silver nanoparticle labels linked to a magnetic microbead support via a ricin immunosandwich. Importantly, ricin was detected at concentrations as low as 34 pM. Additionally, the assay is robust, even in the presence of 100-fold excess hoax materials. Finally, the device is easily remediated after use by incineration. The cost of the device, not including reagents, is just $0.30. The total assay time, including formation of the immunosandwich, is 9.5 min.

  20. Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

    SciTech Connect

    Gonda, Kohsuke; Miyashita, Minoru; Watanabe, Mika; Takahashi, Yayoi; Goda, Hideki; Okada, Hisatake; Nakano, Yasushi; Tada, Hiroshi; Amari, Masakazu; Ohuchi, Noriaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Organic fluorescent material-assembled nanoparticles for IHC were prepared. Black-Right-Pointing-Pointer New nanoparticle fluorescent intensity was 10.2-fold greater than Qdot655. Black-Right-Pointing-Pointer Nanoparticle staining analyzed a wide range of ER expression levels in tissue. Black-Right-Pointing-Pointer Nanoparticle staining enhanced the quantitative sensitivity for ER diagnosis. -- Abstract: The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3 Prime -diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to

  1. Bioimage informatics for understanding spatiotemporal dynamics of cellular processes.

    PubMed

    Yang, Ge

    2013-01-01

    The inner environment of the cell is highly dynamic and heterogeneous yet exquisitely organized. Successful completion of cellular processes within this environment depends on the right molecules or molecular complexes to function at the right place at the right time. Understanding spatiotemporal behaviors of cellular processes is therefore essential to understanding their molecular mechanisms at the systems level. These behaviors are usually visualized and recorded using imaging techniques. However, to infer from them systems-level molecular mechanisms, computational analysis and understanding of recorded image data is crucial, not only for acquiring quantitative behavior measurements but also for comprehending complex interactions among the molecules or molecular complexes involved. The technology of computational analysis and understanding of biological images is often referred to simply as bioimage informatics. This article introduces fundamentals of bioimage informatics for understanding spatiotemporal dynamics of cellular processes and reviews recent advances on this topic. Basic bioimage informatics concepts and techniques for characterizing spatiotemporal cell dynamics are introduced first. Studies on specific cellular processes such as cell migration and signal transduction are then used as examples to analyze and summarize recent advances, with the focus on transforming quantitative measurements of spatiotemporal cellular behaviors into knowledge of underlying molecular mechanisms. Despite the advances made, substantial technological challenges remain, especially in representation of spatiotemporal cellular behaviors and inference of systems-level molecular mechanisms. These challenges are briefly discussed. Overall, understanding spatiotemporal cell dynamics will provide critical insights into how specific cellular processes as well as the entire inner cellular environment are dynamically organized and regulated.

  2. Influence of the side-chain length on the cellular uptake and the cytotoxicity of rhenium triscarbonyl derivatives: a bimodal infrared and luminescence quantitative study.

    PubMed

    Clède, Sylvain; Lambert, François; Saint-Fort, Rénette; Plamont, Marie-Aude; Bertrand, Hélène; Vessières, Anne; Policar, Clotilde

    2014-07-07

    Rhenium triscarbonyl complexes fac-[Re(CO)3 (N^N)] with appropriate ancillary N^N ligands are relevant for fluorescent bio-imaging. Recently, we have shown that [Re(CO)3 ] cores can also be efficiently mapped inside cells using their IR signature and that they can thus be used in a bimodal approach. To describe them we have coined the term SCoMPIs for single-core multimodal probes for imaging. In the context of the use of these SCoMPIs in bio-imaging, the questions of their cellular uptake and cytotoxicity are critical. We report here a series of compounds derived from the [Re(CO)3 Cl(pyta)] core (pyta=4-(2-pyridyl)-1,2,3-triazole). The pyta ligand is of interest because it can be easily functionalized. Aliphatic side chains (C4 , C8 , and C12 ) were appended to this core. A correlative study involving IR and luminescence was performed to monitor and quantify their cellular internalization. We studied the relationship between lipophilicity (log P(o/w)), cytotoxicity (IC50 ), and cellular uptake, and we showed that both uptake and cytotoxicity increase with the length of the side chain, with a higher uptake for the C12 derivative. This study stresses the distinction that has to be made between apparent toxicity, determined as an incubation concentration IC50 , and intrinsic toxicity. Indeed, the intrinsic toxicity of a compound can remain hidden if it is not cell permeable. Therefore it must be kept in mind that IC50 values are composite values, reflecting both cellular uptake and intrinsic toxicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Quantitative analyses of cellularity and proliferative activity reveals the dynamics of the central canal lining during postnatal development of the rat.

    PubMed

    Alexovič Matiašová, Anna; Ševc, Juraj; Tomori, Zoltán; Gombalová, Zuzana; Gedrová, Štefánia; Daxnerová, Zuzana

    2017-02-15

    According to previous opinion, the derivation of neurons and glia from the central canal (CC) lining of the spinal cord in rodents should occur in the embryonic period. Reports of the mitotic activity observed in the lining during postnatal development have often been contradictory, and proliferation was ascribed to the generation of ependymocytes, which are necessary for the elongation of CC walls. Our study quantifies the intensity of proliferation and determines the cellularity of the CC lining in reference to lumbar spinal segment L4 during the postnatal development of rats. The presence of dividing cells peaks in the CC lining on postnatal day 8 (P8), with division occurring in 19.2% ± 3.2% of cells. In adult rats, 3.6% ± 0.9% of cells still proliferate, whereas, in mice, 10.3% ± 2.3% of cells at P8 and only 0.6% ± 0.2% of cells in the CC lining in adulthood are proliferating. In the rat, the length of the cell cycle increases from 100.3 ± 35.7 hours at P1 to 401.4 ± 80.6 hours at P43, with a sudden extension between P15 and P22. Despite the intensive proliferation, the total cellularity of the CC lining at the L4 spinal segment significantly descended in from P8 to P15. According to our calculations, the estimated cellularity was significantly higher compared with the measured cellularity of the CC lining at P15. Our results indicate that CC lining serves as a source of cells beyond ependymal cells during the first postnatal weeks of the rat. J. Comp. Neurol. 525:693-707, 2017. © 2016 Wiley Periodicals, Inc.

  4. Quantitative and simultaneous non-invasive measurement of skin hydration and sebum levels

    PubMed Central

    Ezerskaia, Anna; Pereira, S. F.; Urbach, H. Paul; Verhagen, Rieko; Varghese, Babu

    2016-01-01

    We report a method on quantitative and simultaneous non-contact in-vivo hydration and sebum measurements of the skin using an infrared optical spectroscopic set-up. The method utilizes differential detection with three wavelengths 1720, 1750, and 1770 nm, corresponding to the lipid vibrational bands that lay “in between” the prominent water absorption bands. We have used an emulsifier containing hydro- and lipophilic components to mix water and sebum in various volume fractions which was applied to the skin to mimic different oily-dry skin conditions. We also measured the skin sebum and hydration values on the forehead under natural conditions and its variations to external stimuli. Good agreement was found between our experimental results and reference values measured using conventional biophysical methods such as Corneometer and Sebumeter. PMID:27375946

  5. Pleiotropy analysis of quantitative traits at gene level by multivariate functional linear models.

    PubMed

    Wang, Yifan; Liu, Aiyi; Mills, James L; Boehnke, Michael; Wilson, Alexander F; Bailey-Wilson, Joan E; Xiong, Momiao; Wu, Colin O; Fan, Ruzong

    2015-05-01

    In genetics, pleiotropy describes the genetic effect of a single gene on multiple phenotypic traits. A common approach is to analyze the phenotypic traits separately using univariate analyses and combine the test results through multiple comparisons. This approach may lead to low power. Multivariate functional linear models are developed to connect genetic variant data to multiple quantitative traits adjusting for covariates for a unified analysis. Three types of approximate F-distribution tests based on Pillai-Bartlett trace, Hotelling-Lawley trace, and Wilks's Lambda are introduced to test for association between multiple quantitative traits and multiple genetic variants in one genetic region. The approximate F-distribution tests provide much more significant results than those of F-tests of univariate analysis and optimal sequence kernel association test (SKAT-O). Extensive simulations were performed to evaluate the false positive rates and power performance of the proposed models and tests. We show that the approximate F-distribution tests control the type I error rates very well. Overall, simultaneous analysis of multiple traits can increase power performance compared to an individual test of each trait. The proposed methods were applied to analyze (1) four lipid traits in eight European cohorts, and (2) three biochemical traits in the Trinity Students Study. The approximate F-distribution tests provide much more significant results than those of F-tests of univariate analysis and SKAT-O for the three biochemical traits. The approximate F-distribution tests of the proposed functional linear models are more sensitive than those of the traditional multivariate linear models that in turn are more sensitive than SKAT-O in the univariate case. The analysis of the four lipid traits and the three biochemical traits detects more association than SKAT-O in the univariate case.

  6. Pleiotropy Analysis of Quantitative Traits at Gene Level by Multivariate Functional Linear Models

    PubMed Central

    Wang, Yifan; Liu, Aiyi; Mills, James L.; Boehnke, Michael; Wilson, Alexander F.; Bailey-Wilson, Joan E.; Xiong, Momiao; Wu, Colin O.; Fan, Ruzong

    2015-01-01

    In genetics, pleiotropy describes the genetic effect of a single gene on multiple phenotypic traits. A common approach is to analyze the phenotypic traits separately using univariate analyses and combine the test results through multiple comparisons. This approach may lead to low power. Multivariate functional linear models are developed to connect genetic variant data to multiple quantitative traits adjusting for covariates for a unified analysis. Three types of approximate F-distribution tests based on Pillai–Bartlett trace, Hotelling–Lawley trace, and Wilks’s Lambda are introduced to test for association between multiple quantitative traits and multiple genetic variants in one genetic region. The approximate F-distribution tests provide much more significant results than those of F-tests of univariate analysis and optimal sequence kernel association test (SKAT-O). Extensive simulations were performed to evaluate the false positive rates and power performance of the proposed models and tests. We show that the approximate F-distribution tests control the type I error rates very well. Overall, simultaneous analysis of multiple traits can increase power performance compared to an individual test of each trait. The proposed methods were applied to analyze (1) four lipid traits in eight European cohorts, and (2) three biochemical traits in the Trinity Students Study. The approximate F-distribution tests provide much more significant results than those of F-tests of univariate analysis and SKAT-O for the three biochemical traits. The approximate F-distribution tests of the proposed functional linear models are more sensitive than those of the traditional multivariate linear models that in turn are more sensitive than SKAT-O in the univariate case. The analysis of the four lipid traits and the three biochemical traits detects more association than SKAT-O in the univariate case. PMID:25809955

  7. Inference of cellular level signaling networks using single-cell gene expression data in Caenorhabditis elegans reveals mechanisms of cell fate specification.

    PubMed

    Huang, Xiao-Tai; Zhu, Yuan; Chan, Lai Hang Leanne; Zhao, Zhongying; Yan, Hong

    2017-05-15

    Cell fate specification plays a key role to generate distinct cell types during metazoan development. However, most of the underlying signaling networks at cellular level are not well understood. Availability of time lapse single-cell gene expression data collected throughout Caenorhabditis elegans embryogenesis provides an excellent opportunity for investigating signaling networks underlying cell fate specification at systems, cellular and molecular levels. We propose a framework to infer signaling networks at cellular level by exploring the single-cell gene expression data. Through analyzing the expression data of nhr-25 , a hypodermis-specific transcription factor, in every cells of both wild-type and mutant C.elegans embryos through RNAi against 55 genes, we have inferred a total of 23 genes that regulate (activate or inhibit) nhr-25 expression in cell-specific fashion. We also infer the signaling pathways consisting of each of these genes and nhr-25 based on a probabilistic graphical model for the selected five founder cells, 'ABarp', 'ABpla', 'ABpra', 'Caa' and 'Cpa', which express nhr-25 and mostly develop into hypodermis. By integrating the inferred pathways, we reconstruct five signaling networks with one each for the five founder cells. Using RNAi gene knockdown as a validation method, the inferred networks are able to predict the effects of the knockdown genes. These signaling networks in the five founder cells are likely to ensure faithful hypodermis cell fate specification in C.elegans at cellular level. All source codes and data are available at the github repository https://github.com/xthuang226/Worm_Single_Cell_Data_and_Codes.git . zhuyuan@cug.edu.cn. Supplementary data are available at Bioinformatics online.

  8. Modulation of cellular Hsp72 levels in undifferentiated and neuron-like SH-SY5Y cells determines resistance to staurosporine-induced apoptosis.

    PubMed

    Cheng, Lesley; Smith, Danielle J; Anderson, Robin L; Nagley, Phillip

    2011-01-01

    Increased expression of Hsp72 accompanies differentiation of human neuroblastoma SH-SY5Y cells to neuron-like cells. By modulating cellular levels of Hsp72, we demonstrate here its anti-apoptotic activity both in undifferentiated and neuron-like cells. Thermal preconditioning (43°C for 30 min) induced Hsp72, leading to cellular protection against apoptosis induced by a subsequent treatment with staurosporine. Preconditioned staurosporine-treated cells displayed decreased Bax recruitment to mitochondria and subsequent activation, as well as reduced cytochrome c redistribution from mitochondria. The data are consistent with Hsp72 blocking apoptosis upstream of Bax recruitment to mitochondria. Neuron-like cells (with elevated Hsp72) were more resistant to staurosporine by all measured indices of apoptotic signaling. Use of stable transfectants ectopically expressing moderately elevated levels of Hsp72 revealed that such cells in the undifferentiated state showed enhanced resistance to staurosporine-induced apoptosis, which was even more robust after differentiation to neuron-like cells. Overall, the protective effects of differentiation, thermal preconditioning and ectopic Hsp72 expression were additive. The strong inverse correlation between cellular Hsp72 levels and susceptibility to apoptosis support the notion that Hsp72 acts as a significant neuroprotective factor, enabling post-mitotic neurons to withstand potentially lethal stress that induces apoptosis.

  9. The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

    SciTech Connect

    Butz, Nicole; Ruetz, Stephan; Natt, Francois; Hall, Jonathan; Weiler, Jan; Mestan, Juergen; Ducarre, Monique; Grossenbacher, Rita; Hauser, Patrick; Kempf, Dominique; Hofmann, Francesco . E-mail: francesco.hofmann@pharma.novartis.com

    2005-02-15

    Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found to be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.

  10. A Quantitative Assessment of the Sea Level Drop in the Messinian Mediterranean

    NASA Astrophysics Data System (ADS)

    Bartol, J.; Topper, R. P.

    2014-12-01

    In the Late Miocene progressive restriction of the gateway(s) between the Mediterranean Sea and the Atlantic Ocean initiated the Messinian Salinity Crisis (MSC). The restriction caused a salinity rise and the formation of extremely thick evaporite deposits within the Mediterranean basin. Subsequently, the closure of the connection between the Mediterranean Sea and Atlantic Ocean resulted in a dramatic sea level drop. The lithosphere adepts to changes in loads (sediments and water) on the surface by flexural adjustment of the Earth's surface. This can have a significant impact on the connectivity of basins and the temporal evolution of the sea level drop in each basin. The Mediterranean Sea is characterized by two deep basins with different amounts of river input (western/eastern Mediterranean basin) separated by the relatively shallow Sicily sill. The temporal evolution of the sea level drop in the western/eastern Mediterranean basin is therefore very sensitive to the temporal and spatial evolution of the Sicily gateway and hence flexure response of the earth surface due to the changes in waterload. However, studies of the MSC sea level drop haven't taken this flexural response into consideration (e.g. Meijer and Krijgsman, 2005). Here we use an elastic model (TISC) coupled with a simple hydrology model to calculate the temporal evolution of the sea level drop in and the flexural response of the western and eastern Mediterranean basins. Preliminary results show that when sea level drops below the Sicily sill, the rate of sea level increases in the western but decreases in the eastern Mediterranean. Following a relative sea level drop of ~1300 m, resulting in a flexural uplift of ~700m, the eastern Mediterranean reaches equilibrium. At the time the western Mediterranean (flexural uplift of ~900m) reaches an equilibrium it is almost completely desiccated. The magnitudes of the sea level drops and flexural response are, however, highly dependent on the hypsometry

  11. A novel method for morphological pleomorphism and heterogeneity quantitative measurement: Named cell feature level co-occurrence matrix

    PubMed Central

    Saito, Akira; Numata, Yasushi; Hamada, Takuya; Horisawa, Tomoyoshi; Cosatto, Eric; Graf, Hans-Peter; Kuroda, Masahiko; Yamamoto, Yoichiro

    2016-01-01

    Background: Recent developments in molecular pathology and genetic/epigenetic analysis of cancer tissue have resulted in a marked increase in objective and measurable data. In comparison, the traditional morphological analysis approach to pathology diagnosis, which can connect these molecular data and clinical diagnosis, is still mostly subjective. Even though the advent and popularization of digital pathology has provided a boost to computer-aided diagnosis, some important pathological concepts still remain largely non-quantitative and their associated data measurements depend on the pathologist's sense and experience. Such features include pleomorphism and heterogeneity. Methods and Results: In this paper, we propose a method for the objective measurement of pleomorphism and heterogeneity, using the cell-level co-occurrence matrix. Our method is based on the widely used Gray-level co-occurrence matrix (GLCM), where relations between neighboring pixel intensity levels are captured into a co-occurrence matrix, followed by the application of analysis functions such as Haralick features. In the pathological tissue image, through image processing techniques, each nucleus can be measured and each nucleus has its own measureable features like nucleus size, roundness, contour length, intra-nucleus texture data (GLCM is one of the methods). In GLCM each nucleus in the tissue image corresponds to one pixel. In this approach the most important point is how to define the neighborhood of each nucleus. We define three types of neighborhoods of a nucleus, then create the co-occurrence matrix and apply Haralick feature functions. In each image pleomorphism and heterogeneity are then determined quantitatively. For our method, one pixel corresponds to one nucleus feature, and we therefore named our method Cell Feature Level Co-occurrence Matrix (CFLCM). We tested this method for several nucleus features. Conclusion: CFLCM is showed as a useful quantitative method for pleomorphism

  12. Ultratrace Level Determination and Quantitative Analysis of Kidney Injury Biomarkers in Patient Samples Attained by Zinc Oxide Nanorods

    PubMed Central

    Singh, Manpreet; Alabanza, Anginelle; Gonzalez, Lorelis E.; Wang, Weiwei; Reeves, W. Brian; Hahm, Jong-in

    2016-01-01

    Determining ultratrace amounts of protein biomarkers in patient samples in a straightforward and quantitative manner is extremely important for early disease diagnosis and treatment. Here, we successfully demonstrate the novel use of zinc oxide nanorods (ZnO NRs) in the ultrasensitive and quantitative detection of two acute kidney injury (AKI)-related protein biomarkers, tumor necrosis factor (TNF)-α and interleukin (IL)-8, directly from patient samples. We first validate the ZnO NRs-based IL-8 results via comparison with those obtained from using a conventional enzyme-linked immunosorbent method in samples from 38 individuals. We further assess the full detection capability of the ZnO NRs-based technique by quantifying TNF-α, whose levels in human urine are often below the detection limits of conventional methods. Using the ZnO NR platforms, we determine the TNF-α concentrations of all 46 patient samples tested, down to the fg/mL level. Subsequently, we screen for TNF-α levels in approximately 50 additional samples collected from different patient groups in order to demonstrate a potential use of the ZnO NRs-based assay in assessing cytokine levels useful for further clinical monitoring. Our research efforts demonstrate that ZnO NRs can be straightforwardly employed in the rapid, ultrasensitive, quantitative, and simultaneous detection of multiple AKI-related biomarkers directly in patient urine samples, providing an unparalleled detection capability beyond those of conventional analysis methods. Additional key advantages of the ZnO NRs-based approach include a fast detection speed, low-volume assay condition, multiplexing ability, and easy automation/integration capability to existing fluorescence instrumentation. Therefore, we anticipate that our ZnO NRs-based detection method will be highly beneficial for overcoming the frequent challenges in early biomarker development and treatment assessment, pertaining to the facile and ultrasensitive quantification of

  13. The Na+/Glucose Cotransporter Inhibitor Canagliflozin Activates AMPK by Inhibiting Mitochondrial Function and Increasing Cellular AMP Levels.

    PubMed

    Hawley, Simon A; Ford, Rebecca J; Smith, Brennan K; Gowans, Graeme J; Mancini, Sarah J; Pitt, Ryan D; Day, Emily A; Salt, Ian P; Steinberg, Gregory R; Hardie, D Grahame

    2016-09-01

    Canagliflozin, dapagliflozin, and empagliflozin, all recently approved for treatment of type 2 diabetes, were derived from the natural product phlorizin. They reduce hyperglycemia by inhibiting glucose reuptake by sodium/glucose cotransporter (SGLT) 2 in the kidney, without affecting intestinal glucose uptake by SGLT1. We now report that canagliflozin also activates AMPK, an effect also seen with phloretin (the aglycone breakdown product of phlorizin), but not to any significant extent with dapagliflozin, empagliflozin, or phlorizin. AMPK activation occurred at canagliflozin concentrations measured in human plasma in clinical trials and was caused by inhibition of Complex I of the respiratory chain, leading to increases in cellular AMP or ADP. Although canagliflozin also inhibited cellular glucose uptake independently of SGLT2, this did not account for AMPK activation. Canagliflozin also inhibited lipid synthesis, an effect that was absent in AMPK knockout cells and that required phosphorylation of acetyl-CoA carboxylase (ACC) 1 and/or ACC2 at the AMPK sites. Oral administration of canagliflozin activated AMPK in mouse liver, although not in muscle, adipose tissue, or spleen. Because phosphorylation of ACC by AMPK is known to lower liver lipid content, these data suggest a potential additional benefit of canagliflozin therapy compared with other SGLT2 inhibitors. © 2016 by the American Diabetes Association.

  14. Quantitative relationship between salivary level of Streptococcus mutans and Candida albicans in children with Down's syndrome.

    PubMed

    Linossier, A; Vargas, A; Villegas, R; Chimenos, E

    2002-01-01

    The actual work intends to establish a correlation between quantitative counts for Streptococcus mutans and Candida albicans in the saliva of patients with Down syndrome (DS), and in mentally deficient (MD) patients, with respect to a normal group (C ). This study was performed in 166 children (49 DS, 60MD and 57C), whose saliva samples were grown on TYCSB agar and Sabouraud agar. Subsequently, microbiological analysis, scanning electron microscopy and aggregation assays were made. The results were statistically evaluated using the variance analysis (ANOVA) and the Student "t" test for unpaired data. A correlation of 0.45 was found for group C, 0.55 in DS patients and 0.37 in MD patients, when comparing counts of S. mutans and C. albicans in saliva. Scanning electron microscopy analysis showed coaggregation between S. mutans and C. albicans and this was confirmed by in vitro experiments of S. mutans and C. albicans made on nitrocellulose filters. This kind of association between bacteria and fungi contributes to justify a pathogenic synergy among several microorganisms, as well as some infectious local or systemic manifestations, depending on the immunity status of the patient.

  15. Quantitative Proteomics of Zea mays Hybrids Exhibiting Different Levels of Heterosis.

    PubMed

    Dahal, Diwakar; Newton, Kathleen J; Mooney, Brian P

    2016-08-05

    Maize hybrids exhibiting heterosis (hybrid vigor) were generated from inbred parents with increasing genetic distance. B73 was used as the common female parent in crosses with N192 (low heterosis), MO17 (high-heterosis 1), and NC350 (high-heterosis 2). Total and mitochondria-enriched proteomes were analyzed from ear shoots of field-grown hybrids and their inbred parents. GeLCMS (1D SDS-PAGE fractionation, trypsin digestion, LTQ Orbitrap nano-RP-LC MS/MS) was used to analyze proteins, and spectral counting was used for quantitation. In total, 3,568 proteins were identified and quantified in hybrids including 2,489 in the mitochondria-enriched fraction and 2,162 in the total protein fraction. Sixty-one proteins were differentially abundant (p < 0.05) in one or both of the high-heterosis hybrids compared with the low-heterosis hybrid. For the total proteome, eight of these showed similar trends in abundance in both of the higher-heterosis hybrids. Nine proteins showed this heterosis-correlated pattern in the mitochondrial proteome, including a mitochondria-associated target of rapamycin (TOR) protein. Although differentially abundant proteins belong to various pathways, protein, and RNA metabolism, and stress responsive proteins were the major classes changed in response to increasing heterosis.

  16. Association of Serum MiR-142-3p and MiR-101-3p Levels with Acute Cellular Rejection after Heart Transplantation

    PubMed Central

    Sukma Dewi, Ihdina; Hollander, Zsuzsanna; Lam, Karen K.; McManus, Janet-Wilson; Tebbutt, Scott J.; Ng, Raymond T.; Keown, Paul A.; McMaster, Robert W.; McManus, Bruce M.; Gidlöf, Olof; Öhman, Jenny

    2017-01-01

    Background Identifying non-invasive and reliable blood-derived biomarkers for early detection of acute cellular rejection in heart transplant recipients is of great importance in clinical practice. MicroRNAs are small molecules found to be stable in serum and their expression patterns reflect both physiological and underlying pathological conditions in human. Methods We compared a group of heart transplant recipients with histologically-verified acute cellular rejection (ACR, n = 26) with a control group of heart transplant recipients without allograft rejection (NR, n = 37) by assessing the levels of a select set of microRNAs in serum specimens. Results The levels of seven microRNAs, miR-142-3p, miR-101-3p, miR-424-5p, miR-27a-3p, miR-144-3p, miR-339-3p and miR-326 were significantly higher in ACR group compared to the control group and could discriminate between patients with and without allograft rejection. MiR-142-3p and miR-101-3p had the best diagnostic test performance among the microRNAs tested. Serum levels of miR-142-3p and miR-101-3p were independent of calcineurin inhibitor levels, as measured by tacrolimus and cyclosporin; kidney function, as measured by creatinine level, and general inflammation state, as measured by CRP level. Conclusion This study demonstrated two microRNAs, miR-142-3p and miR-101-3p, that could be relevant as non-invasive diagnostic tools for identifying heart transplant patients with acute cellular rejection. PMID:28125729

  17. The relationship between serum vitamin D levels and spinal fusion success: a quantitative analysis.

    PubMed

    Metzger, Melodie F; Kanim, Linda E A; Zhao, Li; Robinson, Samuel T; Delamarter, Rick B

    2015-04-15

    An in vivo dosing study of vitamin D in a rat posterolateral spinal fusion model with autogenous bone grafting. Rats randomized to 4 levels of vitamin D-adjusted rat chow, longitudinal serum validation, surgeons/observers blinded to dietary conditions, and rats followed prospectively for fusion endpoint. To assess the impact of dietary and serum levels of vitamin D on fusion success, consolidation of fusion mass, and biomechanical stiffness after posterolateral spinal fusion procedure. Metabolic risk factors, including vitamin D insufficiency, are often overlooked by spine surgeons. Currently, there are no published data on the causal effect of insufficient or deficient vitamin D levels on the success of establishing solid bony union after a spinal fusion procedure. Fifty rats were randomized to 4 experimentally controlled rat chow diets: normal control, vitamin D-deficient, vitamin D-insufficient, and a nontoxic high dose of vitamin D, 4 weeks prior to surgery and maintained postsurgery until sacrifice. Serum levels of 25(OH)D were determined at surgery and sacrifice using radioimmunoassay. Posterolateral fusion surgery with tail autograft was performed. Rats were sacrificed 12 weeks postoperatively, and fusion was evaluated via manual palpation, high-resolution radiographs, micro-computed tomographic scans, and biomechanical testing. Serum 25(OH)D and calcium levels were significantly correlated with vitamin D-adjusted chow (P < 0.001). There was a dose-dependent relationship between vitamin D-adjusted chow and manual palpation fusion, with greatest differences found in measures of radiographical density between high and deficient vitamin D (P < 0.05). Adequate levels of vitamin D (high and normal control) yielded stiffer fusion than inadequate levels (insufficient and deficient) (P < 0.05). Manual palpation fusion rates increased with supplementation of dietary vitamin D. Biomechanical stiffness, bone volume, and density were also positively related to vitamin D

  18. KymographClear and KymographDirect: two tools for the automated quantitative analysis of molecular and cellular dynamics using kymographs

    PubMed Central

    Mangeol, Pierre; Prevo, Bram; Peterman, Erwin J. G.

    2016-01-01

    Dynamic processes are ubiquitous and essential in living cells. To properly understand these processes, it is imperative to measure them in a time-dependent way and analyze the resulting data quantitatively, preferably with automated tools. Kymographs are single images that represent the motion of dynamic processes and are widely used in live-cell imaging. Although they contain the full range of dynamics, it is not straightforward to extract this quantitative information in a reliable way. Here we present two complementary, publicly available software tools, KymographClear and KymographDirect, that have the power to reveal detailed insight in dynamic processes. KymographClear is a macro toolset for ImageJ to generate kymographs that provides automatic color coding of the different directions of movement. KymographDirect is a stand-alone tool to extract quantitative information from kymographs obtained from a wide range of dynamic processes in an automated way, with high accuracy and reliability. We discuss the concepts behind these software tools, validate them using simulated data, and test them on experimental data. We show that these tools can be used to extract motility parameters from a diverse set of cell-biological experiments in an automated and user-friendly way. PMID:27099372

  19. Quantitative and spatio-temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing.

    PubMed

    Winkler, Juliane; Seybert, Anja; König, Lars; Pruggnaller, Sabine; Haselmann, Uta; Sourjik, Victor; Weiss, Matthias; Frangakis, Achilleas S; Mogk, Axel; Bukau, Bernd

    2010-03-03

    The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re-localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re-targeted to alternative sites such as the inner membrane in the presence of site-specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage-free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.

  20. Quantitative in vivo assessment of lung microstructure at the alveolar level with hyperpolarized 3He diffusion MRI

    NASA Astrophysics Data System (ADS)

    Yablonskiy, Dmitriy A.; Sukstanskii, Alexander L.; Leawoods, Jason C.; Gierada, David S.; Bretthorst, G. Larry; Lefrak, Stephen S.; Cooper, Joel D.; Conradi, Mark S.

    2002-03-01

    The study of lung emphysema dates back to the beginning of the 17th century. Nevertheless, a number of important questions remain unanswered because a quantitative localized characterization of emphysema requires knowledge of lung structure at the alveolar level in the intact living lung. This information is not available from traditional imaging modalities and pulmonary function tests. Herein, we report the first in vivo measurements of lung geometrical parameters at the alveolar level obtained with 3He diffusion MRI in healthy human subjects and patients with severe emphysema. We also provide the first experimental data demonstrating that 3He gas diffusivity in the acinus of human lung is highly anisotropic. A theory of anisotropic diffusion is presented. Our results clearly demonstrate substantial differences between healthy and emphysematous lung at the acinar level and may provide new insights into emphysema progression. The technique offers promise as a clinical tool for early diagnosis of emphysema.

  1. Efficient Quantitative Analysis of Carboxyalkylpyrrole Ethanolamine Phospholipids: Elevated Levels in Sickle Cell Disease Blood.

    PubMed

    Guo, Junhong; Wang, Hua; Hrinczenko, Borys; Salomon, Robert G

    2016-07-18

    γ-Hydroxy-α,β-unsaturated aldehydes, generated by oxidative damage of polyunsaturated phospholipids, form pyrrole derivatives that incorporate the ethanolamine phospholipid (EP) amino group such as 2-pentylpyrrole (PP)-EP and 2-(ω-carboxyalkyl)pyrrole (CAP)-EP derivatives: 2-(ω-carboxyethyl)pyrrole (CEP)-EP, 2-(ω-carboxypropyl)pyrrole (CPP)-EP, and 2-(ω-carboxyheptyl)pyrrole (CHP)-EP. Because EPs occur in vivo in various forms, a complex mixture of pyrrole-modified EPs with different molecular weights is expected to be generated. To provide a sensitive index of oxidative stress, all of the differences in mass related to the glycerophospholipid moieties were removed by releasing a single CAP-ethanolamine (ETN) or PP-ETN from each mixture by treatment with phospholipase D. Accurate quantization was achieved using the corresponding ethanolamine-d4 pyrroles as internal standards. The product mixture obtained by phospholipolysis of total blood phospholipids from sickle cell disease (SCD) patients was analyzed by LC-MS/MS. The method was applied to measure CAP-EP and PP-EP levels in blood plasma from clinical monitoring of SCD patients. We found uniformly elevated blood levels of CEP-EP (63.9 ± 9.7 nM) similar to mean levels in blood from age-related macular degeneration (AMD) patients (56.3 ± 37.1 nM), and 2-fold lower levels (27.6 ± 3.6 nM, n = 5) were detected in plasma from SCD patients hospitalized to treat a sickle cell crisis, although mean levels remain higher than those (12.1 ± 10.5 nM) detected in blood from healthy controls. Plasma levels of CPP-EPs from SCD clinic patients were 4-fold higher than those of SCD patients hospitalized to treat a sickle cell crisis (45.1 ± 10.9 nM, n = 5 versus 10.9 ± 3.4 nM, n = 6; p < 0.002). PP-EP concentration in plasma from SCD clinic patients is nearly 4.8-fold higher than its level in plasma samples from SCD patients hospitalized to treat a sickle cell crisis (7.06 ± 4.05 vs 1.48 ± 0.92 nM; p < 0.05). Because

  2. [Genetic polymorphism of haptoglobin and quantitative changes in its levels during exposure to asbestos].

    PubMed

    Afanas'eva, I S; Spitsyn, V A; Tsurikova, G V

    1993-11-01

    The polymorphism and serum levels of haptoglobin were studied in asbestosis patients, in the control and the workers exposed to asbest. Hp1-1 has the highest, Hp2-2--the lowest and Hp2-1 has the intermediate concentration of this protein. In the course of contact with asbest, and especially in asbestosis patients, the haptoglobin levels are higher (for all phenotypes). The standard deviation from the Hp concentration in asbestosis patients was significantly higher. The phenotypes Hp1-1 were found more often in asbestosis patients than among asbest-exposed workers.

  3. Qualitative and quantitative detection of human pathogenic Yersinia enterocolitica in different food matrices at retail level in Bavaria.

    PubMed

    Messelhäusser, Ute; Kämpf, Peter; Colditz, Janine; Bauer, Hans; Schreiner, Hermann; Höller, Christiane; Busch, Ulrich

    2011-01-01

    Yersinia enterocolitica is a major foodborne pathogen and the third most important bacteriological cause of diarrhea in Germany. However, studies investigating the occurrence of human pathogenic Y. enterocolitica in food at the retail level are very rare. Most of the studies published so far show qualitative but not quantitative data concerning the prevalence of this human pathogen. In this study the qualitative and quantitative assessment of human pathogenic Y. enterocolitica in different food matrices was investigated. For the qualitative analysis we used an enrichment method according to the International Organisation of Standardization (ISO) standard in combination with a real-time polymerase chain reaction (PCR) method detecting the ail gene of Y. enterocolitica. After detecting Y. enterocolitica in a sample, a quantitative investigation on Cefsulodin-Irgasan-Novobiocin (CIN) Agar was done to get information about the contamination level of the different samples. During the years 2008 and 2009, 446 samples of pork and pork products, 51 samples of game meat, and 61 raw milk samples were investigated for the presence of human pathogenic Y. enterocolitica. The samples were collected at the retail level in Bavaria. From the pork samples investigated, 81 samples (18%) were positive for the ail gene by real-time PCR, but human pathogenic Y. enterocolitica O:3 were found only in 46 (10%) pork samples by culture; the concentration in the samples ranged between 0.04 cfu/g and 2.30 × 10(5) cfu/g. Three game meat samples were positive by real-time PCR, but not by the cultural detection. All raw milk samples were negative by real-time PCR and culture.

  4. Biological (Molecular and Cellular) Markers of Toxicity.

    DTIC Science & Technology

    1991-12-15

    other mammals (Farber, 1987). Therefore, if we are to understand in any depth the processes by which environnental chemicals exhibit genotoxicity ... effectiveness to detect and quantitate biological responses at the molecular level to the action of genotoxic agenst. Particular emphasis was placed on the...Task 3. The short-term responses of the molecular markers as suitable biomarkers to estimate exposure and predict cellular effects to genotoxic

  5. Cellular automata model for citrus variegated chlorosis.

    PubMed

    Martins, M L; Ceotto, G; Alves, S G; Bufon, C C; Silva, J M; Laranjeira, F F

    2000-11-01

    A cellular automata model is proposed to analyze the progress of citrus variegated chlorosis epidemics in São Paulo orange plantations. In this model epidemiological and environmental features, such as motility of sharpshooter vectors that perform Lévy flights, level of plant hydric and nutritional stress, and seasonal climatic effects, are included. The observed epidemic data were quantitatively reproduced by the proposed model on varying the parameters controlling vector motility, plant stress, and initial population of diseased plants.

  6. Quantitative expression analysis of drought responsive genes in clones of Hevea with varying levels of drought tolerance.

    PubMed

    Luke, Lisha P; Mohamed Sathik, M B; Thomas, Molly; Kuruvilla, Linu; Sumesh, K V; Annamalainathan, K

    2015-04-01

    In order to meet the ever rising global demand for natural rubber, cultivation of Hevea is being extended to non-traditional regions of India where extreme climatic conditions like drought and low temperature negatively influence the crop performance. In order to ensure maximum productivity, identification of drought tolerant clones of Hevea which can cope up with stress and give better crop yield is essential. Several attempts have been made previously to identify genes that are associated with drought tolerance in Hevea. In the present study, quantitative expression analysis was made using quantitative PCR for seven drought associated transcripts in four clones of Hevea with varying levels of drought tolerance. Among the seven genes studied, Mitogen Activated Protein (MAP) kinase, Myeloblastosis (Myb) transcription factor, C-repeat responsive element/Dehydration Responsive Element (CRT/DRE) binding factor and Nuclear Factor Y subunit A (NFYA) showed a positive association with drought tolerance. Transcripts of ascorbate peroxidase and heat shock protein 70 (HSP 70) did not show any correlation with drought tolerance. Interestingly, catalase gene was found down regulated in all the clones under drought condition. The possible role of these genes based on their level of gene expression in four different clones of Hevea with varying levels of drought tolerance is discussed.

  7. A chemical biology approach to interrogate quorum-sensing regulated behaviors at the molecular and cellular level.

    PubMed

    Lowery, Colin A; Matamouros, Susana; Niessen, Sherry; Zhu, Jie; Scolnick, Jonathan; Lively, Jenny M; Cravatt, Benjamin F; Miller, Samuel I; Kaufmann, Gunnar F; Janda, Kim D

    2013-07-25

    Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intercellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation.

  8. Cotton defense induction patterns under spatially, temporally and quantitatively varying herbivory levels

    USDA-ARS?s Scientific Manuscript database

    The optimal defense theory (ODT) predicts that plants allocate defense compounds to their tissues depending on its value and the likelihood of herbivore attack. Whereas ODT has been confirmed for static damage levels it remains poorly understood if ODT holds true for defense organization of inducibl...

  9. Quantitative constraints on the sea-level fall that terminated the Littorina Sea Stage, southern Scandinavia

    NASA Astrophysics Data System (ADS)

    Clemmensen, Lars B.; Murray, Andrew S.; Nielsen, Lars

    2012-04-01

    The island of Anholt in the Kattegat sea (southern Scandinavia) is made up largely of an extensive beach-ridge plain. As a result of post-glacial uplift, the earliest beach-ridge and swale deposits are now raised 8-9 m above present mean sea level. It appears that growth of the plain has been almost uninterrupted over the past 7500 years; here we constrain the evolution of this plain between 6300 and 1300 years ago using optically stimulated luminescence dates. The topography and internal architecture of the fossil shoreline deposits were measured on high-resolution maps and in ground-penetrating radar (GPR) reflection data with a vertical resolution of ˜0.25 m. Shoreline topography shows significant changes with time, and it appears that one of the most striking changes took place between 4300 and 3600 years ago; in the shoreline deposits corresponding to this time interval the surface drops by around 3.5 m suggesting a marked fall in relative sea-level. Assuming a constant uplift rate of 1.2 mm/yr, the corresponding drop in absolute sea-level is estimated to be around 2.6 m. This marked sea-level fall in 700 years took place at the transition from the Middle Holocene Thermal Maximum to the Late Holocene Thermal Decline or at the end of the Littorina Sea stage in the Baltic Sea region.

  10. A Quantitative Examination Whether Education Mitigates Stress Levels among Law Enforcement Officers

    ERIC Educational Resources Information Center

    Metts, Gary A.

    2012-01-01

    Stress is damaging if it is continual, overwhelming. and prolonged. Law enforcement officers face stressful events daily. A relationship exists between stress levels and the physical and psychological effects to the human body. Although there is a general understanding of the damage stress can do physically and psychologically, many elements that…

  11. A Quantitative Examination Whether Education Mitigates Stress Levels among Law Enforcement Officers

    ERIC Educational Resources Information Center

    Metts, Gary A.

    2012-01-01

    Stress is damaging if it is continual, overwhelming. and prolonged. Law enforcement officers face stressful events daily. A relationship exists between stress levels and the physical and psychological effects to the human body. Although there is a general understanding of the damage stress can do physically and psychologically, many elements that…

  12. An Interlaboratory Comparative Study on the Quantitative Determination of Glyphosate at Low Levels in Wheat Flour.

    PubMed

    Simonetti, Emanuela; Cartaud, Gérald; Quinn, Robert M; Marotti, Ilaria; Dinelli, Giovanni

    2015-01-01

    In recent years, the use of glyphosate has dramatically increased worldwide, and there is growing concern about contamination of organic products caused by its heavy use on neighboring fields. Glyphosate is found as a residue not only in soil, plants, and groundwater but also in humans and animals. Considering the controversy on glyphosate maximum residue level in foodstuff and the difficulties in its analytical determination, the main purpose of the present paper was to investigate the competence and accuracy of 13 accredited European laboratories in determining glyphosate in wheat flour at a level close to their reporting limit of 10 μg/kg. According to the results of this performance assessment, the laboratories were not able to quantify glyphosate at trace levels. Therefore, their specified reporting limits of 10 μg/kg were not supported by their results, and a reporting limit of around 50 μg/kg of glyphosate in flour seems to be more appropriate to guarantee reliable and robust results. The widespread use of glyphosate and its harmfulness to humans make its detection at trace levels a primary goal for analytical laboratories. This is achievable through the improvement of QA and/or the optimization of the method of analysis used for glyphosate detection.

  13. Effects of high CO₂ levels on fermentation, peroxidation, and cellular water stress in Fragaria vesca stored at low temperature in conditions of unlimited O₂.

    PubMed

    Blanch, Maria; Rosales, Raquel; Mateos, Raquel; Perez-Gago, María B; Sanchez-Ballesta, Maria T; Escribano, María I; Merodio, Carmen

    2015-01-28

    To better understand the tolerance of strawberries (Fragaria vesca L.) to high CO2 in storage atmospheres, fermentation and cellular damage were investigated. Fruits were stored for 3 and 6 days at 0 °C in the presence of different CO2 levels (0, 20, or 40%) with 20% O2. Changes in pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) gene expression and in fermentative metabolites, as well as in bound water and malondialdehyde (MDA) concentrations, were analyzed. In strawberries stored without added CO2, up-regulation of PDC and ADH was not associated with an increase in fermentative metabolites. By contrast, moderate ethanol fermentation in fruits exposed to 20% CO2 seems to be essential to maintain fruit metabolism, reducing both lipid peroxidation and cellular water stress. However, if the CO2 concentration increases (40%), the excess acetaldehyde and ethanol produced were closely correlated with a decrease in bound water and production of MDA.

  14. Changes in rRNA levels during stress invalidates results from mRNA blotting: fluorescence in situ rRNA hybridization permits renormalization for estimation of cellular mRNA levels.

    PubMed

    Hansen, M C; Nielsen, A K; Molin, S; Hammer, K; Kilstrup, M

    2001-08-01

    Regulation of gene expression can be analyzed by a number of different techniques. Some techniques monitor the level of specific mRNA directly, and others monitor indirectly by determining the level of enzymes encoded by the mRNA. Each method has its own inherent way of normalization. When results obtained by these techniques are compared between experiments in which differences in growth rates, strains, or stress treatments occur, the normalization procedure may have a significant impact on the results. In this report we present a solution to the normalization problem in RNA slot blotting experiments, in which mRNA levels routinely are normalized to a fixed amount of extracted total RNA. The cellular levels of specific mRNA species were estimated using a renormalization with the total RNA content per cell. By a combination of fluorescence in situ rRNA hybridization, which estimates the relative level of rRNA per cell, and slot blotting to rRNA probes, which estimates the level of rRNA per extracted total RNA, the amount of RNA per cell was calculated in a series of heat shock experiments with the gram-positive bacterium Lactococcus lactis. It was found that the level of rRNA per cell decreased to 30% in the course of the heat shock. This lowered ribosome level led to a decrease in the total RNA content, resulting in a gradually increasing overestimation of the mRNA levels throughout the experiment. Using renormalized cellular mRNA levels, the HrcA-mediated regulation of the genes in the hrcA-grpE-dnaK operon was analyzed. The hybridization data suggested a complex heat shock regulation indicating that the mRNA levels continued to rise after 30 min, but after renormalization the calculated average cellular levels exhibited a much simpler induction pattern, eventually attaining a moderately increased value.

  15. The Early Aptian planktonic foraminiferal fauna: a taxonomic and quantitative study across the Selli Level (OAE1a)

    NASA Astrophysics Data System (ADS)

    Barchetta, Alessia; Petrizzo, Maria Rose

    2014-05-01

    The mid-Cretaceous period was a critical time on a global scale characterized by an excess of atmospheric CO2, a greenhouse climate and O2 depletion in the ocean. As a consequence, there was a cyclic deposition and preservation of organic carbon-rich sediments (black shales), the so-called Oceanic Anoxic Events (OAEs). The Selli Level (Early Aptian) is a regional marker-bed identified in the Umbria-Marche area (central Italy) at the transition between the Maiolica and Marne a Fucoidi pelagic formations. It consists of laminated black shales rich in organic matter, alternated with radiolarian silts and with a poor carbonate content. The Selli Level is regarded as the sedimentary expression of the OAE1a (Oceanic Anoxic Event 1a) and is marked by a δ13C anomaly, consisting of a pronounced negative shift followed by a long positive excursion. A detailed and quantitative documentation in terms of species identification and distribution, variation of shell size, diversity and abundance of the planktonic foraminifera across the Selli Level equivalent is presented from the 24 m-thick stratigraphic section of the Cismon core (southern Alps, Italy). In general, the planktonic foraminiferal assemblage throughout the stratigraphic interval is rather diverse and mainly composed by pseudo-planispiral taxa with elongate chambers and ampullae (gen. Leupoldina), planispiral taxa with globular and elongate chambers (gen. Globigerinelloides s.l.) and trochospiral taxa with globular (gen. Hedbergella and gen. Gorbachikella) and elongate chambers (gen. Lilliputianella). Foraminiferal taxonomic, quantitative and morphometric analyses were conducted on washed residues and thin sections of about 250 rock samples spaced 5 - 20 cm apart. Species richness and shell size measurements on selected species were performed on washed residues, and absolute abundances were obtained from thin sections. Three intervals were identified (below, within and above the Selli Level) each of them

  16. Low-level arsenic impairs glucose-stimulated insulin secretion in pancreatic beta cells: involvement of cellular adaptive response to oxidative stress.

    PubMed

    Fu, Jingqi; Woods, Courtney G; Yehuda-Shnaidman, Einav; Zhang, Qiang; Wong, Victoria; Collins, Sheila; Sun, Guifan; Andersen, Melvin E; Pi, Jingbo

    2010-06-01

    Chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with incidence of type 2 diabetes (T2D). A key driver in the pathogenesis of T2D is impairment of pancreatic beta-cell function, with the hallmark of beta-cell function being glucose-stimulated insulin secretion (GSIS). Reactive oxygen species (ROS) derived from glucose metabolism serve as one of the metabolic signals for GSIS. Nuclear factor-erythroid 2-related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. We tested the hypothesis that activation of Nrf2 and induction of antioxidant enzymes in response to arsenic exposure impedes glucose-triggered ROS signaling and thus GSIS. Exposure of INS-1(832/13) cells to low levels of arsenite led to decreased GSIS in a dose- and time-dependent fashion. Consistent with our hypothesis, a significantly enhanced Nrf2 activity, determined by its nuclear accumulation and induction of its target genes, was observed in arsenite-exposed cells. In keeping with the activation of Nrf2-mediated antioxidant response, intracellular glutathione and intracellular hydrogen peroxide-scavenging activity was dose dependently increased by arsenite exposure. Although the basal cellular peroxide level was significantly enhanced, the net percentage increase in glucose-stimulated intracellular peroxide production was markedly inhibited in arsenite-exposed cells. In contrast, insulin synthesis and the consensus GSIS pathway, including glucose transport and metabolism, were not significantly reduced by arsenite exposure. Our studies suggest that low levels of arsenic provoke a cellular adaptive oxidative stress response that increases antioxidant levels, dampens ROS signaling involved in GSIS, and thus disturbs beta-cell function.

  17. Two Independent Regions of Simian Virus 40 T Antigen Increase CBP/p300 Levels, Alter Patterns of Cellular Histone Acetylation, and Immortalize Primary Cells

    PubMed Central

    Sáenz Robles, Maria Teresa; Shivalila, Chikdu; Wano, Jeremy; Sorrells, Shelly; Roos, Alison

    2013-01-01

    Simian virus 40 (SV40) large T antigen (SVT) interferes with normal cell regulation and thus has been used to identify cellular components controlling proliferation and homeostasis. We have previously shown that SVT-mediated transformation requires interaction with the histone acetyltransferases (HATs) CBP/p300 and now report that the ectopic expression of SVT in several cell types in vivo and in vitro results in a significant increase in the steady-state levels of CBP/p300. Furthermore, SVT-expressing cells contain higher levels of acetylated CBP/p300, a modification that has been linked to increased HAT activity. Concomitantly, the acetylation levels of histone residues H3K56 and H4K12 are markedly increased in SVT-expressing cells. Other polyomavirus-encoded large T antigens also increase the levels of CBP/p300 and sustain a rise in the acetylation levels of H3K56 and H4K12. SVT does not affect the transcription of CBP/p300, but rather, alters their overall levels through increasing the loading of CBP/p300 mRNAs onto polysomes. Two distinct regions within SVT, one located in the amino terminus and one in the carboxy terminus, can independently alter both the levels of CBP/p300 and the loading of CBP/p300 transcripts onto polysomes. Within the amino-terminal fragment, a functional J domain is necessary for increasing CBP/p300 and specific histone acetylation levels, as well as for immortalizing primary cells. These studies uncover the action of polyomavirus T antigens on cellular CBP/p300 and suggest that additional mechanisms are used by T antigens to induce cell immortalization and transformation. PMID:24089570

  18. Quantitative study of stress levels in AT and BT cut quartz crystal microbalances associated with surface laser irradiation

    NASA Astrophysics Data System (ADS)

    Goodman, L. H.; Bililign, E. S.; McCann, B. J.; Keller, B. W.; Stevens, K.; Kenny, S. G.; Krim, J.

    The frequency response of an AT cut Quartz Crystal Microbalance (QCM) to laser irradiation has been increasingly studied in recent years, as the combination of photons with materials on a QCM's electrodes enables fundamental studies of topics that span biophysics to photovoltaics. In order for such studies to advance, however, the impact of heating effects associated with laser irradiation of the QCM must be accounted for. Prior studies reached qualitative conclusions that laser irradiation induces stress QCM's arising from non-uniform thermal expansion, but did not quantitatively measure the degree of stress. Secondary effects such as surface film desorption and/or changes in temperature were also reported to be present. We report here a study of the frequency response of AT and BT cut QCM's to laser irradiation. AT and BT cut QCM's have similar response to mass adsorption, but opposite frequency response to stress levels, allowing the stress levels induced by the laser light to be quantitatively measured when the results are compared. Studies were performed in both vacuum and air, to control for the presence of adsorbed films. As expected, system designs that minimize temperature gradients result in less of an effect. Work supported by NSF DMR-1310456.

  19. AquaLite, a bioluminescent label for immunoassay and nucleic acid detection: quantitative analyses at the attomol level

    NASA Astrophysics Data System (ADS)

    Smith, David F.; Stults, Nancy L.

    1996-04-01

    AquaLiteR is a direct, bioluminescent label capable of detecting attomol levels of analyte in clinical immunoassays and assays for the quantitative measurement of nucleic acids. Bioluminescent immunoassays (BIAs) require no radioisotopes and avoid complex fluorescent measurements and many of the variables of indirect enzyme immunoassays (EIAs). AquaLite, a recombinant form of the photoprotein aequorin from a bioluminescent jellyfish, is coupled directly to antibodies to prepare bioluminescent conjugates for assay development. When the AquaLite-antibody complex is exposed to a solution containing calcium ions, a flash of blue light ((lambda) max equals 469 nm) is generated. The light signal is measured in commercially available luminometers that simultaneously inject a calcium solution and detect subattomol photoprotein levies in either test tubes or microtiter plates. Immunometric or 'sandwich' type assays are available for the quantitative measurement of human endocrine hormones and nucleic acids. The AquaLite TSH assay can detect 1 attomol of thyroid stimulating hormone (TSH) in 0.2 mL of human serum and is a useful clinical tool for diagnosing hyperthyroid patients. AquaLite-based nucleic acid detection permits quantifying attomol levels of specific nucleic acid markers and represents possible solution to the difficult problem of quantifying the targets of nucleic acid amplification methods.

  20. Influence of oxygen on lovastatin biosynthesis by Aspergillus terreus ATCC 20542 quantitatively studied on the level of individual pellets.

    PubMed

    Bizukojc, Marcin; Gonciarz, Joanna

    2015-07-01

    Despite oxygen is believed to be the most important environmental factor for any aerobic microbial process, the quantitative studies of its influence on growth and metabolite formation on the level of individual pellets formed by filamentous fungi were seldom performed. Never was it made for lovastatin producer Aspergillus terreus ATCC20542. Thus, this work is a quantitative study of oxygen transfer into A. terreus pellets during lovastatin biosynthesis in the shake flask culture. The basic measurement tool was an oxygen microprobe allowing for obtaining oxygen concentration profiles in the pellets. The pellets of various sizes from 1,600 to 6,400 μm exerting different oxygen transfer conditions were studied. Also various initial concentrations of carbon source were applied to change the conditions of biological reaction running in the pellets. Effective diffusivities in A. terreus pellets ranged from 643 to 1,342 μm s(-1) dependent on their size and structure. It occurred that only the smallest pellets of diameter equal to about 1,400 μm were fully penetrated by oxygen. What is more, apart from the size of pellets, the appropriate lactose concentration was required to effectively produce lovastatin. Its value was correlated with oxygen concentration on the surface of the pellet and could not be either too high, as the aforementioned oxygen level tended then to zero, or too low, as despite high oxygen concentration no biological reaction ran in the pellet and no lovastatin was formed.

  1. Diatom Abundance in Surface Sediments: A Quantitative Proxy for Primary Productivity at the Global Level?

    NASA Astrophysics Data System (ADS)

    Abrantes, F. F. G.; Lopes, C.; Romero, O. E.; Matos, L.; Rufino, M. M.; Magalhaes, V. H.; Cermeno, P.

    2014-12-01

    Diatom abundance and assemblage composition has for long been considered one of the best proxies for primary production, in particular for low latitude coastal upwelling areas, where they constitute the dominant phytoplankton. To investigate productivity conditions at those upwelling systems at any time and at the global level would be of great use for C export estimations and climate modeling, since primary production and C export in those systems is of major importance in controlling Earth's climate. To assess the value of the diatom sediment record at the global level, total abundance of marine diatoms was determined for 730 sites distributed by the five most important coastal upwelling systems of the modern ocean, and compared to several meaningful ecological parameters. Investigations of the satellite estimated primary productivity; upwelling index; water column physical properties and nutrient content, reveal a clear relation between sediment diatom abundance and primary production - although different between areas. Furthermore, upwelled waters [Si] appear as a determinant factor of the observed global diatom distribution.

  2. Estimation of tea catechin levels using micellar electrokinetic chromatography: a quantitative approach.

    PubMed

    Liu, Chao-Ming; Chen, Chung-Yu; Lin, Yang-Wei

    2014-05-01

    A simple, inexpensive micellar electrokinetic chromatography (MEKC) method with UV detection was used to determine seven catechins and one xanthine (caffeine) in tea. All the compounds were successfully separated (15kV) within a 15-min migration period with a high number of theoretical plates (>8.0×10(4)) in a running buffer (pH 7) containing 10mmoll(-1) sodium tetraborate, 4mmoll(-1) sodium phosphate, and 25mmoll(-1) SDS. The regression lines of all standard catechins were linear within the range of 0.03-4μgml(-1). Green tea infused at 95°C for 10min showed higher levels of catechins (especially epigallocatechin galate, epicatechin gallate, and epicatechin) than tea infused at 80°C. In addition, major differences were observed in the levels of catechins in the first and second infusions (both brewed at 95°C for 10min). Finally, green tea leaves were infused separately with tap water, deionised water, spring water, reverse osmosis water, and distilled water at 95°C, and the catechin content of the infusions was investigated by the proposed method. In the infusion brewed with tap water, catechins appeared to be epimerisation from the epistructure to the nonepistructure. This epimerisation may take place more readily in tap water than in distilled water owing to the complexity of the ions present in tap water.

  3. Cellular and molecular research to reduce uncertainties in estimates of health effects from low-level radiation

    SciTech Connect

    Elkind, M.M.; Bedford, J.; Benjamin, S.A.; Waldren, C.A. ); Gotchy, R.L. )

    1990-10-01

    A study was undertaken by five radiation scientists to examine the feasibility of reducing the uncertainties in the estimation of risk due to protracted low doses of ionizing radiation. In addressing the question of feasibility, a review was made by the study group: of the cellular, molecular, and mammalian radiation data that are available; of the way in which altered oncogene properties could be involved in the loss of growth control that culminates in tumorigenesis; and of the progress that had been made in the genetic characterizations of several human and animal neoplasms. On the basis of this analysis, the study group concluded that, at the present time, it is feasible to mount a program of radiation research directed at the mechanism(s) of radiation-induced cancer with special reference to risk of neoplasia due to protracted, low doses of sparsely ionizing radiation. To implement a program of research, a review was made of the methods, techniques, and instruments that would be needed. This review was followed by a survey of the laboratories and institutions where scientific personnel and facilities are known to be available. A research agenda of the principal and broad objectives of the program is also discussed. 489 refs., 21 figs., 14 tabs.

  4. Discrimination of liver cancer in cellular level based on backscatter micro-spectrum with PCA algorithm and BP neural network

    NASA Astrophysics Data System (ADS)

    Yang, Jing; Wang, Cheng; Cai, Gan; Dong, Xiaona

    2016-10-01

    The incidence and mortality rate of the primary liver cancer are very high and its postoperative metastasis and recurrence have become important factors to the prognosis of patients. Circulating tumor cells (CTC), as a new tumor marker, play important roles in the early diagnosis and individualized treatment. This paper presents an effective method to distinguish liver cancer based on the cellular scattering spectrum, which is a non-fluorescence technique based on the fiber confocal microscopic spectrometer. Combining the principal component analysis (PCA) with back propagation (BP) neural network were utilized to establish an automatic recognition model for backscatter spectrum of the liver cancer cells from blood cell. PCA was applied to reduce the dimension of the scattering spectral data which obtained by the fiber confocal microscopic spectrometer. After dimensionality reduction by PCA, a neural network pattern recognition model with 2 input layer nodes, 11 hidden layer nodes, 3 output nodes was established. We trained the network with 66 samples and also tested it. Results showed that the recognition rate of the three types of cells is more than 90%, the relative standard deviation is only 2.36%. The experimental results showed that the fiber confocal microscopic spectrometer combining with the algorithm of PCA and BP neural network can automatically identify the liver cancer cell from the blood cells. This will provide a better tool for investigating the metastasis of liver cancers in vivo, the biology metabolic characteristics of liver cancers and drug transportation. Additionally, it is obviously referential in practical application.

  5. Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels.

    PubMed Central

    Ramalho, C B; Hastings, J W; Colepicolo, P

    1995-01-01

    A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1) isolated from the marine dinoflagellate Gonyaulax polyedra is shown to be attributable to the daily synthesis and destruction of the protein. The enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 310 kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmus braunii. The experiments reported indicate that the cellular expression of NR is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase. PMID:7870813

  6. Modelling Molecular Mechanisms: A Framework of Scientific Reasoning to Construct Molecular-Level Explanations for Cellular Behaviour

    ERIC Educational Resources Information Center

    van Mil, Marc H. W.; Boerwinkel, Dirk Jan; Waarlo, Arend Jan

    2013-01-01

    Although molecular-level details are part of the upper-secondary biology curriculum in most countries, many studies report that students fail to connect molecular knowledge to phenomena at the level of cells, organs and organisms. Recent studies suggest that students lack a framework to reason about complex systems to make this connection. In this…

  7. Modelling Molecular Mechanisms: A Framework of Scientific Reasoning to Construct Molecular-Level Explanations for Cellular Behaviour

    ERIC Educational Resources Information Center

    van Mil, Marc H. W.; Boerwinkel, Dirk Jan; Waarlo, Arend Jan

    2013-01-01

    Although molecular-level details are part of the upper-secondary biology curriculum in most countries, many studies report that students fail to connect molecular knowledge to phenomena at the level of cells, organs and organisms. Recent studies suggest that students lack a framework to reason about complex systems to make this connection. In this…

  8. Quantitation of the mRNA levels of Epo and EpoR in various tissues in the ovine fetus.

    PubMed

    David, R Bruce; Lim, Gaik Bee; Moritz, Karen M; Koukoulas, Irene; Wintour, E Marelyn

    2002-02-25

    A partial cDNA of the sheep erythropoietin receptor (EpoR) was obtained and used in real-time PCR to quantitate mRNA levels in placenta, liver and kidney throughout development (term=150 days). This was compared with Epo mRNA levels in the same tissues. Both Epo and EpoR mRNA were present in the placenta throughout gestation at low levels from 66 days onwards and these did not vary throughout gestation. Compared with the expression levels in the placenta, the levels of EpoR gene expression in the liver at 66, 99 and 140 days were, median (range)-288 (120-343), 278 (63-541) and 7 (3-15), respectively, reflecting the disappearance of erythropoiesis after 130 days. Low levels of EpoR gene expression were seen in the kidney at 3 (2-5), 5 (2-7), and 7 (2-10) times that in the placenta at 66, 99, and 140 days, respectively. By hybridization histochemistry the EpoR mRNA was located in the proximal tubular cells of the mesonephros and metanephros at 42 days. Epo mRNA levels in the kidney were 215 (116-867), 528 (113-765) and 46 (15-204) times those in the placenta at 69, 99, and 140 days, respectively. In the liver at the same ages the concentrations of mRNA were lower than in the kidney, the liver/placenta ratios being 50 (11-90), 17 (3-39), 9 (5-14). At 130 days Epo/EpoR levels in the hippocampus were 6+/-3 and 8+/-3 times that in the term placenta, respectively. These studies demonstrate that the ovine placenta expresses the Epo gene from at least 66 days of gestation. However, gene expression levels are very low compared with those in the liver and kidney, and even the hippocampus.

  9. Quantitative investigation of bacterial chemotaxis at the single-cell level

    NASA Astrophysics Data System (ADS)

    Min, Taejin

    Living cells sense and respond to constantly changing environmental conditions. Depending on the type of stimuli, the cell may response by altering gene expression pattern, secreting molecules, or migrating to a different environment. Directed movement of cells in response to chemical stimuli is called chemotaxis. In bacterial chemotaxis, small extracellular molecules bind receptor proteins embedded in the cell membrane, which then transmit the signal inside the cell through a cascade of protein-protein interactions. This chain of events influences the behavior of motor proteins that drive the rotation of helical filaments called flagella. Individual cells of the gut-dwelling bacteria Escherichia coli (E. coli) have many such flagella, whose collective action results in the swimming behavior of the cell. A recent study found that in absence of chemical stimuli, fluctuations in the protein cascade can cause non-Poissonian switching behavior in the flagellar motor (2). A corollary was that extension of such behavior to the whole-cell swimming level would have implications for E. coli's foraging strategy. However, existence of such behavior at the swimming cell level could not be predicted a priori, since the mapping from single flagellum behavior to the swimming behavior of a multi-flagellated cell is complex and poorly understood (3, 4). Here we characterize the chemotactic behavior of swimming E. coli cells using a novel optical trap-based measurement technique. This technique allows us to trap individual cells and monitor their swimming behavior over long time periods with high temporal resolution. We find that swimming cells exhibit non-Poissonian switching statistics between different swimming states, in a manner similar to the rotational direction-switching behavior seen in individual flagella. Furthermore, we develop a data analysis routine that allows us to characterize higher order swimming features such as reversal of swimming direction and existence of

  10. Spatially resolved quantitative in-situ phase analysis of a self-leveling compound

    SciTech Connect

    Seifert, Severin; Neubauer, Juergen; Goetz-Neunhoeffer, Friedlinde

    2012-07-15

    The development of the crystalline microstructure of a hydrating self-leveling compound (SLC) was analyzed using a two-dimensional XRD (GADDS). The application of non-destructive micro-diffraction with the GADDS, combined with a custom-made sample holder, made it possible to carry out position-sensitive in-situ measurements of a Calcium-Aluminate-Cement-(CAC)-dominated SLC. Different substrates were used in the measurement procedures so as to acquire data regarding the influence of the properties of the ground surface on the process of hydration. The results show that the crystalline microstructure is strongly affected by the availability of free water. The strongly vertically-fluctuating water-content of the hydrating mortar, which is mainly influenced by outside conditions, has a very significant effect upon the resulting ettringite content. This fact is also reflected in the resulting microstructure of the cured SLC.

  11. QUANTITATIVE CELL MOTILITY FOR IN VITRO WOUND HEALING USING LEVEL SET-BASED ACTIVE CONTOUR TRACKING.

    PubMed

    Bunyak, Filiz; Palaniappan, Kannappan; Nath, Sumit K; Baskin, Tobias I; Dong, Gang

    2006-04-06

    Quantifying the behavior of cells individually, and in clusters as part of a population, under a range of experimental conditions, is a challenging computational task with many biological applications. We propose a versatile algorithm for segmentation and tracking of multiple motile epithelial cells during wound healing using time-lapse video. The segmentation part of the proposed method relies on a level set-based active contour algorithm that robustly handles a large number of cells. The tracking part relies on a detection-based multiple-object tracking method with delayed decision enabled by multi-hypothesis testing. The combined method is robust to complex cell behavior including division and apoptosis, and to imaging artifacts such as illumination changes.

  12. Novice nurses' level of global interdependence identity: a quantitative research study.

    PubMed

    Kozlowski-Gibson, Maria

    2015-01-01

    Often, therapeutic relationships are cross-cultural in nature, which places both nurses and patients at risk for stress, depression, and anxiety. The purpose of this investigation was to describe novice nurses' level of global interdependence identity, as manifested by worldminded attitudes, and identify the strongest predictors of worldminded attitudes. Prospective descriptive with multiple regression study. The various nursing units of a large hospital in the great Cleveland, OH, area. The participants were novice nurses up to two years after graduation from nursing school and employed as hospital clinicians. Descriptive statistics with the mean and standard deviation of the scores was used for the delineation of the development of the participants. The study relied on a survey instrument, the Scale to Measure Worldminded Attitudes developed by Sampson and Smith (1957). The numerical data was scored and organized on a Microsoft Excel spreadsheet. The Statistical Package for Social Sciences (SPSS) version 21 was the program used to assist with analysis. The assessment of the models created through regression was completed using the model summary and analysis of variance (ANOVA). The nurses' mean level of global interdependence identity was slightly above the neutral point between extreme national-mindedness and full development of global interdependence identity. The best predictors of worldminded attitudes were immigration, patriotism, and war conceptualized under a global frame of reference. Novice nurses did not demonstrate an optimum developmental status of global interdependence identity to safeguard cross-cultural encounters with patients. The recommendation is the inclusion of immigration, patriotism, and war in the nursing curriculum and co-curriculum to promote student development and a turnaround improvement in patient experience. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction.

    PubMed

    Sudiro, T M; Zivny, J; Ishiko, H; Green, S; Vaughn, D W; Kalayanarooj, S; Nisalak, A; Norman, J E; Ennis, F A; Rothman, A L

    2001-01-01

    There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections.

  14. An image analysis method for the precise selection and quantitation of fluorescently labeled cellular constituents: application to the measurement of human muscle cells in culture.

    PubMed

    Agley, Chibeza C; Velloso, Cristiana P; Lazarus, Norman R; Harridge, Stephen D R

    2012-06-01

    The accurate measurement of the morphological characteristics of cells with nonuniform conformations presents difficulties. We report here a straightforward method using immunofluorescent staining and the commercially available imaging program Adobe Photoshop, which allows objective and precise information to be gathered on irregularly shaped cells. We have applied this measurement technique to the analysis of human muscle cells and their immunologically marked intracellular constituents, as these cells are prone to adopting a highly branched phenotype in culture. Use of this method can be used to overcome many of the long-standing limitations of conventional approaches for quantifying muscle cell size in vitro. In addition, wider applications of Photoshop as a quantitative and semiquantitative tool in immunocytochemistry are explored.

  15. Muscle disuse alters skeletal muscle contractile function at the molecular and cellular levels in older adult humans in a sex-specific manner

    PubMed Central

    Callahan, Damien M; Miller, Mark S; Sweeny, Andrew P; Tourville, Timothy W; Slauterbeck, James R; Savage, Patrick D; Maugan, David W; Ades, Philip A; Beynnon, Bruce D; Toth, Michael J

    2014-01-01

    Physical inactivity that accompanies ageing and disease may hasten disability by reducing skeletal muscle contractility. To characterize skeletal muscle functional adaptations to muscle disuse, we compared contractile performance at the molecular, cellular and whole-muscle levels in healthy active older men and women (n = 15) and inactive older men and women with advanced-stage, symptomatic knee osteoarthritis (OA) (n = 16). OA patients showed reduced (P < 0.01) knee extensor function. At the cellular level, single muscle fibre force production was reduced in OA patients in myosin heavy chain (MHC) I and IIA fibres (both P < 0.05) and differences in IIA fibres persisted after adjustments for fibre cross-sectional area (P < 0.05). Although no group differences in contractile velocity or power output were found for any fibre type, sex was found to modify the effect of OA, with a reduction in MHC IIA power output and a trend towards reduced shortening velocity in women, but increases in both variables in men (P < 0.05 and P = 0.07, respectively). At the molecular level, these adaptations in MHC IIA fibre function were explained by sex-specific differences (P ≤ 0.05) in myosin–actin cross-bridge kinetics. Additionally, cross-bridge kinetics were slowed in MHC I fibres in OA patients (P < 0.01), attributable entirely to reductions in women with knee OA (P < 0.05), a phenotype that could be reproduced in vitro by chemical modification of protein thiol residues. Our results identify molecular and cellular functional adaptations in skeletal muscle that may contribute to reduced physical function with knee OA-associated muscle disuse, with sex-specific differences that may explain a greater disposition towards disability in women. PMID:25038243

  16. Quantitative analysis of the postcontractile blood-oxygenation-level-dependent (BOLD) effect in skeletal muscle

    PubMed Central

    Towse, Theodore F.; Slade, Jill M.; Ambrose, Jeffrey A.; DeLano, Mark C.

    2011-01-01

    Previous studies show that transient increases in both blood flow and magnetic resonance image signal intensity (SI) occur in human muscle after brief, single contractions, and that the SI increases are threefold larger in physically active compared with sedentary subjects. This study examined the relationship between these transient changes by measuring anterior tibial artery flow (Doppler ultrasound), anterior muscle SI (3T, one-shot echo-planar images, TR/TE = 1,000/35), and muscle blood volume and hemoglobin saturation [near-infrared spectroscopy (NIRS)] in the same subjects after 1-s-duration maximum isometric ankle dorsiflexion contractions. Arterial flow increased to a peak 5.9 ± 0.7-fold above rest (SE, n = 11, range 2.6–10.2) within 7 s and muscle SI increased to a peak 2.7 ± 0.6% (range 0.0–6.0%) above rest within 12 s after the contractions. The peak postcontractile SI change was significantly correlated with both peak postcontractile flow (r = 0.61, n = 11) and with subject activity level (r = 0.63, n = 10) estimated from 7-day accelerometer recordings. In a subset of 7 subjects in which NIRS data acquisition was successful, the peak magnitude of the postcontractile SI change agreed well with SI calculated from the NIRS blood volume and saturation changes (r = 0.80, slope = 1.02, intercept = 0.16), confirming the blood-oxygenation-level-dependent (BOLD) mechanism underlying the SI change. The magnitudes of postcontractile changes in blood saturation and SI were reproduced by a simple one-compartment muscle vascular model that incorporated the observed pattern of postcontractile flow, and which assumed muscle O2 consumption peaks within 2 s after a brief contraction. The results show that muscle postcontractile BOLD SI changes depend critically on the balance between O2 delivery and O2 consumption, both of which can be altered by chronic physical activity. PMID:21330621

  17. Effects of low level laser therapy (LLLT) on pressured human osteoblasts: A histomorphologic and quantitative study

    NASA Astrophysics Data System (ADS)

    Pyo, S. J.; Song, W. W.; Kim, I. R.; Park, B. S.; Kim, C. H.; Kim, S. S.; Chung, I. K.; Kim, Y. D.

    2012-03-01

    Previous research has investigated the effects of LLLT during titanium implantation, tooth movement and bone graft using deproteinized bovine bone and recognized that these circumstances were nothing more than intentional controlled overpressure against static cells since this controlled trauma could affect cell function/malfunction, or cell recovery/apoptosis. The present preliminary study was conducted to prove if LLL would influence cell viability and cell function after excessive damage, which is enough to diminish cell numbers and distort the features of cells. Our aim is to evaluate whether low level laser irradiation (LLLi) could be helpful in the recovery of traumatized osteoblasts (pressure damaged cells) by observing the morphology and the survival rate of those cells. This model used bone cell cultures which were traumatized by a pressure with 250 G of centripetal force and observed their response to such trauma and low level laser irradiation. In this experiment, a Ga-Al-As diode LLL (IMPRA-ORT, NDLux, Seoul, KOREA) was used with a wavelength of 808 nm, a focus of 14 × 24 mm, which was wide enough to cover the whole dish surface or well within at least 2 times radiation, and an output of 100 mW. Statistical analysis showed a higher recovery rate of damaged osteoblasts in the radiation group than the non-radiation group ( p < 0.05). The nonradiation group had a very poor proliferation rate in comparison to the control group ( p < 0.05) in every time period. In the control group, actin filaments showed a random orientation and cell process branched variously around each cell. In contrast, compressed cells, these patterns were turned into thicker and shorter cytoskeletons. As time progressed, every living cell recovered from the severe stress and recovered both form and function. In summary, the present study showed the capacity of LLLT to aid the recovery of the cell skeleton and affect cell viability on overpressured osteoblasts. These results may

  18. Understanding Cellular Respiration in Terms of Matter & Energy within Ecosystems

    ERIC Educational Resources Information Center

    White, Joshua S.; Maskiewicz, April C.

    2014-01-01

    Using a design-based research approach, we developed a data-rich problem (DRP) set to improve student understanding of cellular respiration at the ecosystem level. The problem tasks engage students in data analysis to develop biological explanations. Several of the tasks and their implementation are described. Quantitative results suggest that…

  19. Understanding Cellular Respiration in Terms of Matter & Energy within Ecosystems

    ERIC Educational Resources Information Center

    White, Joshua S.; Maskiewicz, April C.

    2014-01-01

    Using a design-based research approach, we developed a data-rich problem (DRP) set to improve student understanding of cellular respiration at the ecosystem level. The problem tasks engage students in data analysis to develop biological explanations. Several of the tasks and their implementation are described. Quantitative results suggest that…

  20. Quantitative evaluation of waste prevention on the level of small and medium sized enterprises (SMEs).

    PubMed

    Laner, David; Rechberger, Helmut

    2009-02-01

    Waste prevention is a principle means of achieving the goals of waste management and a key element for developing sustainable economies. Small and medium sized enterprises (SMEs) contribute substantially to environmental degradation, often not even being aware of their environmental effects. Therefore, several initiatives have been launched in Austria aimed at supporting waste prevention measures on the level of SMEs. To promote the most efficient projects, they have to be evaluated with respect to their contribution to the goals of waste management. It is the aim of this paper to develop a methodology for evaluating waste prevention measures in SMEs based on their goal orientation. At first, conceptual problems of defining and delineating waste prevention activities are briefly discussed. Then an approach to evaluate waste prevention activities with respect to their environmental performance is presented and benchmarks which allow for an efficient use of the available funds are developed. Finally the evaluation method is applied to a number of former projects and the calculated results are analysed with respect to shortcomings and limitations of the model. It is found that the developed methodology can provide a tool for a more objective and comprehensible evaluation of waste prevention measures.

  1. In vivo localization at the cellular level of stilbene fluorescence induced by Plasmopara viticola in grapevine leaves

    PubMed Central

    Bellow, Sébastien; Latouche, Gwendal; Brown, Spencer C.; Poutaraud, Anne; Cerovic, Zoran G.

    2012-01-01

    Accurate localization of phytoalexins is a key for better understanding their role. This work aims to localize stilbenes, the main phytoalexins of grapevine. The cellular localization of stilbene fluorescence induced by Plasmopara viticola, the agent of downy mildew, was determined in grapevine leaves of very susceptible, susceptible, and partially resistant genotypes during infection. Laser scanning confocal microscopy and microspectrofluorimetry were used to acquire UV-excited autofluorescence three-dimensional images and spectra of grapevine leaves 5–6 days after inoculation. This noninvasive technique of investigation in vivo was completed with in vitro spectrofluorimetric studies on pure stilbenes as their fluorescence is largely affected by the physicochemical environment in various leaf compartments. Viscosity was the major physicochemical factor influencing stilbene fluorescence intensity, modifying fluorescence yield by more than two orders of magnitude. Striking differences in the localization of stilbene fluorescence induced by P. viticola were observed between the different genotypes. All inoculated genotypes displayed stilbene fluorescence in cell walls of guard cells and periclinal cell walls of epidermal cells. Higher fluorescence intensity was observed in guard-cell walls than in any other compartment due to increased local viscosity. In addition stilbene fluorescence was found in epidermal cell vacuoles of the susceptible genotype and in the infected spongy parenchyma of the partially resistant genotype. The very susceptible genotype was devoid of fluorescence both in the epidermal vacuoles and the mesophyll. This strongly suggests that the resistance of grapevine leaves to P. viticola is correlated with the pattern of localization of induced stilbenes in host tissues. PMID:22412183

  2. Myofibroblasts Electrotonically Coupled to Cardiomyocytes Alter Conduction: Insights at the Cellular Level from a Detailed In silico Tissue Structure Model

    PubMed Central

    Jousset, Florian; Maguy, Ange; Rohr, Stephan; Kucera, Jan P.

    2016-01-01

    Fibrotic myocardial remodeling is typically accompanied by the appearance of myofibroblasts (MFBs). In vitro, MFBs were shown to slow conduction and precipitate ectopic activity following gap junctional coupling to cardiomyocytes (CMCs). To gain further mechanistic insights into this arrhythmogenic MFB-CMC crosstalk, we performed numerical simulations in cell-based high-resolution two-dimensional tissue models that replicated experimental conditions. Cell dimensions were determined using confocal microscopy of single and co-cultured neonatal rat ventricular CMCs and MFBs. Conduction was investigated as a function of MFB density in three distinct cellular tissue architectures: CMC strands with endogenous MFBs, CMC strands with coating MFBs of two different sizes, and CMC strands with MFB inserts. Simulations were performed to identify individual contributions of heterocellular gap junctional coupling and of the specific electrical phenotype of MFBs. With increasing MFB density, both endogenous and coating MFBs slowed conduction. At MFB densities of 5–30%, conduction slowing was most pronounced in strands with endogenous MFBs due to the MFB-dependent increase in axial resistance. At MFB densities >40%, very slow conduction and spontaneous activity was primarily due to MFB-induced CMC depolarization. Coating MFBs caused non-uniformities of resting membrane potential, which were more prominent with large than with small MFBs. In simulations of MFB inserts connecting two CMC strands, conduction delays increased with increasing insert lengths and block appeared for inserts >1.2 mm. Thus, electrophysiological properties of engineered CMC-MFB co-cultures depend on MFB density, MFB size and their specific positioning in respect to CMCs. These factors may influence conduction characteristics in the heterocellular myocardium. PMID:27833567

  3. Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels.

    PubMed

    Migliorini, Elisa; Thakar, Dhruv; Sadir, Rabia; Pleiner, Tino; Baleux, Françoise; Lortat-Jacob, Hugues; Coche-Guerente, Liliane; Richter, Ralf P

    2014-10-01

    Glycosaminoglycans (GAGs) are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell-cell and cell-matrix interactions. The supramolecular presentation of GAG chains, along with other matrix components, is likely to be functionally important but remains challenging to control and to characterize, both in vivo and in vitro. We present a method to create well-defined biomimetic surfaces that display GAGs, either alone or together with other cell ligands, in a background that suppresses non-specific binding. Through the design of the immobilization platform - a streptavidin monolayer serves as a molecular breadboard for the attachment of various biotinylated ligands - and a set of surface-sensitive in situ analysis techniques (including quartz crystal microbalance and spectroscopic ellipsometry), the biomimetic surfaces are tailor made with tight control on biomolecular orientation, surface density and lateral mobility. Analysing the interactions between a selected GAG (heparan sulphate, HS) and the HS-binding chemokine CXCL12α (also called SDF-1α), we demonstrate that these surfaces are versatile for biomolecular and cellular interaction studies. T-lymphocytes are found to adhere specifically to surfaces presenting CXCL12α, both when reversibly bound through HS and when irreversibly immobilized on the inert surface, even in the absence of any bona fide cell adhesion ligand. Moreover, surfaces which present both HS-bound CXCL12α and the intercellular adhesion molecule 1 (ICAM-1) synergistically promote cell adhesion. Our surface biofunctionalization strategy should be broadly applicable for functional studies that require a well-defined supramolecular presentation of GAGs along with other matrix or cell-surface components.

  4. Investigation of microgravity effects on basic imune functions on the cellular level - The TRIPLELUX-B experiment

    NASA Astrophysics Data System (ADS)

    Unruh, Eckehardt; Hansen, Peter-Diedrich

    Hemocytes are the primary defence of the Blue Mussel against invading microorganisms and foreign particles. The hemocytes of mussels as part of the immune system of invertebrates has not been studied so far in space. The choice of the phagocytes from invertebrates is justified by the claim to study the universal validity of innate immune responses. The hemocytes of mussels have a lot in common with macrophages of higher organisms. They are able to detect the presence of microorganisms and kill these microorganisms by phagocytosis. The phagocy-tosis related production of ROS will be stimulated with opsonised zymosan. The hemocytes will be stored frozen and reconstituted in-flight for the experiment. The signals of the im-muno cellular responses are translated into luminescence as a rapid optical reporter system. The primary aim of Triplelux B is to investigate under space flight conditions the effect of microgravity on the ability of isolated Blue Mussel hemocytes to perform phagocytosis. As a secondery objectiv, the results expected will allow to conclude whether the observed responses are caused by microgravity and/or radiation (change in permeability, endpoints in genotoxicity: DNA unwinding). The TRIPLELUX-B Experiment contributes to risk assessment concerning immunotoxicity under space flight conditions. The components of the fully automated AEC (Advanced Experimental Containment) will be demonstrated. The AEC of the TRIPLELUX-B experiment will contribute to a real time operational monitoring for immunotoxicity testing for earth. Blue mussels have been used repeatedly for monitoring imunotoxicity and genotoxicity in coastal waters. Based on the AEC an automatet measuring device will allow "real time monitoring" providing observations of immunotoxicity in coastal and inland waters.

  5. Gravity dependency of the gramicidin A channel conductivity. A model for gravity perception on the cellular level.

    PubMed

    Schatz, A; Linke-Hommes, A; Neubert, J

    1996-01-01

    Theoretical investigations involving the membrane-solution interface have revealed that the density of the solution varies appreciably within interfacial layers adjacent to charged membrane surfaces. The hypothesis that gravity interacts with this configuration and modifies transport rates across horizontal and vertical membranes differently was supported by initial experiments with gramicidin A channels in phosphatidylserine (PS) membranes in 0.1 M KCl. Channel conductivity was found to be about 1.6 times higher in horizontal membranes than in vertical membranes. Here we present the results of further experiments with gramicidin A channels (incorporated into charged PS- and uncharged phosphatidylcholine (PC) membranes in KCl- and CsCl-solutions) to demonstrate that the hypothesis is more generally applicable. Again, channel conductivity was found to be higher in horizontal PS membranes by a factor of between 1.20 and 1.75 in 0.1 M CsCl. No difference in channel conductivity was found for uncharged PC membranes in 0.1 M KCl and in 0.1 M CsCl. However, for PC membranes in 0.05 M KCl the channel conductivity was significantly higher in horizontal membranes by a factor of between 1.07 and 1.14. These results are consistent with the results of our model calculations of layer density and extension, which showed that the layer formation is enhanced by increasing membrane surface charge and decreasing electrolyte ion concentration. The mechanism of gravity interaction with membrane transport processes via interface reactions might be utilized by biological systems for orientational behaviour in the gravity field, which has been observed even for cellular systems.

  6. Myofibroblasts Electrotonically Coupled to Cardiomyocytes Alter Conduction: Insights at the Cellular Level from a Detailed In silico Tissue Structure Model.

    PubMed

    Jousset, Florian; Maguy, Ange; Rohr, Stephan; Kucera, Jan P

    2016-01-01

    Fibrotic myocardial remodeling is typically accompanied by the appearance of myofibroblasts (MFBs). In vitro, MFBs were shown to slow conduction and precipitate ectopic activity following gap junctional coupling to cardiomyocytes (CMCs). To gain further mechanistic insights into this arrhythmogenic MFB-CMC crosstalk, we performed numerical simulations in cell-based high-resolution two-dimensional tissue models that replicated experimental conditions. Cell dimensions were determined using confocal microscopy of single and co-cultured neonatal rat ventricular CMCs and MFBs. Conduction was investigated as a function of MFB density in three distinct cellular tissue architectures: CMC strands with endogenous MFBs, CMC strands with coating MFBs of two different sizes, and CMC strands with MFB inserts. Simulations were performed to identify individual contributions of heterocellular gap junctional coupling and of the specific electrical phenotype of MFBs. With increasing MFB density, both endogenous and coating MFBs slowed conduction. At MFB densities of 5-30%, conduction slowing was most pronounced in strands with endogenous MFBs due to the MFB-dependent increase in axial resistance. At MFB densities >40%, very slow conduction and spontaneous activity was primarily due to MFB-induced CMC depolarization. Coating MFBs caused non-uniformities of resting membrane potential, which were more prominent with large than with small MFBs. In simulations of MFB inserts connecting two CMC strands, conduction delays increased with increasing insert lengths and block appeared for inserts >1.2 mm. Thus, electrophysiological properties of engineered CMC-MFB co-cultures depend on MFB density, MFB size and their specific positioning in respect to CMCs. These factors may influence conduction characteristics in the heterocellular myocardium.

  7. In Vivo Bystander Effect: Cranial X-Irradiation Leads to Elevated DNA Damage, Altered Cellular Proliferation and Apoptosis, and Increased p53 Levels in Shielded Spleen

    SciTech Connect

    Koturbash, Igor; Loree, Jonathan; Kutanzi, Kristy; Koganow, Clayton; Pogribny, Igor; Kovalchuk, Olga

    2008-02-01

    Purpose: It is well accepted that irradiated cells may 'forward' genome instability to nonirradiated neighboring cells, giving rise to the 'bystander effect' phenomenon. Although bystander effects were well studied by using cell cultures, data for somatic bystander effects in vivo are relatively scarce. Methods and Materials: We set out to analyze the existence and molecular nature of bystander effects in a radiation target-organ spleen by using a mouse model. The animal's head was exposed to X-rays while the remainder of the body was completely protected by a medical-grade shield. Using immunohistochemistry, we addressed levels of DNA damage, cellular proliferation, apoptosis, and p53 protein in the spleen of control animals and completely exposed and head-exposed/body bystander animals. Results: We found that localized head radiation exposure led to the induction of bystander effects in the lead-shielded distant spleen tissue. Namely, cranial irradiation led to increased levels of DNA damage and p53 expression and also altered levels of cellular proliferation and apoptosis in bystander spleen tissue. The observed bystander changes were not caused by radiation scattering and were observed in two different mouse strains; C57BL/6 and BALB/c. Conclusion: Our study proves that bystander effects occur in the distant somatic organs on localized exposures. Additional studies are required to characterize the nature of an enigmatic bystander signal and analyze the long-term persistence of these effects and possible contribution of radiation-induced bystander effects to secondary radiation carcinogenesis.

  8. [The experimental evaluation with flow cytofluorimetry technique of the level of cellular immunologic memory in persons vaccinated against plague and anthrax].

    PubMed

    Bogacheva, N V; Kriuchkov, A V; Darmov, I V; Vorob'ev, K A; Pechenkin, D V; Elagin, G D; Kolesnikiov, D P

    2013-11-01

    The article deals with experimental evaluation with flow cytofluorimetry technique of the level of cellular immunologic memory in persons vaccinated with plague and anthrax live dry vaccines. It is established that the introduction of plague and anthrax live dry vaccines into organism of vaccinated persons ignites immunologic rearrangement manifested by reliable increase of level of blood concentration of Th1-lymphocytes (immunologic memory cells) against the background of vaccination. The higher correlation coefficient is detected between leucocytes lysis coefficient and stimulation coefficient according blood concentration level of T-lymphocytes predominantly at the expense of Th1-lymphocytes. The values of stimulation coefficient were calculated for corresponding blood cells of vaccinated persons. This data testifies the effectiveness of application of vaccination against plague and anthrax.

  9. Application of Quantitative NDE Techniques to High Level Waste Storage Tanks

    SciTech Connect

    Thompson, R. B.; Rehbein, D. K.; Bastiaans, G.; Terry, M.; Alers, R.

    2002-02-25

    As various issues make the continued usage of high-level waste storage tanks attractive, there is an increasing need to sharpen the assessment of their structural integrity. One aspect of a structural integrity program, nondestructive evaluation, is the focus of this paper. In September 2000, a program to support the sites was initiated jointly by Tanks Focus Area and Characterization, Monitoring, and Sensor Technologies Crosscutting Program of the Office of Environmental Management, Department of Energy (DOE). The vehicle was the Center for Nondestructive Evaluation, one of the National Science Foundation's Industry/University Cooperative Research Centers that is operated in close collaboration with the Ames Laboratory, USDOE. The support activities that have been provided by the center will be reviewed. Included are the organization of a series of annual workshops to allow the sites to share experiences and develop coordinated approaches to common problems, the development of an electronic source of relevant information, and assistance of the sites on particular technical problems. Directions and early results on some of these technical assistance projects are emphasized. Included are the discussion of theoretical analysis of ultrasonic wave propagation in curved plates to support the interpretation of tandem synthetic aperture focusing data to detect flaws in the knuckle region of double shell tanks; the evaluation of guided ultrasonic waves, excited by couplant free, electromagnetic acoustic transducers, to rapidly screen for inner wall corrosion in tanks; the use of spread spectrum techniques to gain information about the structural integrity of concrete domes; and the use of magnetic techniques to identify the alloys used in the construction of tanks.

  10. Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue.

    PubMed

    Tong, Huaxiang; Wiemer, Andrew J; Neighbors, Jeffrey D; Hohl, Raymond J

    2008-07-15

    Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at -80 degrees C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355+/-0.030 and 0.827+/-0.082 units of nmol/g wet tissues in brain, 0.320+/-0.019 and 0.293+/-0.035 units of nmol/g wet tissues in kidney, 0.326+/-0.064 and 0.213+/-0.029 units of nmol/g wet tissues in liver, and 0.364+/-0.015 and 0.349+/-0.023 units of nmol/g wet tissues in heart (means+/-SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.

  11. Quantitative trace-level speciation of arsenite and arsenate in drinking water by ion chromatography.

    PubMed

    Johnson, Rebecca L; Aldstad, Joseph H

    2002-10-01

    We describe an improved method for the determination of inorganic arsenic in drinking water. The method is based on comprehensive optimization of the anion-exchange ion chromatographic (IC) separation of arsenite and arsenate with post-column generation and detection of the arsenate-molybdate heteropoly acid (AMHPA) complex ion. The arsenite capacity factor was improved from 0.081 to 0.13 by using a mobile phase (2.0 mL min(-1)) composed of 2.5 mM Na2CO3 and 0.91 mM NaHCO3 (pH 10.5). A post-column photo-oxidation reactor (2.5 m x 0.7 mm) was optimized (0.37 microM potassium persulfate at 0.50 mL min(-1)) such that arsenite was converted to arsenate with 99.8 +/- 4.2% efficiency. Multi-variate optimization of the complexation reaction conditions yielded the following levels: 1.3 mM ammonium molybdate, 7.7 mM ascorbic acid, 0.48 M nitric acid, 0.17 mM potassium antimony tartrate, and 1.0% (v/v) glycerol. A long-path length flow cell (Teflon AF, 100-cm) was used to measure the absorption of the AMHPA complex (818 +/- 2 nm). Figures of merit for arsenite/arsenate include: limit of detection (1.6/0.40 microg L(-1)): standard error in absorbance (5.1 x 10(-3)/3.5 x 10(-3)); and sensitivity (2.9 x 10(-3)/2.2 x 10(-3) absorbance units per ppb). Successful application of the method to fortified surface and ground waters (100 microL samples) is also described.

  12. DNA-dependent homodimerization, sub-cellular partitioning, and protein destabilization control WUSCHEL levels and spatial patterning.

    PubMed

    Rodriguez, Kevin; Perales, Mariano; Snipes, Stephen; Yadav, Ram Kishor; Diaz-Mendoza, Mercedes; Reddy, G Venugopala

    2016-10-11

    The homeodomain transcription factor WUSCHEL (WUS) promotes stem cell maintenance in inflorescence meristems of Arabidopsis thaliana WUS, which is synthesized in the rib meristem, migrates and accumulates at lower levels in adjacent cells. Maintenance of WUS protein levels and spatial patterning distribution is not well-understood. Here, we show that the last 63-aa stretch of WUS is necessary for maintaining different levels of WUS protein in the rib meristem and adjacent cells. The 63-aa region contains the following transcriptional regulatory domains: the acidic region, the WUS-box, which is conserved in WUS-related HOMEOBOX family members, and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR-like) domain. Our analysis reveals that the opposing functions of WUS-box, which is required for nuclear retention, and EAR-like domain, which participates in nuclear export, are necessary to maintain higher nuclear levels of WUS in cells of the rib meristem and lower nuclear levels in adjacent cells. We also show that the N-terminal DNA binding domain, which is required for both DNA binding and homodimerization, along with the homodimerization sequence located in the central part of the protein, restricts WUS from spreading excessively and show that the homodimerization is critical for WUS function. Our analysis also reveals that a higher level of WUS outside the rib meristem leads to protein destabilization, suggesting a new tier of regulation in WUS protein regulation. Taken together our data show that processes that influence WUS protein levels and spatial distribution are highly coupled to its transcriptional activity.

  13. Quantitative determinations of anti-Kaposi sarcoma-associated herpesvirus antibody levels in men who have sex with men.

    PubMed

    Gogineni, Emile; Marshall, Vickie; Miley, Wendell; Bayat, Ahmad; Whitby, Denise; Kovacs, Joseph A; Burbelo, Peter D

    2013-05-01

    Infection with Kaposi sarcoma-associated herpesvirus (KSHV; also called human herpesvirus-8) is common among men who have sex with men (MSM). Here, quantitative anti-KSHV antibody levels were measured using luciferase immunoprecipitation systems (LIPS) in an MSM cohort with and without HIV from the NIH Clinical Center. Antibodies were detected using a mixture of 4 KSHV antigens in the MSM cohort and in Kaposi sarcoma (KS) patients. Along with HIV status, these results were compared with K8.1 and ORF73 ELISA, PCR virus detection, and additional LIPS testing. LIPS revealed that 25% (76/307) of the MSM cohort were KSHV seropositive, including 59 HIV+ and 17 HIV- subjects. The anti-KSHV antibody levels detected by LIPS were not statistically different between the KSHV+/HIV+ and KSHV+/HIV- subgroups but were lower than the KS patients (P < 0.0001). ELISA analysis of the MSM cohort detected a 35.5% frequency of KSHV infection and showed agreement with 81% of the samples evaluated by LIPS. Further LIPS testing with v-cyclin, a second ORF73 fragment and ORF38 reconciled some of the differences observed between LIPS and the ELISA immunoassays, and the revised LIPS seroprevalence in the MSM cohort was increased to 31%. Additional quantitative antibody analysis demonstrated statistically lower KSHV antibody levels in MSM compared to KS patients, but no difference was found between KSHV infected with and without HIV coinfection. These findings also suggest that antibodies against v-cyclin and ORF38 are useful for identifying patients with asymptomatic KSHV infection.

  14. Quantitation of signal transduction.

    PubMed

    Krauss, S; Brand, M D

    2000-12-01

    Conventional qualitative approaches to signal transduction provide powerful ways to explore the architecture and function of signaling pathways. However, at the level of the complete system, they do not fully depict the interactions between signaling and metabolic pathways and fail to give a manageable overview of the complexity that is often a feature of cellular signal transduction. Here, we introduce a quantitative experimental approach to signal transduction that helps to overcome these difficulties. We present a quantitative analysis of signal transduction during early mitogen stimulation of lymphocytes, with steady-state respiration rate as a convenient marker of metabolic stimulation. First, by inhibiting various key signaling pathways, we measure their relative importance in regulating respiration. About 80% of the input signal is conveyed via identifiable routes: 50% through pathways sensitive to inhibitors of protein kinase C and MAP kinase and 30% through pathways sensitive to an inhibitor of calcineurin. Second, we quantify how each of these pathways differentially stimulates functional units of reactions that produce and consume a key intermediate in respiration: the mitochondrial membrane potential. Both the PKC and calcineurin routes stimulate consumption more strongly than production, whereas the unidentified signaling routes stimulate production more than consumption, leading to no change in membrane potential despite increased respiration rate. The approach allows a quantitative description of the relative importance of signal transduction pathways and the routes by which they activate a specific cellular process. It should be widely applicable.

  15. Estimation of quantitative levels of diesel exhaust exposure and the health impact in the contemporary Australian mining industry.

    PubMed

    Peters, Susan; de Klerk, Nicholas; Reid, Alison; Fritschi, Lin; Musk, Aw Bill; Vermeulen, Roel

    2017-03-01

    To estimate quantitative levels of exposure to diesel exhaust expressed by elemental carbon (EC) in the contemporary mining industry and to describe the excess risk of lung cancer that may result from those levels. EC exposure has been monitored in Western Australian miners since 2003. Mixed-effects models were used to estimate EC levels for five surface and five underground occupation groups (as a fixed effect) and specific jobs within each group (as a random effect). Further fixed effects included sampling year and duration, and mineral mined. On the basis of published risk functions, we estimated excess lifetime risk of lung cancer mortality for several employment scenarios. Personal EC measurements (n=8614) were available for 146 different jobs at 124 mine sites. The mean estimated EC exposure level for surface occupations in 2011 was 14 µg/m(3) for 12 hour shifts. Levels for underground occupation groups ranged from 18 to 44 µg/m(3). Underground diesel loader operators had the highest exposed specific job: 59 µg/m(3). A lifetime career (45 years) as a surface worker or underground miner, experiencing exposure levels as estimated for 2011 (14 and 44 µg/m(3) EC), was associated with 5.5 and 38 extra lung cancer deaths per 1000 males, respectively. EC exposure levels in the contemporary Australian mining industry are still substantial, particularly for underground workers. The estimated excess numbers of lung cancer deaths associated with these exposures support the need for implementation of stringent occupational exposure limits for diesel exhaust. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  16. Synthesizing qualitative and quantitative evidence on non-financial access barriers: implications for assessment at the district level.

    PubMed

    O'Connell, Thomas S; Bedford, K Juliet A; Thiede, Michael; McIntyre, Di

    2015-06-09

    A key element of the global drive to universal health coverage is ensuring access to needed health services for everyone, and to pursue this goal in an equitable way. This requires concerted efforts to reduce disparities in access through understanding and acting on barriers facing communities with the lowest utilisation levels. Financial barriers dominate the empirical literature on health service access. Unless the full range of access barriers are investigated, efforts to promote equitable access to health care are unlikely to succeed. This paper therefore focuses on exploring the nature and extent of non-financial access barriers. We draw upon two structured literature reviews on barriers to access and utilization of maternal, newborn and child health services in Ghana, Bangladesh, Vietnam and Rwanda. One review analyses access barriers identified in published literature using qualitative research methods; the other in published literature using quantitative analysis of household survey data. We then synthesised the key qualitative and quantitative findings through a conjoint iterative analysis. Five dominant themes on non-financial access barriers were identified: ethnicity; religion; physical accessibility; decision-making, gender and autonomy; and knowledge, information and education. The analysis highlighted that non-financial factors pose considerable barriers to access, many of which relate to the acceptability dimension of access and are challenging to address. Another key finding is that quantitative research methods, while yielding important findings, are inadequate for understanding non-financial access barriers in sufficient detail to develop effective responses. Qualitative research is critical in filling this gap. The analysis also indicates that the nature of non-financial access barriers vary considerably, not only between countries but also between different communities within individual countries. To adequately understand access barriers as a

  17. Protein-based identification of quantitative trait loci associated with malignant transformation in two HER2+ cellular models of breast cancer

    PubMed Central

    2012-01-01

    Background A contemporary view of the cancer genome reveals extensive rearrangement compared to normal cells. Yet how these genetic alterations translate into specific proteomic changes that underpin acquiring the hallmarks of cancer remains unresolved. The objectives of this study were to quantify alterations in protein expression in two HER2+ cellular models of breast cancer and to infer differentially regulated signaling pathways in these models associated with the hallmarks of cancer. Results A proteomic workflow was used to identify proteins in two HER2 positive tumorigenic cell lines (BT474 and SKBR3) that were differentially expressed relative to a normal human mammary epithelial cell line (184A1). A total of 64 (BT474-184A1) and 69 (SKBR3-184A1) proteins were uniquely identified that were differentially expressed by at least 1.5-fold. Pathway inference tools were used to interpret these proteins in terms of functionally enriched pathways in the tumor cell lines. We observed "protein ubiquitination" and "apoptosis signaling" pathways were both enriched in the two breast cancer models while "IGF signaling" and "cell motility" pathways were enriched in BT474 and "amino acid metabolism" were enriched in the SKBR3 cell line. Conclusion While "protein ubiquitination" and "apoptosis signaling" pathways were common to both the cell lines, the observed patterns of protein expression suggest that the evasion of apoptosis in each tumorigenic cell line occurs via different mechanisms. Evidently, apoptosis is regulated in BT474 via down regulation of Bid and in SKBR3 via up regulation of Calpain-11 as compared to 184A1. PMID:22357162

  18. QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics.

    PubMed

    Choi, Hyungwon; Kim, Sinae; Fermin, Damian; Tsou, Chih-Chiang; Nesvizhskii, Alexey I

    2015-11-03

    We introduce QPROT, a statistical framework and computational tool for differential protein expression analysis using protein intensity data. QPROT is an extension of the QSPEC suite, originally developed for spectral count data, adapted for the analysis using continuously measured protein-level intensity data. QPROT offers a new intensity normalization procedure and model-based differential expression analysis, both of which account for missing data. Determination of differential expression of each protein is based on the standardized Z-statistic based on the posterior distribution of the log fold change parameter, guided by the false discovery rate estimated by a well-known Empirical Bayes method. We evaluated the classification performance of QPROT using the quantification calibration data from the clinical proteomic technology assessment for cancer (CPTAC) study and a recently published Escherichia coli benchmark dataset, with evaluation of FDR accuracy in the latter. QPROT is a statistical framework with computational software tool for comparative quantitative proteomics analysis. It features various extensions of QSPEC method originally built for spectral count data analysis, including probabilistic treatment of missing values in protein intensity data. With the increasing popularity of label-free quantitative proteomics data, the proposed method and accompanying software suite will be immediately useful for many proteomics laboratories. This article is part of a Special Issue entitled: Computational Proteomics. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Integrated Smartphone-App-Chip System for On-Site Parts-Per-Billion-Level Colorimetric Quantitation of Aflatoxins.

    PubMed

    Li, Xiaochun; Yang, Fan; Wong, Jessica X H; Yu, Hua-Zhong

    2017-09-05

    We demonstrate herein an integrated, smartphone-app-chip (SPAC) system for on-site quantitation of food toxins, as demonstrated with aflatoxin B1 (AFB1), at parts-per-billion (ppb) level in food products. The detection is based on an indirect competitive immunoassay fabricated on a transparent plastic chip with the assistance of a microfluidic channel plate. A 3D-printed optical accessory attached to a smartphone is adapted to align the assay chip and to provide uniform illumination for imaging, with which high-quality images of the assay chip are captured by the smartphone camera and directly processed using a custom-developed Android app. The performance of this smartphone-based detection system was tested using both spiked and moldy corn samples; consistent results with conventional enzyme-linked immunosorbent assay (ELISA) kits were obtained. The achieved detection limit (3 ± 1 ppb, equivalent to μg/kg) and dynamic response range (0.5-250 ppb) meet the requested testing standards set by authorities in China and North America. We envision that the integrated SPAC system promises to be a simple and accurate method of food toxin quantitation, bringing much benefit for rapid on-site screening.

  20. An Emerging Method for Rapid Characterization of Feed Structures and Feed Component Matrix at a Cellular Level and Relation to Feed Quality and Nutritive Value

    SciTech Connect

    Yu,P.

    2006-01-01

    Feed quality, feed characteristics, nutrient utilization and digestive behavior are closely related to: (i) total feed composition, (ii) feed intrinsic structures, and (iii) biological component matrix (such as protein to starch matrix, protein to carbohydrate matrix). Conventional 'wet' chemical analysis can determine total chemical composition, but fails to detect the feed intrinsic structures and biological component matrix due to destruction of feed samples during the processing for chemical analysis and the 'wet' chemical analysis cannot link structural information to chemical information within intact feed tissue. Recently, advanced synchrotron-based Fourier transform infrared (FTIR) microspectroscopy has been developed as a non-destructive and non-invasive structural-chemical analytical technique. This technique can link chemical information to structural information of biological samples within intact tissue within cellular dimensions. It can provide four kinds of information simultaneously: tissue composition, tissue structure, tissue chemistry and tissue environment. However, this novel technique has been found mainly for medical science research, extremely rare for feed science and nutrition research. The objective of this review article was to illustrate synchrotron-based FTIR microspectroscopy as a novel research tool for rapid characterization of feed structures at a cellular level and for detection of chemical features and molecular chemical make-up of feed biological component matrix and nutrient interaction. The emphasis of this article was to show that feed structural-chemical features at a cellular level are closely related to feed characteristics, feed quality and nutritive value in animals. The synchrotron-based technology will provide us with a greater understanding of the plant-animal interface.

  1. Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods

    NASA Astrophysics Data System (ADS)

    Singh, Manpreet; Alabanza, Anginelle; Gonzalez, Lorelis E.; Wang, Weiwei; Reeves, W. Brian; Hahm, Jong-In

    2016-02-01

    Determining ultratrace amounts of protein biomarkers in patient samples in a straightforward and quantitative manner is extremely important for early disease diagnosis and treatment. Here, we successfully demonstrate the novel use of zinc oxide nanorods (ZnO NRs) in the ultrasensitive and quantitative detection of two acute kidney injury (AKI)-related protein biomarkers, tumor necrosis factor (TNF)-α and interleukin (IL)-8, directly from patient samples. We first validate the ZnO NRs-based IL-8 results via comparison with those obtained from using a conventional enzyme-linked immunosorbent method in samples from 38 individuals. We further assess the full detection capability of the ZnO NRs-based technique by quantifying TNF-α, whose levels in human urine are often below the detection limits of conventional methods. Using the ZnO NR platforms, we determine the TNF-α concentrations of all 46 patient samples tested, down to the fg per mL level. Subsequently, we screen for TNF-α levels in approximately 50 additional samples collected from different patient groups in order to demonstrate a potential use of the ZnO NRs-based assay in assessing cytokine levels useful for further clinical monitoring. Our research efforts demonstrate that ZnO NRs can be straightforwardly employed in the rapid, ultrasensitive, quantitative, and simultaneous detection of multiple AKI-related biomarkers directly in patient urine samples, providing an unparalleled detection capability beyond those of conventional analysis methods. Additional key advantages of the ZnO NRs-based approach include a fast detection speed, low-volume assay condition, multiplexing ability, and easy automation/integration capability to existing fluorescence instrumentation. Therefore, we anticipate that our ZnO NRs-based detection method will be highly beneficial for overcoming the frequent challenges in early biomarker development and treatment assessment, pertaining to the facile and ultrasensitive quantification

  2. The effects of gamma radiation, UV and visible light on ATP levels in yeast cells depend on cellular melanization.

    PubMed

    Bryan, Ruth; Jiang, Zewei; Friedman, Matthew; Dadachova, Ekaterina

    2011-10-01

    Previously we have shown that growth of melanized fungi is stimulated by low levels of gamma radiation. The goal of this study was to examine the effects of visible light, UV light, and gamma radiation on the energy level (ATP concentration) in melanized Cryptococcus neoformans cells. Melanized C. neoformans cells as well as non-melanized controls were subjected to visible, UV or gamma radiation, and ATP was quantified by measuring the amount of light emitted by the ATP-dependent reaction of luciferase with luciferin. We found that all three forms of radiation led to a reduction in the ATP levels in melanized C. neoformans cells. This points to a universal melanin-related mechanism underlying observation of ATP decrease in irradiated melanized cells. In contrast, in non-melanized cells visible light led to increase in ATP levels; gamma radiation did not cause any changes while UV exposure resulted in some ATP decrease, however, much less pronounced than in melanized cells. Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.

  3. Molecular Mechanistic Reasoning: Toward Bridging the Gap between the Molecular and Cellular Levels in Life Science Education

    ERIC Educational Resources Information Center

    van Mil, Marc H. W.; Postma, Paulien A.; Boerwinkel, Dirk Jan; Klaassen, Kees; Waarlo, Arend Jan

    2016-01-01

    Although learning about DNA, RNA, and proteins is part of the upper secondary biology curriculum in most countries, many studies report that students fail to connect molecular knowledge to phenomena at the higher level of cells, organs, and organisms. As a result, many students use memorization and rote learning as a coping strategy when presented…

  4. Molecular Mechanistic Reasoning: Toward Bridging the Gap between the Molecular and Cellular Levels in Life Science Education

    ERIC Educational Resources Information Center

    van Mil, Marc H. W.; Postma, Paulien A.; Boerwinkel, Dirk Jan; Klaassen, Kees; Waarlo, Arend Jan

    2016-01-01

    Although learning about DNA, RNA, and proteins is part of the upper secondary biology curriculum in most countries, many studies report that students fail to connect molecular knowledge to phenomena at the higher level of cells, organs, and organisms. As a result, many students use memorization and rote learning as a coping strategy when presented…

  5. Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels.

    PubMed

    Zhou, Sheng; Zong, Yang; Ney, Paul A; Nair, Geeta; Stewart, Clinton F; Sorrentino, Brian P

    2005-03-15

    ABCG2/BCRP is a member of the adenosine triphosphate-binding cassette (ABC) transporter family and is expressed in intestine, kidney, and liver, where it modulates the absorption and excretion of xenobiotic compounds. ABCG2 is also expressed in hematopoietic stem cells and erythroid cells; however, little is known regarding its role in hematopoiesis. Abcg2 null mice have increased levels of protoporphyrin IX (PPIX) in erythroid cells, yet the mechanism for this remains uncertain. We have found that Abcg2 mRNA expression was up-regulated in differentiating erythroid cells, coinciding with increased expression of other erythroid-specific genes. This expression pattern was associated with significant amounts of ABCG2 protein on the membrane of mature peripheral blood erythrocytes. Erythroid cells engineered to express ABCG2 had significantly lower intracellular levels of PPIX, suggesting the modulation of PPIX level by ABCG2. This modulating activity was abrogated by treatment with a specific ABCG2 inhibitor, Ko143, implying that PPIX may be a direct substrate for the transporter. Taken together, our results demonstrate that ABCG2 plays a role in regulating PPIX levels during erythroid differentiation and suggest a potential role for ABCG2 as a genetic determinant in erythropoietic protoporphyria.

  6. Effect of garlic oil on neutrophil infiltration in the small intestine of endotoxin-injected rats and its association with levels of soluble and cellular adhesion molecules.

    PubMed

    Kuo, Chia-Hao; Lee, Shih-Hua; Chen, Ke-Ming; Lii, Chong-Kuei; Liu, Cheng-Tzu

    2011-07-27

    Garlic ( Allium sativum ) possesses anti-inflammatory effects. This study investigated the effects of garlic oil on endotoxin-induced neutrophil infiltration in the small intestine. Wistar rats received by gavage 10, 50, or 100 mg/kg body wt garlic oil (GO) or the vehicle (corn oil; 2 mL/kg body wt) every other day for 2 weeks before being injected with endotoxin (ip, 5 mg/kg body wt). Control rats were administered corn oil and injected with sterile saline. Blood samples for the measurement of soluble adhesion molecules were collected at various time points after injection, and all other samples were collected 18 h after injection. The 10 and 50 mg/kg doses suppressed endotoxin-induced neutrophilia, serum levels of sL-selectin and sICAM-1, cellular CD11b on neutrophils, intestinal ICAM-1 content, and neutrophil infiltration (P < 0.05). The 100 mg/kg dose significantly lowered local ICAM-1 and cellular CD11b on neutrophils (P < 0.05) but did not have a beneficial effect on neutrophil infiltration. In addition, 100 mg/kg of GO worsened the elevation of the local TNF-α level and neutrophilia. Appropriate doses of garlic oil have a preventive effect on endotoxin-induced neutrophil infiltration and damage to the small intestine.

  7. In vitro discrimination of the role of LRP1 at the BBB cellular level: focus on brain capillary endothelial cells and brain pericytes.

    PubMed

    Candela, Pietra; Saint-Pol, Julien; Kuntz, Mélanie; Boucau, Marie-Christine; Lamartiniere, Yordenca; Gosselet, Fabien; Fenart, Laurence

    2015-01-12

    Several studies have demonstrated that the blood-brain barrier (BBB) (dynamic cellular complex composed by brain capillary endothelial cells (BCECs) and surrounded by astrocytic end feet and pericytes) regulates the exchanges of amyloid β (Aβ) peptide between the blood and the brain. Deregulation of these exchanges seems to be a key trigger for the brain accumulation of Aβ peptide observed in Alzheimer's disease (AD). Whereas the involvement of receptor for advanced glycation end-products in Aβ peptide transcytosis has been demonstrated in our laboratory, low-density lipoprotein receptor's role at the cellular level needs to be clarified. For this, we used an in vitro BBB model that consists of a co-culture of bovine BCECs and rat glial cells. This model has already been used to characterize low-density lipoprotein receptor-related peptide (LRP)'s involvement in the transcytosis of molecules such as tPA and angiopep-2. Our results suggest that Aβ peptide efflux across the BCEC monolayer involves a transcellular transport. However, the experiments with RAP discard an involvement of LRP family members at BCECs level. In contrast, our results show a strong transcriptional expression of LRP1 in pericytes and suggest its implication in Aβ endocytosis. Moreover, the observations of pericytes contraction and local downregulation of LRP1 in response to Aβ treatment opens up perspectives for studying this cell type with respect to Aβ peptide metabolism and AD. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Massive bowel resection upregulates the intestinal mRNA expression levels of cellular retinol-binding protein II and apolipoprotein A-IV and alters the intestinal vitamin A status in rats.

    PubMed

    Hebiguchi, Taku; Mezaki, Yoshihiro; Morii, Mayako; Watanabe, Ryo; Yoshikawa, Kiwamu; Miura, Mitsutaka; Imai, Katsuyuki; Senoo, Haruki; Yoshino, Hiroaki

    2015-03-01

    Short bowel (SB) syndrome causes the malabsorption of various nutrients. Among these, vitamin A is important for a number of physiological activities. Vitamin A is absorbed by epithelial cells of the small intestine and is discharged into the lymphatic vessels as a component of chylomicrons and is delivered to the liver. In the present study, we used a rat model of SB syndrome in order to assess its effects on the expression of genes associated with the absorption, transport and metabolism of vitamin A. In the rats with SB, the intestinal mRNA expression levels of cellular retinol-binding protein II (CRBP II, gene symbol Rbp2) and apolipoprotein A-IV (gene symbol Apoa4) were higher than those in the sham-operated rats, as shown by RT-qPCR. Immunohistochemical analysis revealed that absorptive epithelial cells stained positive for both CRBP II and lecithin retinol acyltransferase, which are both required for the effective esterification of vitamin A. In the rats with SB, the retinol content in the ileum and the retinyl ester content in the jejunum were lower than those in the sham-operated rats, as shown by quantitative analysis of retinol and retinyl esters by high performance liquid chromatography. These results suggest that the elevated mRNA expression levels of Rbp2 and Apoa4 in the rats with SB contribute to the effective esterification and transport of vitamin A.

  9. Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

    PubMed Central

    Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

    2013-01-01

    Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

  10. Cellular detection of 50 Hz magnetic fields and weak blue light: effects on superoxide levels and genotoxicity.

    PubMed

    Höytö, Anne; Herrala, Mikko; Luukkonen, Jukka; Juutilainen, Jukka; Naarala, Jonne

    2017-06-01

    We tested the hypothesis that the effects of 50 Hz magnetic fields (MFs) on superoxide levels and genotoxicity depend on the presence of blue light. Human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 100 μT MF with or without non-phototoxic level of blue light for 24 h. We also studied whether these treatments alter responses to menadione, an agent that induces mitochondrial superoxide (O2(• -)) production and DNA damage. Micronuclei, proliferation, viability, cytosolic and mitochondrial O2(• -) levels were assessed. MF (without blue light) increased cytosolic O2(• -) production and blue light suppressed this effect. Mitochondrial O2(• -) production was reduced by both MF and blue light, but these effects were not additive. Micronucleus frequency was not affected by blue light or MF alone, but blue light (significantly when combined with MF) enhanced menadione-induced micronuclei. The original simple hypothesis (blue light is needed for MF effects) was not supported, but interaction of MF and blue light was nevertheless observed. The results are consistent with MF effects on light-independent radical reactions.

  11. Medawar's legacy to cellular immunology and clinical transplantation: a commentary on Billingham, Brent and Medawar (1956) ‘Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance’

    PubMed Central

    Simpson, Elizabeth

    2015-01-01

    Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance’, published in Philosophical Transactions B in 1956 by Peter Medawar and his colleagues, PhD graduate Leslie Brent and postdoctoral fellow Rupert Billingham, is a full description of the concept of acquired transplantation tolerance. Their 1953 Nature paper (Billingham RE et al. 1953 Nature 172, 603–606. (doi:10.1038/172603a0)) had provided initial evidence with experimental results from a small number of neonatal mice, with mention of similar findings in chicks. The Philosophical Transactions B 1956 paper is clothed with an astonishing amount of further experimental detail. It is written in Peter Medawar's landmark style: witty, perceptive and full of images that can be recalled even when details of the supporting information have faded. Those images are provided not just by a series of 20 colour plates showing skin graft recipient mice, rats, rabbits, chickens and duck, bearing fur or plumage of donor origin, but by his choice of metaphor, simile and analogy to express the questions being addressed and the interpretation of their results, along with those of relevant published data and his prescient ideas of what the results might portend. This work influenced both immunology researchers and clinicians and helped to lay the foundations for successful transplantation programmes. It led to the award of a Nobel prize in 1960 to Medawar, and subsequently to several scientists who advanced these areas. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society. PMID:25750245

  12. Medawar's legacy to cellular immunology and clinical transplantation: a commentary on Billingham, Brent and Medawar (1956) 'Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance'.

    PubMed

    Simpson, Elizabeth

    2015-04-19

    'Quantitative studies on tissue transplantation immunity. III. Actively acquired tolerance', published in Philosophical Transactions B in 1956 by Peter Medawar and his colleagues, PhD graduate Leslie Brent and postdoctoral fellow Rupert Billingham, is a full description of the concept of acquired transplantation tolerance. Their 1953 Nature paper (Billingham RE et al. 1953 Nature 172, 603-606. (doi:10.1038/172603a0)) had provided initial evidence with experimental results from a small number of neonatal mice, with mention of similar findings in chicks. The Philosophical Transactions B 1956 paper is clothed with an astonishing amount of further experimental detail. It is written in Peter Medawar's landmark style: witty, perceptive and full of images that can be recalled even when details of the supporting information have faded. Those images are provided not just by a series of 20 colour plates showing skin graft recipient mice, rats, rabbits, chickens and duck, bearing fur or plumage of donor origin, but by his choice of metaphor, simile and analogy to express the questions being addressed and the interpretation of their results, along with those of relevant published data and his prescient ideas of what the results might portend. This work influenced both immunology researchers and clinicians and helped to lay the foundations for successful transplantation programmes. It led to the award of a Nobel prize in 1960 to Medawar, and subsequently to several scientists who advanced these areas. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society.

  13. Segregation and linkage analysis of serum angiotensin I-converting enzyme levels: Evidence for two quantitative-trait loci

    SciTech Connect

    McKenzie, C.A.; Keavney, B.; Farrall, M.

    1995-12-01

    Human serum angiotensin I-converting enzyme (ACE) levels vary substantially between individuals and are highly heritable. Segregation analysis in European families has shown that more than half of the total variability in ACE levels is influenced by quantitative-trait loci (QTL). One of these QTLs is located within or close to the ACE locus itself. Combined segregation/linkage analysis in a series of African Caribbean families from Jamaica shows that the ACE insertion-deletion polymorphism is in moderate linkage disequilibrium with an ACE-linked QTL. Linkage analysis with a highly informative polymorphism at the neighboring growth hormone gene (GH) shows surprisingly little support for linkage (LOD score [Z] = 0.12). An extended analysis with a two-QTL model, where an ACE-linked QTL interacts additively with an unlinked QTL, significantly improves both the fit of the model (P = .002) and the support for linkage between the ACE-linked QTL and GH polymorphism (Z = 5.0). We conclude that two QTLs jointly influence serum ACE levels in this population. One QTL is located within or close to the ACE locus and explains 27% of the total variability; the second QTL is unlinked to the ACE locus and explains 52% of the variability. The identification of the molecular mechanisms underlying both QTLs is necessary in order to interpret the role of ACE in cardiovascular disease. 44 refs., 7 tabs.

  14. Cellular proteomic analysis of porcine circovirus type 2 and classical swine fever virus coinfection in porcine kidney-15 cells using isobaric tags for relative and absolute quantitation-coupled LC-MS/MS.

    PubMed

    Zhou, Niu; Fan, Chunmei; Liu, Song; Zhou, Jianwei; Jin, Yulan; Zheng, Xiaojuan; Wang, Qin; Liu, Jue; Yang, Hanchun; Gu, Jinyan; Zhou, Jiyong

    2017-05-01

    Viral coinfection or superinfection in host has caused public health concern and huge economic losses of farming industry. The influence of viral coinfection on cellular protein abundance is essential for viral pathogenesis. Based on a coinfection model for porcine circovirus type 2 (PCV2) and classical swine fever virus (CSFV) developed previously by our laboratory, isobaric tags for relative and absolute quantitation (iTRAQ)-coupled LC-MS/MS proteomic profiling was performed to explore the host cell responses to PCV2-CSFV coinfection. Totally, 3932 proteins were identified in three independent mass spectrometry analyses. Compared with uninfected cells, 304 proteins increased (fold change >1.2) and