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

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

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

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.

Effects of methotrexate on the viscoelastic properties of single cells probed by atomic force microscopy.

PubMed

Li, Mi; Liu, Lianqing; Xiao, Xiubin; Xi, Ning; Wang, Yuechao

2016-10-01

Methotrexate is a commonly used anti-cancer chemotherapy drug. Cellular mechanical properties are fundamental parameters that reflect the physiological state of a cell. However, so far the role of cellular mechanical properties in the actions of methotrexate is still unclear. In recent years, probing the behaviors of single cells with the use of atomic force microscopy (AFM) has contributed much to the field of cell biomechanics. In this work, with the use of AFM, the effects of methotrexate on the viscoelastic properties of four types of cells were quantitatively investigated. The inhibitory and cytotoxic effects of methotrexate on the proliferation of cells were observed by optical and fluorescence microscopy. AFM indenting was used to measure the changes of cellular viscoelastic properties (Young's modulus and relaxation time) by using both conical tip and spherical tip, quantitatively showing that the stimulation of methotrexate resulted in a significant decrease of both cellular Young's modulus and relaxation times. The morphological changes of cells induced by methotrexate were visualized by AFM imaging. The study improves our understanding of methotrexate action and offers a novel way to quantify drug actions at the single-cell level by measuring cellular viscoelastic properties, which may have potential impacts on developing label-free methods for drug evaluation.

Research highlights: microfluidics meets big data.

PubMed

Tseng, Peter; Weaver, Westbrook M; Masaeli, Mahdokht; Owsley, Keegan; Di Carlo, Dino

2014-03-07

In this issue we highlight a collection of recent work in which microfluidic parallelization and automation have been employed to address the increasing need for large amounts of quantitative data concerning cellular function--from correlating microRNA levels to protein expression, increasing the throughput and reducing the noise when studying protein dynamics in single-cells, and understanding how signal dynamics encodes information. The painstaking dissection of cellular pathways one protein at a time appears to be coming to an end, leading to more rapid discoveries which will inevitably translate to better cellular control--in producing useful gene products and treating disease at the individual cell level. From these studies it is also clear that development of large scale mutant or fusion libraries, automation of microscopy, image analysis, and data extraction will be key components as microfluidics contributes its strengths to aid systems biology moving forward.

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.

Regulation of BolA abundance mediates morphogenesis in Fremyella diplosiphon

DOE PAGES

Singh, Shailendra P.; Montgomery, Beronda L.

2015-11-05

Filamentous cyanobacterium Fremyella diplosiphon is known to alter its pigmentation and morphology during complementary chromatic acclimation (CCA) to efficiently harvest available radiant energy for photosynthesis. F. diplosiphon cells are rectangular and filaments are longer under green light (GL), whereas smaller, spherical cells and short filaments are prevalent under red light (RL). Light regulation of bolA morphogene expression is correlated with photoregulation of cellular morphology in F. diplosiphon. Here, we investigate a role for quantitative regulation of cellular BolA protein levels in morphology determination. Overexpression of bolA in WT was associated with induction of RL-characteristic spherical morphology even when cultures weremore » grown under GL. Overexpression of bolA in a ΔrcaE background, which lacks cyanobacteriochrome photosensor RcaE and accumulates lower levels of BolA than WT, partially reverted the cellular morphology of the strain to a WT-like state. Overexpression of BolA in WT and ΔrcaE backgrounds was associated with decreased cellular reactive oxygen species (ROS) levels and an increase in filament length under both GL and RL. Morphological defects and high ROS levels commonly observed in ΔrcaE could, thus, be in part due to low accumulation of BolA. Together, these findings support an emerging model for RcaE-dependent photoregulation of BolA in controlling the cellular morphology of F. diplosiphon during CCA.« less

Metabolic Mapping: Quantitative Enzyme Cytochemistry and Histochemistry to Determine the Activity of Dehydrogenases in Cells and Tissues.

PubMed

Molenaar, Remco J; Khurshed, Mohammed; Hira, Vashendriya V V; Van Noorden, Cornelis J F

2018-05-26

Altered cellular metabolism is a hallmark of many diseases, including cancer, cardiovascular diseases and infection. The metabolic motor units of cells are enzymes and their activity is heavily regulated at many levels, including the transcriptional, mRNA stability, translational, post-translational and functional level. This complex regulation means that conventional quantitative or imaging assays, such as quantitative mRNA experiments, Western Blots and immunohistochemistry, yield incomplete information regarding the ultimate activity of enzymes, their function and/or their subcellular localization. Quantitative enzyme cytochemistry and histochemistry (i.e., metabolic mapping) show in-depth information on in situ enzymatic activity and its kinetics, function and subcellular localization in an almost true-to-nature situation. We describe a protocol to detect the activity of dehydrogenases, which are enzymes that perform redox reactions to reduce cofactors such as NAD(P) + and FAD. Cells and tissue sections are incubated in a medium that is specific for the enzymatic activity of one dehydrogenase. Subsequently, the dehydrogenase that is the subject of investigation performs its enzymatic activity in its subcellular site. In a chemical reaction with the reaction medium, this ultimately generates blue-colored formazan at the site of the dehydrogenase's activity. The formazan's absorbance is therefore a direct measure of the dehydrogenase's activity and can be quantified using monochromatic light microscopy and image analysis. The quantitative aspect of this protocol enables researchers to draw statistical conclusions from these assays. Besides observational studies, this technique can be used for inhibition studies of specific enzymes. In this context, studies benefit from the true-to-nature advantages of metabolic mapping, giving in situ results that may be physiologically more relevant than in vitro enzyme inhibition studies. In all, metabolic mapping is an indispensable technique to study metabolism at the cellular or tissue level. The technique is easy to adopt, provides in-depth, comprehensive and integrated metabolic information and enables rapid quantitative analysis.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells.

PubMed

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-02-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer.

Cellular density-dependent down-regulation of EP4 prostanoid receptors via the up-regulation of hypoxia-inducible factor-1α in HCA-7 human colon cancer cells

PubMed Central

Otake, Sho; Yoshida, Kenji; Seira, Naofumi; Sanchez, Christopher M; Regan, John W; Fujino, Hiromichi; Murayama, Toshihiko

2015-01-01

Increases in prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) levels are features of colon cancer. Among the different E-type prostanoid receptor subtypes, EP4 receptors are considered to play a crucial role in carcinogenesis by, for example, inducing COX-2 when stimulated with PGE2. However, EP4 receptor levels and PGE2-induced cellular responses are inconsistent among the cellular conditions. Therefore, the connections responsible for the expression of EP4 receptors were investigated in the present study by focusing on cell density-induced hypoxia-inducible factor-1α (HIF-1α). The expression of EP4 receptors was examined using immunoblot analysis, quantitative polymerase chain reaction, and reporter gene assays in HCA-7 human colon cancer cells with different cellular densities. The involvement of HIF-1α and its signaling pathways were also examined by immunoblot analysis, reporter gene assays, and with siRNA. We here demonstrated that EP4 receptors as well as EP4 receptor-mediated COX-2 expression levels decreased with an increase in cellular density. In contrast, HIF-1α levels increased in a cellular density-dependent manner. The knockdown of HIF-1α by siRNA restored the expression of EP4 receptors and EP4 receptor-mediated COX-2 in cells at a high density. Thus, the cellular density-dependent increase observed in HIF-1α expression levels reduced the expression of COX-2 by decreasing EP4 receptor levels. This novel regulation mechanism for the expression of EP4 receptors by HIF-1α may provide an explanation for the inconsistent actions of PGE2. The expression levels of EP4 receptors may vary depending on cellular density, which may lead to the differential activation of their signaling pathways by PGE2. Thus, cellular density-dependent PGE2-mediated signaling may determine the fate/stage of cancer cells, i.e., the surrounding environments could define the fate/stage of malignancies associated with colon cancer. PMID:25692008

Parallel labeling experiments for pathway elucidation and (13)C metabolic flux analysis.

PubMed

Antoniewicz, Maciek R

2015-12-01

Metabolic pathway models provide the foundation for quantitative studies of cellular physiology through the measurement of intracellular metabolic fluxes. For model organisms metabolic models are well established, with many manually curated genome-scale model reconstructions, gene knockout studies and stable-isotope tracing studies. However, for non-model organisms a similar level of knowledge is often lacking. Compartmentation of cellular metabolism in eukaryotic systems also presents significant challenges for quantitative (13)C-metabolic flux analysis ((13)C-MFA). Recently, innovative (13)C-MFA approaches have been developed based on parallel labeling experiments, the use of multiple isotopic tracers and integrated data analysis, that allow more rigorous validation of pathway models and improved quantification of metabolic fluxes. Applications of these approaches open new research directions in metabolic engineering, biotechnology and medicine. Copyright © 2015 Elsevier Ltd. All rights reserved.

Effects of fade distribution on a mobile satellite downlink and uplink performance in a frequency reuse cellular configuration

NASA Technical Reports Server (NTRS)

Boutin, Karl; Lecours, Michel; Pelletier, Marcel; Delisle, Gilles Y.

1990-01-01

In a mobile satellite system with a frequency reuse cellular configuration, significant co-channel interference can be experienced due to the antenna sidelobe level. The signal will be subjected not only to its own fading, but also to the effect of the varying degree of fading on co-channel interferer, and this interference will behave differently in the up and in the down link. This paper presents a quantitative evaluation of the combined effects of fades and co-channel interference on a mobile satellite link.

Bioelectronic Sensors and Devices

NASA Astrophysics Data System (ADS)

Reed, Mark

Nanoscale electronic devices have recently enabled the ability to controllably probe biological systems, from the molecular to the cellular level, opening up new applications and understanding of biological function and response. This talk reviews some of the advances in the field, ranging from diagnostic and therapeutic applications, to cellular manipulation and response, to the emulation of biological response. In diagnostics, integrated nanodevice biosensors compatible with CMOS technology have achieved unprecedented sensitivity, enabling a wide range of label-free biochemical and macromolecule sensing applications down to femtomolar concentrations. These systems have demonstrated integrated assays of biomarkers at clinically important concentrations for both diagnostics and as a quantitative tool for drug design and discovery. Cellular level response can also be observed, including immune response function and dynamics. Finally, the field is beginning to create devices that emulate function, and the demonstration of a solid state artificial ion channel will be discussed.

Semi-quantitative estimation of cellular SiO2 nanoparticles using flow cytometry combined with X-ray fluorescence measurements.

PubMed

Choi, Seo Yeon; Yang, Nuri; Jeon, Soo Kyung; Yoon, Tae Hyun

2014-09-01

In this study, we have demonstrated feasibility of a semi-quantitative approach for the estimation of cellular SiO2 nanoparticles (NPs), which is based on the flow cytometry measurements of their normalized side scattering intensity. In order to improve our understanding on the quantitative aspects of cell-nanoparticle interactions, flow cytometry, transmission electron microscopy, and X-ray fluorescence experiments were carefully performed for the HeLa cells exposed to SiO2 NPs with different core diameters, hydrodynamic sizes, and surface charges. Based on the observed relationships among the experimental data, a semi-quantitative cellular SiO2 NPs estimation method from their normalized side scattering and core diameters was proposed, which can be applied for the determination of cellular SiO2 NPs within their size-dependent linear ranges. © 2014 International Society for Advancement of Cytometry.

[The blood glucose value not necessarily indicates correctly the cellular metabolic state].

PubMed

Simon, Kornél; Wittmann, István

2017-03-01

In clinical recommendations the normalized blood glucose level is declared as the main target in therapy of diabetes mellitus, i.e. the achievement of euglycemia is the main therapeutic goal. This approach suggests, that the normal blood glucose value is the marker of the normal carbohydrate metabolism (eumetabolism), and vice versa: hyperglycemia is associated with abnormal metabolism (dysmetabolism). However the question arises, whether identical blood glucose values do reflect the same intracellular biochemical mechanisms? On the basis of data published in the literature authors try to answer these questions by studying the relations between the short/longterm blood glucose level and the cellular metabolism in different clinical settings characterized by divergent pathophysiological parameters. The correlations between blood glucose level and cellular metabolism in development of micro-, and macroangiopathy, in the breakthrough phenomenon, as well as during administration of metabolic promoters, the discrepancies of relation between blood glucose values and cellular metabolism in type 1, and type 2 diabetes mellitus, furthermore association between blood glucose value and myocardial metabolism in acute and chronic stress were analyzed. Authors conclude, that the actual blood glucose values reveal the actual cellular metabolism in a very variable manner: neither euglycemia does mandatorily indicate eumetabolism (balance of cellular energy production), nor hyperglycemia is necessarily a marker of abnormal metabolic state (dept of cellular energy production). Moreover, at the same actual blood glucose level both the metabolic efficacy of the same organ may sharply vary, and the intracellular biochemical machinery could also be very different. In case of the very same longterm blood glucose level the metabolic state of the different organs could be very variable: some organs show an energetically balanced metabolism, while others produce a significant deficit. These inconsistencies between blood glucose level and cellular metabolism can be explained by the fact, that blood glucose value is a transport parameter, reflecting the actual steady state of glucose transport from the carbohydrate pools into the blood, and that from the blood into the tissues. Without knowing the speed of these transports of opposite direction, the blood glucose value per se can not reveal the quantitative and qualitative characteristics of cellular metabolism. Orv. Hetil., 2017, 158(11), 409-417.

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

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers.

PubMed

Lichten, Catherine A; White, Rachel; Clark, Ivan B N; Swain, Peter S

2014-02-03

To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour.

Unmixing of fluorescence spectra to resolve quantitative time-series measurements of gene expression in plate readers

PubMed Central

2014-01-01

Background To connect gene expression with cellular physiology, we need to follow levels of proteins over time. Experiments typically use variants of Green Fluorescent Protein (GFP), and time-series measurements require specialist expertise if single cells are to be followed. Fluorescence plate readers, however, a standard in many laboratories, can in principle provide similar data, albeit at a mean, population level. Nevertheless, extracting the average fluorescence per cell is challenging because autofluorescence can be substantial. Results Here we propose a general method for correcting plate reader measurements of fluorescent proteins that uses spectral unmixing and determines both the fluorescence per cell and the errors on that fluorescence. Combined with strain collections, such as the GFP fusion collection for budding yeast, our methodology allows quantitative measurements of protein levels of up to hundreds of genes and therefore provides complementary data to high throughput studies of transcription. We illustrate the method by following the induction of the GAL genes in Saccharomyces cerevisiae for over 20 hours in different sugars and argue that the order of appearance of the Leloir enzymes may be to reduce build-up of the toxic intermediate galactose-1-phosphate. Further, we quantify protein levels of over 40 genes, again over 20 hours, after cells experience a change in carbon source (from glycerol to glucose). Conclusions Our methodology is sensitive, scalable, and should be applicable to other organisms. By allowing quantitative measurements on a per cell basis over tens of hours and over hundreds of genes, it should increase our understanding of the dynamic changes that drive cellular behaviour. PMID:24495318

The Next Frontier: Quantitative Biochemistry in Living Cells.

PubMed

Honigmann, Alf; Nadler, André

2018-01-09

Researchers striving to convert biology into an exact science foremost rely on structural biology and biochemical reconstitution approaches to obtain quantitative data. However, cell biological research is moving at an ever-accelerating speed into areas where these approaches lose much of their edge. Intrinsically unstructured proteins and biochemical interaction networks composed of interchangeable, multivalent, and unspecific interactions pose unique challenges to quantitative biology, as do processes that occur in discrete cellular microenvironments. Here we argue that a conceptual change in our way of conducting biochemical experiments is required to take on these new challenges. We propose that reconstitution of cellular processes in vitro should be much more focused on mimicking the cellular environment in vivo, an approach that requires detailed knowledge of the material properties of cellular compartments, essentially requiring a material science of the cell. In a similar vein, we suggest that quantitative biochemical experiments in vitro should be accompanied by corresponding experiments in vivo, as many newly relevant cellular processes are highly context-dependent. In essence, this constitutes a call for chemical biologists to convert their discipline from a proof-of-principle science to an area that could rightfully be called quantitative biochemistry in living cells. In this essay, we discuss novel techniques and experimental strategies with regard to their potential to fulfill such ambitious aims.

Quantitative Proteomic Analysis of BHK-21 Cells Infected with Foot-and-Mouth Disease Virus Serotype Asia 1.

PubMed

Guo, Hui-Chen; Jin, Ye; Han, Shi-Chong; Sun, Shi-Qi; Wei, Yan-Quan; Liu, Xian-Ji; Feng, Xia; Liu, Ding Xiang; Liu, Xiang-Tao

2015-01-01

Stable isotope labeling with amino acids in cell culture (SILAC) was used to quantitatively study the host cell gene expression profile, in order to achieve an unbiased overview of the protein expression changes in BHK-21 cells infected with FMDV serotype Asia 1. The SILAC-based approach identified overall 2,141 proteins, 153 of which showed significant alteration in the expression level 6 h post FMDV infection (57 up-regulated and 96 down-regulated). Among these proteins, six cellular proteins, including three down-regulated (VPS28, PKR, EVI5) and three up-regulated (LYPLA1, SEC62 and DARs), were selected according to the significance of the changes and/or the relationship with PKR. The expression level and pattern of the selected proteins were validated by immunoblotting and confocal microscopy. Furthermore, the functions of these cellular proteins were assessed by small interfering RNA-mediated depletion, and their functional importance in the replication of FMDV was demonstrated by western blot, reverse transcript PCR (RT-PCR) and 50% Tissue Culture Infective Dose (TCID50). The results suggest that FMDV infection may have effects on the expression of specific cellular proteins to create more favorable conditions for FMDV infection. This study provides novel data that can be utilized to understand the interactions between FMDV and the host cell.

Cellular automata with object-oriented features for parallel molecular network modeling.

PubMed

Zhu, Hao; Wu, Yinghui; Huang, Sui; Sun, Yan; Dhar, Pawan

2005-06-01

Cellular automata are an important modeling paradigm for studying the dynamics of large, parallel systems composed of multiple, interacting components. However, to model biological systems, cellular automata need to be extended beyond the large-scale parallelism and intensive communication in order to capture two fundamental properties characteristic of complex biological systems: hierarchy and heterogeneity. This paper proposes extensions to a cellular automata language, Cellang, to meet this purpose. The extended language, with object-oriented features, can be used to describe the structure and activity of parallel molecular networks within cells. Capabilities of this new programming language include object structure to define molecular programs within a cell, floating-point data type and mathematical functions to perform quantitative computation, message passing capability to describe molecular interactions, as well as new operators, statements, and built-in functions. We discuss relevant programming issues of these features, including the object-oriented description of molecular interactions with molecule encapsulation, message passing, and the description of heterogeneity and anisotropy at the cell and molecule levels. By enabling the integration of modeling at the molecular level with system behavior at cell, tissue, organ, or even organism levels, the program will help improve our understanding of how complex and dynamic biological activities are generated and controlled by parallel functioning of molecular networks. Index Terms-Cellular automata, modeling, molecular network, object-oriented.

Label-free assessment of endothelial cell metabolic state using autofluorescent microscopy

NASA Astrophysics Data System (ADS)

Pullen, Benjamin J.; Nguyen, Tam; Gosnell, Martin; Anwer, Ayad G.; Goldys, Ewa; Nicholls, Stephen J.; Psaltis, Peter J.

2016-12-01

To examine the process of endothelial cell aging we utilised hyperspectral imaging to collect broad autofluorescence emission at the individual cellular level and mathematically isolate the characteristic spectra of nicotinamide and flavin adenine dinucleotides (NADH and FAD, respectively). Quantitative analysis of this data provides the basis for a non-destructive spatial imaging method for cells and tissue. FAD and NADH are important factors in cellular metabolism and have been shown to be involved with the redox state of the cell; with the ratio between the two providing the basis for an `optical redox ratio'.

FRET-based genetically-encoded sensors for quantitative monitoring of metabolites.

PubMed

Mohsin, Mohd; Ahmad, Altaf; Iqbal, Muhammad

2015-10-01

Neighboring cells in the same tissue can exist in different states of dynamic activities. After genomics, proteomics and metabolomics, fluxomics is now equally important for generating accurate quantitative information on the cellular and sub-cellular dynamics of ions and metabolite, which is critical for functional understanding of organisms. Various spectrometry techniques are used for monitoring ions and metabolites, although their temporal and spatial resolutions are limited. Discovery of the fluorescent proteins and their variants has revolutionized cell biology. Therefore, novel tools and methods targeting sub-cellular compartments need to be deployed in specific cells and targeted to sub-cellular compartments in order to quantify the target-molecule dynamics directly. We require tools that can measure cellular activities and protein dynamics with sub-cellular resolution. Biosensors based on fluorescence resonance energy transfer (FRET) are genetically encoded and hence can specifically target sub-cellular organelles by fusion to proteins or targetted sequences. Since last decade, FRET-based genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of cellular physiology. This review, describing the design and principles of sensors, presents a database of sensors for different analytes/processes, and illustrate examples of application in quantitative live cell imaging.

A quantitative image cytometry technique for time series or population analyses of signaling networks.

PubMed

Ozaki, Yu-ichi; Uda, Shinsuke; Saito, Takeshi H; Chung, Jaehoon; Kubota, Hiroyuki; Kuroda, Shinya

2010-04-01

Modeling of cellular functions on the basis of experimental observation is increasingly common in the field of cellular signaling. However, such modeling requires a large amount of quantitative data of signaling events with high spatio-temporal resolution. A novel technique which allows us to obtain such data is needed for systems biology of cellular signaling. We developed a fully automatable assay technique, termed quantitative image cytometry (QIC), which integrates a quantitative immunostaining technique and a high precision image-processing algorithm for cell identification. With the aid of an automated sample preparation system, this device can quantify protein expression, phosphorylation and localization with subcellular resolution at one-minute intervals. The signaling activities quantified by the assay system showed good correlation with, as well as comparable reproducibility to, western blot analysis. Taking advantage of the high spatio-temporal resolution, we investigated the signaling dynamics of the ERK pathway in PC12 cells. The QIC technique appears as a highly quantitative and versatile technique, which can be a convenient replacement for the most conventional techniques including western blot, flow cytometry and live cell imaging. Thus, the QIC technique can be a powerful tool for investigating the systems biology of cellular signaling.

Reliable measurement of E. coli single cell fluorescence distribution using a standard microscope set-up.

PubMed

Cortesi, Marilisa; Bandiera, Lucia; Pasini, Alice; Bevilacqua, Alessandro; Gherardi, Alessandro; Furini, Simone; Giordano, Emanuele

2017-01-01

Quantifying gene expression at single cell level is fundamental for the complete characterization of synthetic gene circuits, due to the significant impact of noise and inter-cellular variability on the system's functionality. Commercial set-ups that allow the acquisition of fluorescent signal at single cell level (flow cytometers or quantitative microscopes) are expensive apparatuses that are hardly affordable by small laboratories. A protocol that makes a standard optical microscope able to acquire quantitative, single cell, fluorescent data from a bacterial population transformed with synthetic gene circuitry is presented. Single cell fluorescence values, acquired with a microscope set-up and processed with custom-made software, are compared with results that were obtained with a flow cytometer in a bacterial population transformed with the same gene circuitry. The high correlation between data from the two experimental set-ups, with a correlation coefficient computed over the tested dynamic range > 0.99, proves that a standard optical microscope- when coupled with appropriate software for image processing- might be used for quantitative single-cell fluorescence measurements. The calibration of the set-up, together with its validation, is described. The experimental protocol described in this paper makes quantitative measurement of single cell fluorescence accessible to laboratories equipped with standard optical microscope set-ups. Our method allows for an affordable measurement/quantification of intercellular variability, whose better understanding of this phenomenon will improve our comprehension of cellular behaviors and the design of synthetic gene circuits. All the required software is freely available to the synthetic biology community (MUSIQ Microscope flUorescence SIngle cell Quantification).

Quantitative optical imaging of paracetamol-induced metabolism changes in the liver

NASA Astrophysics Data System (ADS)

Liang, Xiaowen; Wang, Haolu; Liu, Xin; Roberts, Michael

2016-12-01

Paracetamol is the most readily available and widely used painkiller. However, its toxicity remains the most common cause of liver injury. The toxicity of paracetamol has been attributing to its toxic metabolite, which depletes cellular glutathione (GSH) stores and reacts within cells to increase oxidative stress, leading to mitochondrial dysfunction and cell necrosis. Multiphoton microscopy (MPM) and fluorescence lifetime imaging (FLIM) can provide quantitative imaging of biological tissues and organs in vivo and allow direct visualization of cellular events, which were used to monitor cellular metabolism in paracetamol-induced toxicity in this study. To better understand mechanisms of paracetamol induced liver injury, the redox ratio of NADH/FAD in liver cells were detected and quantified by MPM imaging to represent the relative rates of glycolysis and oxidative phosphorylation within cells. Compared to normal liver, average fluorescence lifetime of NADH and redox ratio of NADH/FAD in hepatocytes was significantly decreased after paracetamol overdose for 12 and 24 hrs, reflecting impaired metabolic activity. GSH levels of treatment groups were significantly lower than those of normal livers, with gradually decreasing from periportal to centrilobular zonation. This imaging technique has significant implications for investigating metabolic mechanisms of paracetamol toxicity.

Universality and predictability in molecular quantitative genetics.

PubMed

Nourmohammad, Armita; Held, Torsten; Lässig, Michael

2013-12-01

Molecular traits, such as gene expression levels or protein binding affinities, are increasingly accessible to quantitative measurement by modern high-throughput techniques. Such traits measure molecular functions and, from an evolutionary point of view, are important as targets of natural selection. We review recent developments in evolutionary theory and experiments that are expected to become building blocks of a quantitative genetics of molecular traits. We focus on universal evolutionary characteristics: these are largely independent of a trait's genetic basis, which is often at least partially unknown. We show that universal measurements can be used to infer selection on a quantitative trait, which determines its evolutionary mode of conservation or adaptation. Furthermore, universality is closely linked to predictability of trait evolution across lineages. We argue that universal trait statistics extends over a range of cellular scales and opens new avenues of quantitative evolutionary systems biology. Copyright © 2013. Published by Elsevier Ltd.

Cellular MYCro economics: Balancing MYC function with MYC expression.

PubMed

Levens, David

2013-11-01

The expression levels of the MYC oncoprotein have long been recognized to be associated with the outputs of major cellular processes including proliferation, cell growth, apoptosis, differentiation, and metabolism. Therefore, to understand how MYC operates, it is important to define quantitatively the relationship between MYC input and expression output for its targets as well as the higher-order relationships between the expression levels of subnetwork components and the flow of information and materials through those networks. Two different views of MYC are considered, first as a molecular microeconomic manager orchestrating specific positive and negative responses at individual promoters in collaboration with other transcription and chromatin components, and second, as a macroeconomic czar imposing an overarching rule onto all active genes. In either case, c-myc promoter output requires multiple inputs and exploits diverse mechanisms to tune expression to the appropriate levels relative to the thresholds of expression that separate health and disease.

Emerging Imaging and Genomic Tools for Developmental Systems Biology.

PubMed

Liu, Zhe; Keller, Philipp J

2016-03-21

Animal development is a complex and dynamic process orchestrated by exquisitely timed cell lineage commitment, divisions, migration, and morphological changes at the single-cell level. In the past decade, extensive genetic, stem cell, and genomic studies provided crucial insights into molecular underpinnings and the functional importance of genetic pathways governing various cellular differentiation processes. However, it is still largely unknown how the precise coordination of these pathways is achieved at the whole-organism level and how the highly regulated spatiotemporal choreography of development is established in turn. Here, we discuss the latest technological advances in imaging and single-cell genomics that hold great promise for advancing our understanding of this intricate process. We propose an integrated approach that combines such methods to quantitatively decipher in vivo cellular dynamic behaviors and their underlying molecular mechanisms at the systems level with single-cell, single-molecule resolution. Copyright © 2016 Elsevier Inc. All rights reserved.

Correlation of transforming growth factor-β messenger RNA (TGF-β mRNA) expression with cellular immunoassays in Triamcinolone-treated captive hybrid striped bass

USGS Publications Warehouse

Harms, Craig A.; Ottinger, Christopher A.; Kennedy-Stoskopf, S.

2000-01-01

Assessing fish immune status with molecular markers has been hampered by a lack of specific reagents. A quantitative polymerase chain reaction (PCR) method (reverse transcription quantitative–competitive PCR, RT-qcPCR) for measuring transforming growth factor-β (TGF-β) transcription from a broad range of teleost fish has recently been developed. The quantitative PCR now permits monitoring production of this important immunosuppressive cytokine in response to immunomodulating agents and conditions. We examined anterior kidney and spleen mononuclear cells from hybrid striped bass (female striped bass Morone saxatilis× male white bass M. chrysops) for production of TGF-β messenger RNA (mRNA) in response to administration of the synthetic glucocorticoid triamcinolone. We also compared TGF-β transcription with anterior kidney macrophage bactericidal activity and splenic lymphocyte blastogenesis. Anterior kidney mononuclear cell TGF-β mRNA levels decreased, whereas bactericidal activity increased. Spleen TGF-β mRNA levels did not change significantly, and splenic lymphocyte pokeweed mitogen stimulation index increased in triamcinolone-treated fish. Since triamcinolone is used therapeutically as a suppressive immunomodulator, the enhanced immune functions indicated by the cellular immunoassays were unexpected; however, the inverse response of TGF-β production and macrophage bactericidal activity was consistent with the known relationship between TGF-β and macrophage activation in mammals. Induced immunomodulation in hybrid striped bass was detectable by both traditional cellular immunoassays and the new RT-qcPCR for TGF-β.

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

PubMed

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. Copyright © 2013 Wiley Periodicals, Inc.

Intraspecific variation in cellular and biochemical heat response strategies of Mediterranean Xeropicta derbentina [Pulmonata, Hygromiidae].

PubMed

Troschinski, Sandra; Di Lellis, Maddalena A; Sereda, Sergej; Hauffe, Torsten; Wilke, Thomas; Triebskorn, Rita; Köhler, Heinz-R

2014-01-01

Dry and hot environments challenge the survival of terrestrial snails. To minimize overheating and desiccation, physiological and biochemical adaptations are of high importance for these animals. In the present study, seven populations of the Mediterranean land snail species Xeropicta derbentina were sampled from their natural habitat in order to investigate the intraspecific variation of cellular and biochemical mechanisms, which are assigned to contribute to heat resistance. Furthermore, we tested whether genetic parameters are correlated with these physiological heat stress response patterns. Specimens of each population were individually exposed to elevated temperatures (25 to 52°C) for 8 h in the laboratory. After exposure, the health condition of the snails' hepatopancreas was examined by means of qualitative description and semi-quantitative assessment of histopathological effects. In addition, the heat-shock protein 70 level (Hsp70) was determined. Generally, calcium cells of the hepatopancreas were more heat resistant than digestive cells - this phenomenon was associated with elevated Hsp70 levels at 40°C.We observed considerable variation in the snails' heat response strategy: Individuals from three populations invested much energy in producing a highly elevated Hsp70 level, whereas three other populations invested energy in moderate stress protein levels - both strategies were in association with cellular functionality. Furthermore, one population kept cellular condition stable despite a low Hsp70 level until 40°C exposure, whereas prominent cellular reactions were observed above this thermal limit. Genetic diversity (mitochondrial cytochrome c oxidase subunit I gene) within populations was low. Nevertheless, when using genetic indices as explanatory variables in a multivariate regression tree (MRT) analysis, population structure explained mean differences in cellular and biochemical heat stress responses, especially in the group exposed to 40°C. Our study showed that, even in similar habitats within a close range, populations of the same species use different stress response strategies that all rendered survival possible.

Intraspecific Variation in Cellular and Biochemical Heat Response Strategies of Mediterranean Xeropicta derbentina [Pulmonata, Hygromiidae

PubMed Central

Troschinski, Sandra; Di Lellis, Maddalena A.; Sereda, Sergej; Hauffe, Torsten; Wilke, Thomas; Triebskorn, Rita; Köhler, Heinz-R.

2014-01-01

Dry and hot environments challenge the survival of terrestrial snails. To minimize overheating and desiccation, physiological and biochemical adaptations are of high importance for these animals. In the present study, seven populations of the Mediterranean land snail species Xeropicta derbentina were sampled from their natural habitat in order to investigate the intraspecific variation of cellular and biochemical mechanisms, which are assigned to contribute to heat resistance. Furthermore, we tested whether genetic parameters are correlated with these physiological heat stress response patterns. Specimens of each population were individually exposed to elevated temperatures (25 to 52°C) for 8 h in the laboratory. After exposure, the health condition of the snails' hepatopancreas was examined by means of qualitative description and semi-quantitative assessment of histopathological effects. In addition, the heat-shock protein 70 level (Hsp70) was determined. Generally, calcium cells of the hepatopancreas were more heat resistant than digestive cells - this phenomenon was associated with elevated Hsp70 levels at 40°C.We observed considerable variation in the snails' heat response strategy: Individuals from three populations invested much energy in producing a highly elevated Hsp70 level, whereas three other populations invested energy in moderate stress protein levels - both strategies were in association with cellular functionality. Furthermore, one population kept cellular condition stable despite a low Hsp70 level until 40°C exposure, whereas prominent cellular reactions were observed above this thermal limit. Genetic diversity (mitochondrial cytochrome c oxidase subunit I gene) within populations was low. Nevertheless, when using genetic indices as explanatory variables in a multivariate regression tree (MRT) analysis, population structure explained mean differences in cellular and biochemical heat stress responses, especially in the group exposed to 40°C. Our study showed that, even in similar habitats within a close range, populations of the same species use different stress response strategies that all rendered survival possible. PMID:24475158

A Model of Risk Analysis in Analytical Methodology for Biopharmaceutical Quality Control.

PubMed

Andrade, Cleyton Lage; Herrera, Miguel Angel De La O; Lemes, Elezer Monte Blanco

2018-01-01

One key quality control parameter for biopharmaceutical products is the analysis of residual cellular DNA. To determine small amounts of DNA (around 100 pg) that may be in a biologically derived drug substance, an analytical method should be sensitive, robust, reliable, and accurate. In principle, three techniques have the ability to measure residual cellular DNA: radioactive dot-blot, a type of hybridization; threshold analysis; and quantitative polymerase chain reaction. Quality risk management is a systematic process for evaluating, controlling, and reporting of risks that may affects method capabilities and supports a scientific and practical approach to decision making. This paper evaluates, by quality risk management, an alternative approach to assessing the performance risks associated with quality control methods used with biopharmaceuticals, using the tool hazard analysis and critical control points. This tool provides the possibility to find the steps in an analytical procedure with higher impact on method performance. By applying these principles to DNA analysis methods, we conclude that the radioactive dot-blot assay has the largest number of critical control points, followed by quantitative polymerase chain reaction, and threshold analysis. From the analysis of hazards (i.e., points of method failure) and the associated method procedure critical control points, we conclude that the analytical methodology with the lowest risk for performance failure for residual cellular DNA testing is quantitative polymerase chain reaction. LAY ABSTRACT: In order to mitigate the risk of adverse events by residual cellular DNA that is not completely cleared from downstream production processes, regulatory agencies have required the industry to guarantee a very low level of DNA in biologically derived pharmaceutical products. The technique historically used was radioactive blot hybridization. However, the technique is a challenging method to implement in a quality control laboratory: It is laborious, time consuming, semi-quantitative, and requires a radioisotope. Along with dot-blot hybridization, two alternatives techniques were evaluated: threshold analysis and quantitative polymerase chain reaction. Quality risk management tools were applied to compare the techniques, taking into account the uncertainties, the possibility of circumstances or future events, and their effects upon method performance. By illustrating the application of these tools with DNA methods, we provide an example of how they can be used to support a scientific and practical approach to decision making and can assess and manage method performance risk using such tools. This paper discusses, considering the principles of quality risk management, an additional approach to the development and selection of analytical quality control methods using the risk analysis tool hazard analysis and critical control points. This tool provides the possibility to find the method procedural steps with higher impact on method reliability (called critical control points). Our model concluded that the radioactive dot-blot assay has the larger number of critical control points, followed by quantitative polymerase chain reaction and threshold analysis. Quantitative polymerase chain reaction is shown to be the better alternative analytical methodology in residual cellular DNA analysis. © PDA, Inc. 2018.

Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth.

PubMed

Sankar, Martial; Nieminen, Kaisa; Ragni, Laura; Xenarios, Ioannis; Hardtke, Christian S

2014-02-11

Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001.

Automated quantitative histology reveals vascular morphodynamics during Arabidopsis hypocotyl secondary growth

PubMed Central

Sankar, Martial; Nieminen, Kaisa; Ragni, Laura; Xenarios, Ioannis; Hardtke, Christian S

2014-01-01

Among various advantages, their small size makes model organisms preferred subjects of investigation. Yet, even in model systems detailed analysis of numerous developmental processes at cellular level is severely hampered by their scale. For instance, secondary growth of Arabidopsis hypocotyls creates a radial pattern of highly specialized tissues that comprises several thousand cells starting from a few dozen. This dynamic process is difficult to follow because of its scale and because it can only be investigated invasively, precluding comprehensive understanding of the cell proliferation, differentiation, and patterning events involved. To overcome such limitation, we established an automated quantitative histology approach. We acquired hypocotyl cross-sections from tiled high-resolution images and extracted their information content using custom high-throughput image processing and segmentation. Coupled with automated cell type recognition through machine learning, we could establish a cellular resolution atlas that reveals vascular morphodynamics during secondary growth, for example equidistant phloem pole formation. DOI: http://dx.doi.org/10.7554/eLife.01567.001 PMID:24520159

Quantifying lipid changes in various membrane compartments using lipid binding protein domains.

PubMed

Várnai, Péter; Gulyás, Gergő; Tóth, Dániel J; Sohn, Mira; Sengupta, Nivedita; Balla, Tamas

2017-06-01

One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms. Published by Elsevier Ltd.

Quantitative Proteomic Analysis of Duck Ovarian Follicles Infected with Duck Tembusu Virus by Label-Free LC-MS

PubMed Central

Han, Kaikai; Zhao, Dongmin; Liu, Yuzhuo; Liu, Qingtao; Huang, Xinmei; Yang, Jing; An, Fengjiao; Li, Yin

2016-01-01

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that has caused massive economic losses to the duck industry in China. DTMUV infection mainly results in significant decreases in egg production in egg-laying ducks within 1–2 weeks post infection. However, information on the comparative protein expression of host tissues in response to DTMUV infection is limited. In the present study, the cellular protein response to DTMUV infection in duck ovarian follicles was analyzed using nano-flow high-performance liquid chromatography-electrospray tandem mass spectrometry. Quantitative proteomic analysis revealed 131 differentially expressed proteins, among which 53 were up regulated and 78 were down regulated. The identified proteins were involved in the regulation of essential processes such as cellular structure and integrity, RNA processing, protein biosynthesis and modification, vesicle transport, signal transduction, and mitochondrial pathway. Some selected proteins that were found to be regulated in DTMUV-infected tissues were screened by quantitative real-time PCR to examine their regulation at the transcriptional level, western blot analysis was used to validate the changes of some selected proteins on translational level. To our knowledge, this study is the first to analyze the proteomic changes in duck ovarian follicles following DTMUV infection. The protein-related information obtained in this study may be useful to understand the host response to DTMUV infection and the inherent mechanism of DTMUV replication and pathogenicity. PMID:27066001

Impact of inflammation on iron stores in involved and non-involved psoriatic skin

NASA Astrophysics Data System (ADS)

Pinheiro, T.; Ynsa, M. D.; Alves, L. C.; Teixeira, P.; Ferreira, J.; Filipe, P.

2015-04-01

Accumulating evidence supports a role for cellular Fe in cell proliferation, inflammation, and disease tolerance. Psoriasis is a severe inflammatory and hyper proliferative condition of human skin whose aetiology remains poorly understood. Herein, we performed nuclear microscopy techniques to quantify with cellular resolution and high sensitivity the concentration of Fe in lesional (psoriatic plaques) and non-lesional adjacent skin of psoriatic patients. Fe contents were measured across skin depth and along epidermal strata either by quantitatively imaging Fe distribution in regions of interest, or by determining Fe profiles through analysis of sequential points along selected transepts. Both procedures require deconvolution of spectra to project quantitative elemental data through the application of different software codes. Using these approaches a detailed quantitative distribution of Fe was resolved. We show that in both lesional and non-lesional skin, the epidermal profiles of Fe contents showed a peak at the basal layer and that Fe concentration along the basal layer was not uniformly distributed. Typically, Fe levels were significantly higher in epidermal ridges relative to regions above dermal papillae. Lesional skin displayed excess Fe over extended regions above basal layer. In conclusion, we found significantly increased Fe deposits in the epidermis of psoriatic patients, particularly in areas of epidermal hyper proliferation. These findings suggest an important role for Fe in the pathogenesis of psoriasis. They also raise the possibility that manipulation of Fe levels in the skin may become relevant for the clinical management of psoriasis.

Low vitamin D levels predict clinical features of schizophrenia.

PubMed

Cieslak, Kristina; Feingold, Jordyn; Antonius, Daniel; Walsh-Messinger, Julie; Dracxler, Roberta; Rosedale, Mary; Aujero, Nicole; Keefe, David; Goetz, Deborah; Goetz, Raymond; Malaspina, Dolores

2014-11-01

Vitamin D plays crucial roles in neuroprotection and neurodevelopment, and low levels are commonly associated with schizophrenia. We considered if the association was spurious or causal by examining the association of Vitamin D with Leukocyte Telomere Length (LTL), a marker of cellular aging. Vitamin D levels in 22 well-characterized schizophrenia cases were examined with respect to symptoms, cognition, and functioning. LTL was assessed using quantitative polymerase chain reaction (qPCR). The results showed that 91% (20) had deficient or insufficient Vitamin D levels, which were associated with excitement and grandiosity, social anhedonia, and poverty of speech. Sex-specific analyses showed strong associations of hypovitamintosis D to negative symptoms and decreased premorbid adjustment in males, and to lesser hallucinations and emotional withdrawal, but increased anti-social aggression in females. In females LTL was furthermore associated with Vitamin D levels. This study demonstrates a relationship of low vitamin D levels with increased cellular aging in females. It is also the first study to demonstrate potential sex-specific profiles among schizophrenia cases with hypovitaminosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Quantifying the correlation between spatially defined oxygen gradients and cell fate in an engineered three-dimensional culture model.

PubMed

Ardakani, Amir G; Cheema, Umber; Brown, Robert A; Shipley, Rebecca J

2014-09-06

A challenge in three-dimensional tissue culture remains the lack of quantitative information linking nutrient delivery and cellular distribution. Both in vivo and in vitro, oxygen is delivered by diffusion from its source (blood vessel or the construct margins). The oxygen level at a defined distance from its source depends critically on the balance of diffusion and cellular metabolism. Cells may respond to this oxygen environment through proliferation, death and chemotaxis, resulting in spatially resolved gradients in cellular density. This study extracts novel spatially resolved and simultaneous data on tissue oxygenation, cellular proliferation, viability and chemotaxis in three-dimensional spiralled, cellular collagen constructs. Oxygen concentration gradients drove preferential cellular proliferation rates and viability in the higher oxygen zones and induced chemotaxis along the spiral of the collagen construct; an oxygen gradient of 1.03 mmHg mm(-1) in the spiral direction induced a mean migratory speed of 1015 μm day(-1). Although this movement was modest, it was effective in balancing the system to a stable cell density distribution, and provided insights into the natural cell mechanism for adapting cell number and activity to a prevailing oxygen regime.

System-wide organization of actin cytoskeleton determines organelle transport in hypocotyl plant cells

PubMed Central

Nowak, Jacqueline; Ivakov, Alexander; Somssich, Marc; Persson, Staffan; Nikoloski, Zoran

2017-01-01

The actin cytoskeleton is an essential intracellular filamentous structure that underpins cellular transport and cytoplasmic streaming in plant cells. However, the system-level properties of actin-based cellular trafficking remain tenuous, largely due to the inability to quantify key features of the actin cytoskeleton. Here, we developed an automated image-based, network-driven framework to accurately segment and quantify actin cytoskeletal structures and Golgi transport. We show that the actin cytoskeleton in both growing and elongated hypocotyl cells has structural properties facilitating efficient transport. Our findings suggest that the erratic movement of Golgi is a stable cellular phenomenon that might optimize distribution efficiency of cell material. Moreover, we demonstrate that Golgi transport in hypocotyl cells can be accurately predicted from the actin network topology alone. Thus, our framework provides quantitative evidence for system-wide coordination of cellular transport in plant cells and can be readily applied to investigate cytoskeletal organization and transport in other organisms. PMID:28655850

Living with high putrescine: expression of ornithine and arginine biosynthetic pathway genes in high and low putrescine producing poplar cells

Treesearch

Andrew F. Page; Rakesh Minocha; Subhash C. Minocha

2012-01-01

Arginine (Arg) and ornithine (Orn), both derived from glutamate (Glu), are the primary substrates for polyamine (PA) biosynthesis, and also play important roles as substrates and intermediates of overall N metabolism in plants. Their cellular homeostasis is subject to multiple levels of regulation. Using reverse transcription quantitative PCR (RT-qPCR), we studied...

Coupling biomechanics to a cellular level model: an approach to patient-specific image driven multi-scale and multi-physics tumor simulation.

PubMed

May, Christian P; Kolokotroni, Eleni; Stamatakos, Georgios S; Büchler, Philippe

2011-10-01

Modeling of tumor growth has been performed according to various approaches addressing different biocomplexity levels and spatiotemporal scales. Mathematical treatments range from partial differential equation based diffusion models to rule-based cellular level simulators, aiming at both improving our quantitative understanding of the underlying biological processes and, in the mid- and long term, constructing reliable multi-scale predictive platforms to support patient-individualized treatment planning and optimization. The aim of this paper is to establish a multi-scale and multi-physics approach to tumor modeling taking into account both the cellular and the macroscopic mechanical level. Therefore, an already developed biomodel of clinical tumor growth and response to treatment is self-consistently coupled with a biomechanical model. Results are presented for the free growth case of the imageable component of an initially point-like glioblastoma multiforme tumor. The composite model leads to significant tumor shape corrections that are achieved through the utilization of environmental pressure information and the application of biomechanical principles. Using the ratio of smallest to largest moment of inertia of the tumor material to quantify the effect of our coupled approach, we have found a tumor shape correction of 20% by coupling biomechanics to the cellular simulator as compared to a cellular simulation without preferred growth directions. We conclude that the integration of the two models provides additional morphological insight into realistic tumor growth behavior. Therefore, it might be used for the development of an advanced oncosimulator focusing on tumor types for which morphology plays an important role in surgical and/or radio-therapeutic treatment planning. Copyright © 2011 Elsevier Ltd. All rights reserved.

Quantitative Differences in a Single Maternal Factor Determine Survival Probabilities among Drosophila Germ Cells.

PubMed

Slaidina, Maija; Lehmann, Ruth

2017-01-23

Germ cell death occurs in many species [1-3] and has been proposed as a mechanism by which the fittest, strongest, or least damaged germ cells are selected for transmission to the next generation. However, little is known about how the choice is made between germ cell survival and death. Here, we focus on the mechanisms that regulate germ cell survival during embryonic development in Drosophila. We find that the decision to die is a germ cell-intrinsic process linked to quantitative differences in germ plasm inheritance, such that higher germ plasm inheritance correlates with higher primordial germ cell (PGC) survival probability. We demonstrate that the maternal factor lipid phosphate phosphatase Wunen-2 (Wun2) regulates PGC survival in a dose-dependent manner. Since wun2 mRNA levels correlate with the levels of other maternal determinants at the single-cell level, we propose that Wun2 is used as a readout of the overall germ plasm quantity, such that only PGCs with the highest germ plasm quantity survive. Furthermore, we demonstrate that Wun2 and p53, another regulator of PGC survival, have opposite yet independent effects on PGC survival. Since p53 regulates cell death upon DNA damage and various cellular stresses, we hypothesize that together they ensure selection of the PGCs with highest germ plasm quantity and least cellular damage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery.

PubMed

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

2011-08-01

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. © The Author(s) 2011

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

Metabolic pathway profiling of mitochondrial respiratory chain mutants in C. elegans

PubMed Central

MJ, Falk; Z, Zhang; Rosenjack; Nissim; E, Daikhin; Nissim; MM, Sedensky; M, Yudkoff; PG, Morgan

2008-01-01

C. elegans affords a model of primary mitochondrial dysfunction that provides insight into cellular adaptations which accompany mutations in nuclear gene that encode mitochondrial proteins. To this end, we characterized genome-wide expression profiles of C. elegans strains with mutations in nuclear-encoded subunits of respiratory chain complexes. Our goal was to detect concordant changes among clusters of genes that comprise defined metabolic pathways. Results indicate that respiratory chain mutants significantly upregulate a variety of basic cellular metabolic pathways involved in carbohydrate, amino acid, and fatty acid metabolism, as well as cellular defense pathways such as the metabolism of P450 and glutathione. To further confirm and extend expression analysis findings, quantitation of whole worm free amino acid levels was performed in C. elegans mitochondrial mutants for subunits of complexes I, II, and III. Significant differences were seen for 13 of 16 amino acid levels in complex I mutants compared with controls, as well as overarching similarities among profiles of complex I, II, and III mutants compared with controls. The specific pattern of amino acid alterations observed provides novel evidence to suggest that an increase in glutamate-linked transamination reactions caused by the failure of NAD+ dependent oxidation of ketoacids occurs in primary mitochondrial respiratory chain mutants. Recognition of consistent alterations among patterns of nuclear gene expression for multiple biochemical pathways and in quantitative amino acid profiles in a translational genetic model of mitochondrial dysfunction allows insight into the complex pathogenesis underlying primary mitochondrial disease. Such knowledge may enable the development of a metabolomic profiling diagnostic tool applicable to human mitochondrial disease. PMID:18178500

Interface Pattern Selection in Directional Solidification

NASA Technical Reports Server (NTRS)

Trivedi, Rohit; Tewari, Surendra N.

2001-01-01

The central focus of this research is to establish key scientific concepts that govern the selection of cellular and dendritic patterns during the directional solidification of alloys. Ground-based studies have established that the conditions under which cellular and dendritic microstructures form are precisely where convection effects are dominant in bulk samples. Thus, experimental data can not be obtained terrestrially under pure diffusive regime. Furthermore, reliable theoretical models are not yet possible which can quantitatively incorporate fluid flow in the pattern selection criterion. Consequently, microgravity experiments on cellular and dendritic growth are designed to obtain benchmark data under diffusive growth conditions that can be quantitatively analyzed and compared with the rigorous theoretical model to establish the fundamental principles that govern the selection of specific microstructure and its length scales. In the cellular structure, different cells in an array are strongly coupled so that the cellular pattern evolution is controlled by complex interactions between thermal diffusion, solute diffusion and interface effects. These interactions give infinity of solutions, and the system selects only a narrow band of solutions. The aim of this investigation is to obtain benchmark data and develop a rigorous theoretical model that will allow us to quantitatively establish the physics of this selection process.

Apoptotic transition of senescent cells accompanied with mitochondrial hyper-function

PubMed Central

Wang, Danli; Liu, Yang; Zhang, Rui; Zhang, Fen; Sui, Weihao; Chen, Li; Zheng, Ran; Chen, Xiaowen; Wen, Feiqiu; Ouyang, Hong-Wei; Ji, Junfeng

2016-01-01

Defined as stable cell-cycle arrest, cellular senescence plays an important role in diverse biological processes including tumorigenesis, organismal aging, and embryonic development. Although increasing evidence has documented the metabolic changes in senescent cells, mitochondrial function and its potential contribution to the fate of senescent cells remain largely unknown. Here, using two in vitro models of cellular senescence induced by doxorubicin treatment and prolonged passaging of neonatal human foreskin fibroblasts, we report that senescent cells exhibited high ROS level and augmented glucose metabolic rate concomitant with both morphological and quantitative changes of mitochondria. Furthermore, mitochondrial membrane potential depolarized at late stage of senescent cells which eventually led to apoptosis. Our study reveals that mitochondrial hyper-function contributes to the implementation of cellular senescence and we propose a model in which the mitochondrion acts as the key player in promoting fate-determination in senescent cells. PMID:27056883

A comprehensive model of the spatio-temporal stem cell and tissue organisation in the intestinal crypt.

PubMed

Buske, Peter; Galle, Jörg; Barker, Nick; Aust, Gabriela; Clevers, Hans; Loeffler, Markus

2011-01-06

We introduce a novel dynamic model of stem cell and tissue organisation in murine intestinal crypts. Integrating the molecular, cellular and tissue level of description, this model links a broad spectrum of experimental observations encompassing spatially confined cell proliferation, directed cell migration, multiple cell lineage decisions and clonal competition.Using computational simulations we demonstrate that the model is capable of quantitatively describing and predicting the dynamic behaviour of the intestinal tissue during steady state as well as after cell damage and following selective gain or loss of gene function manipulations affecting Wnt- and Notch-signalling. Our simulation results suggest that reversibility and flexibility of cellular decisions are key elements of robust tissue organisation of the intestine. We predict that the tissue should be able to fully recover after complete elimination of cellular subpopulations including subpopulations deemed to be functional stem cells. This challenges current views of tissue stem cell organisation.

Multiplexed mass cytometry profiling of cellular states perturbed by small-molecule regulators

PubMed Central

Bodenmiller, Bernd; Zunder, Eli R.; Finck, Rachel; Chen, Tiffany J.; Savig, Erica S.; Bruggner, Robert V.; Simonds, Erin F.; Bendall, Sean C.; Sachs, Karen; Krutzik, Peter O.; Nolan, Garry P.

2013-01-01

The ability to comprehensively explore the impact of bio-active molecules on human samples at the single-cell level can provide great insight for biomedical research. Mass cytometry enables quantitative single-cell analysis with deep dimensionality, but currently lacks high-throughput capability. Here we report a method termed mass-tag cellular barcoding (MCB) that increases mass cytometry throughput by sample multiplexing. 96-well format MCB was used to characterize human peripheral blood mononuclear cell (PBMC) signaling dynamics, cell-to-cell communication, the signaling variability between 8 donors, and to define the impact of 27 inhibitors on this system. For each compound, 14 phosphorylation sites were measured in 14 PBMC types, resulting in 18,816 quantified phosphorylation levels from each multiplexed sample. This high-dimensional systems-level inquiry allowed analysis across cell-type and signaling space, reclassified inhibitors, and revealed off-target effects. MCB enables high-content, high-throughput screening, with potential applications for drug discovery, pre-clinical testing, and mechanistic investigation of human disease. PMID:22902532

Systems-Level Analysis of Innate Immunity

PubMed Central

Zak, Daniel E.; Tam, Vincent C.; Aderem, Alan

2014-01-01

Systems-level analysis of biological processes strives to comprehensively and quantitatively evaluate the interactions between the relevant molecular components over time, thereby enabling development of models that can be employed to ultimately predict behavior. Rapid development in measurement technologies (omics), when combined with the accessible nature of the cellular constituents themselves, is allowing the field of innate immunity to take significant strides toward this lofty goal. In this review, we survey exciting results derived from systems biology analyses of the immune system, ranging from gene regulatory networks to influenza pathogenesis and systems vaccinology. PMID:24655298

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

NASA Astrophysics Data System (ADS)

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-04-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue.

Single cell analysis in native tissue: Quantification of the retinoid content of hepatic stellate cells

PubMed Central

Galler, Kerstin; Requardt, Robert Pascal; Glaser, Uwe; Markwart, Robby; Bocklitz, Thomas; Bauer, Michael; Popp, Jürgen; Neugebauer, Ute

2016-01-01

Hepatic stellate cells (HSCs) are retinoid storing cells in the liver: The retinoid content of those cells changes depending on nutrition and stress level. There are also differences with regard to a HSC’s anatomical position in the liver. Up to now, retinoid levels were only accessible from bulk measurements of tissue homogenates or cell extracts. Unfortunately, they do not account for the intercellular variability. Herein, Raman spectroscopy relying on excitation by the minimally destructive wavelength 785 nm is introduced for the assessment of the retinoid state of single HSCs in freshly isolated, unprocessed murine liver lobes. A quantitative estimation of the cellular retinoid content is derived. Implications of the retinoid content on hepatic health state are reported. The Raman-based results are integrated with histological assessments of the tissue samples. This spectroscopic approach enables single cell analysis regarding an important cellular feature in unharmed tissue. PMID:27063397

STARD13 promotes hepatocellular carcinoma apoptosis by acting as a ceRNA for Fas.

PubMed

Zhang, Hai; Wang, Fang; Hu, Yahua

2017-02-01

To study the roles of STARD13 in cellular apoptosis of hepatocellular carcinoma (HCC). Quantitative real-time PCR and immunohistochemistry analyses showed that the expression levels of STARD13 and Fas were lower in clinical HCC tissues than in normal tissues and were positively correlated, which is consistent with the results analyzed by The Cancer Genome Atlas (TCGA) data. Patients with higher STARD13 or Fas expression levels had longer overall survival. Additionally, STARD13 3'-UTR enhanced cellular apoptosis and the 3'-UTRs of STARD13 and Fas were predicted to harbor nine similar miRNA binding sites. And STARD13 3'-UTR promoted Fas expression in a 3'-UTR- and miRNA-dependent way and increased the sensitivity of HCC cells to chemotherapy. Importantly, the coding sequence of STARD13 did not increase Fas expression. STARD13 3'-UTR promotes HCC apoptosis through acting as a ceRNA for Fas.

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.

Wavelet-based multiscale analysis of bioimpedance data measured by electric cell-substrate impedance sensing for classification of cancerous and normal cells.

PubMed

Das, Debanjan; Shiladitya, Kumar; Biswas, Karabi; Dutta, Pranab Kumar; Parekh, Aditya; Mandal, Mahitosh; Das, Soumen

2015-12-01

The paper presents a study to differentiate normal and cancerous cells using label-free bioimpedance signal measured by electric cell-substrate impedance sensing. The real-time-measured bioimpedance data of human breast cancer cells and human epithelial normal cells employs fluctuations of impedance value due to cellular micromotions resulting from dynamic structural rearrangement of membrane protrusions under nonagitated condition. Here, a wavelet-based multiscale quantitative analysis technique has been applied to analyze the fluctuations in bioimpedance. The study demonstrates a method to classify cancerous and normal cells from the signature of their impedance fluctuations. The fluctuations associated with cellular micromotion are quantified in terms of cellular energy, cellular power dissipation, and cellular moments. The cellular energy and power dissipation are found higher for cancerous cells associated with higher micromotions in cancer cells. The initial study suggests that proposed wavelet-based quantitative technique promises to be an effective method to analyze real-time bioimpedance signal for distinguishing cancer and normal cells.

Growing Up Or Growing Old? Cellular Aging Linked With Testosterone Reactivity To Stress In Youth

PubMed Central

Drury, Stacy S.; Shirtcliff, Elizabeth A.; Shachet, Andrew; Phan, Jenny; Mabile, Emily; Brett, Zoë H.; Wren, Michael; Esteves, Kyle; Theall, Katherine P.

2014-01-01

Background Given the established relation between testosterone and aging in older adults, we tested whether buccal telomere length (TL), an established cellular biomarker of aging, was associated with testosterone levels in youth. Methods Children, mean age 10.2 years, were recruited from the greater New Orleans area and salivary testosterone was measured during both an acute stressor and diurnally. Buccal TL was measured using monochrome multiplex quantitative real-time PCR (MMQ-PCR). Testosterone and telomere length data was available on 77 individuals. The association between buccal TL and testosterone was tested using multivariate Generalized Estimating Equations (GEE) to account for clustering of children within families. Results Greater peak testosterone levels (β=-0.87, p < 0.01) and slower recovery (β=-0.56, p < 0.01) and reactivity (β = -1.22, p < 0.01) following a social stressor were significantly associated with shorter buccal TL after controlling for parental age at conception, child age, sex, sociodemographic factors and puberty. No association was initially present between diurnal measurements of testosterone or morning basal testosterone levels and buccal TL. Sex significantly moderated the relation between testosterone reactivity and buccal TL. Conclusions The association between testosterone and buccal TL supports gonadal maturation as a developmentally sensitive biomarker of aging within youth. As stress levels of testosterone were significantly associated with buccal TL, these findings are consistent with the growing literature linking stress exposure and accelerated maturation. The lack of association of diurnal testosterone or morning basal levels with buccal TL bolsters the notion of a shared stress-related maturational mechanism between cellular stress and the hypothalamic pituitary gonadal (HPG) axis. These data provide novel evidence supporting the interaction of aging, physiologic stress and cellular processes as an underlying mechanism linking negative health outcomes and early life stress. PMID:25010187

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields.

PubMed

Kuzniar, Arnold; Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A Peter M; Demmers, Jeroen; Lebbink, Joyce H G; Kanaar, Roland

2017-01-01

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.

Semi-quantitative proteomics of mammalian cells upon short-term exposure to non-ionizing electromagnetic fields

PubMed Central

Laffeber, Charlie; Eppink, Berina; Bezstarosti, Karel; Dekkers, Dick; Woelders, Henri; Zwamborn, A. Peter M.; Demmers, Jeroen; Lebbink, Joyce H. G.; Kanaar, Roland

2017-01-01

The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein- and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture. PMID:28234898

Simultaneous quantitation of oxidized and reduced glutathione via LC-MS/MS: An insight into the redox state of hematopoietic stem cells.

PubMed

Carroll, Dustin; Howard, Diana; Zhu, Haining; Paumi, Christian M; Vore, Mary; Bondada, Subbarao; Liang, Ying; Wang, Chi; St Clair, Daret K

2016-08-01

Cellular redox balance plays a significant role in the regulation of hematopoietic stem-progenitor cell (HSC/MPP) self-renewal and differentiation. Unregulated changes in cellular redox homeostasis are associated with the onset of most hematological disorders. However, accurate measurement of the redox state in stem cells is difficult because of the scarcity of HSC/MPPs. Glutathione (GSH) constitutes the most abundant pool of cellular antioxidants. Thus, GSH metabolism may play a critical role in hematological disease onset and progression. A major limitation to studying GSH metabolism in HSC/MPPs has been the inability to measure quantitatively GSH concentrations in small numbers of HSC/MPPs. Current methods used to measure GSH levels not only rely on large numbers of cells, but also rely on the chemical/structural modification or enzymatic recycling of GSH and therefore are likely to measure only total glutathione content accurately. Here, we describe the validation of a sensitive method used for the direct and simultaneous quantitation of both oxidized and reduced GSH via liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) in HSC/MPPs isolated from bone marrow. The lower limit of quantitation (LLOQ) was determined to be 5.0ng/mL for GSH and 1.0ng/mL for GSSG with lower limits of detection at 0.5ng/mL for both glutathione species. Standard addition analysis utilizing mouse bone marrow shows that this method is both sensitive and accurate with reproducible analyte recovery. This method combines a simple extraction with a platform for the high-throughput analysis, allows for efficient determination of GSH/GSSG concentrations within the HSC/MPP populations in mouse, chemotherapeutic treatment conditions within cell culture, and human normal/leukemia patient samples. The data implicate the importance of the modulation of GSH/GSSG redox couple in stem cells related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Blood-brain barrier permeability and monocyte infiltration in experimental allergic encephalomyelitis: a quantitative MRI study.

PubMed

Floris, S; Blezer, E L A; Schreibelt, G; Döpp, E; van der Pol, S M A; Schadee-Eestermans, I L; Nicolay, K; Dijkstra, C D; de Vries, H E

2004-03-01

Enhanced cerebrovascular permeability and cellular infiltration mark the onset of early multiple sclerosis lesions. So far, the precise sequence of these events and their role in lesion formation and disease progression remain unknown. Here we provide quantitative evidence that blood-brain barrier leakage is an early event and precedes massive cellular infiltration in the development of acute experimental allergic encephalomyelitis (EAE), the animal correlate of multiple sclerosis. Cerebrovascular leakage and monocytes infiltrates were separately monitored by quantitative in vivo MRI during the course of the disease. Magnetic resonance enhancement of the contrast agent gadolinium diethylenetriaminepentaacetate (Gd-DTPA), reflecting vascular leakage, occurred concomitantly with the onset of neurological signs and was already at a maximal level at this stage of the disease. Immunohistochemical analysis also confirmed the presence of the serum-derived proteins such as fibrinogen around the brain vessels early in the disease, whereas no cellular infiltrates could be detected. MRI further demonstrated that Gd-DTPA leakage clearly preceded monocyte infiltration as imaged by the contrast agent based on ultra small particles of iron oxide (USPIO), which was maximal only during full-blown EAE. Ultrastructural and immunohistochemical investigation revealed that USPIOs were present in newly infiltrated macrophages within the inflammatory lesions. To validate the use of USPIOs as a non-invasive tool to evaluate therapeutic strategies, EAE animals were treated with the immunomodulator 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor, lovastatin, which ameliorated clinical scores. MRI showed that the USPIO load in the brain was significantly diminished in lovastatin-treated animals. Data indicate that cerebrovascular leakage and monocytic trafficking into the brain are two distinct processes in the development of inflammatory lesions during multiple sclerosis, which can be monitored on-line with MRI using USPIOs and Gd-DTPA as contrast agents. These studies also implicate that USPIOs are a valuable tool to visualize monocyte infiltration in vivo and quantitatively assess the efficacy of new therapeutics like lovastatin.

Physical biology of human brain development.

PubMed

Budday, Silvia; Steinmann, Paul; Kuhl, Ellen

2015-01-01

Neurodevelopment is a complex, dynamic process that involves a precisely orchestrated sequence of genetic, environmental, biochemical, and physical events. Developmental biology and genetics have shaped our understanding of the molecular and cellular mechanisms during neurodevelopment. Recent studies suggest that physical forces play a central role in translating these cellular mechanisms into the complex surface morphology of the human brain. However, the precise impact of neuronal differentiation, migration, and connection on the physical forces during cortical folding remains unknown. Here we review the cellular mechanisms of neurodevelopment with a view toward surface morphogenesis, pattern selection, and evolution of shape. We revisit cortical folding as the instability problem of constrained differential growth in a multi-layered system. To identify the contributing factors of differential growth, we map out the timeline of neurodevelopment in humans and highlight the cellular events associated with extreme radial and tangential expansion. We demonstrate how computational modeling of differential growth can bridge the scales-from phenomena on the cellular level toward form and function on the organ level-to make quantitative, personalized predictions. Physics-based models can quantify cortical stresses, identify critical folding conditions, rationalize pattern selection, and predict gyral wavelengths and gyrification indices. We illustrate that physical forces can explain cortical malformations as emergent properties of developmental disorders. Combining biology and physics holds promise to advance our understanding of human brain development and enable early diagnostics of cortical malformations with the ultimate goal to improve treatment of neurodevelopmental disorders including epilepsy, autism spectrum disorders, and schizophrenia.

Smartphone-based imaging of the corneal endothelium at sub-cellular resolution

NASA Astrophysics Data System (ADS)

Toslak, Devrim; Thapa, Damber; Erol, Muhammet Kazim; Chen, Yanjun; Yao, Xincheng

2017-07-01

This aim of this study was to test the feasibility of smartphone-based specular microscopy of the corneal endothelium at a sub-cellular resolution. Quantitative examination of endothelial cells is essential for evaluating corneal disease such as determining a diagnosis, monitoring progression and assessing treatment. Smartphone-based technology promises a new opportunity to develop affordable devices to foster quantitative examination of endothelial cells in rural and underserved areas. In our study, we incorporated an iPhone 6 and a slit lamp to demonstrate the feasibility of smartphone-based microscopy of the corneal endothelium at a sub-cellular resolution. The sub-cellular resolution images allowed quantitative calculation of the endothelial cell density. Comparative measurements revealed a normal endothelial cell density of 2978 cells/mm2 in the healthy cornea, and a significantly reduced cell density of 1466 cells/mm2 in the diseased cornea with Fuchs' dystrophy. Our ultimate goal is to develop a smartphone-based telemedicine device for low-cost examination of the corneal endothelium, which can benefit patients in rural areas and underdeveloped countries to reduce health care disparities.

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

Endele, Max; Etzrodt, Martin; Schroeder, Timm, E-mail: timm.schroeder@bsse.ethz.ch

Hematopoiesis is the cumulative consequence of finely tuned signaling pathways activated through extrinsic factors, such as local niche signals and systemic hematopoietic cytokines. Whether extrinsic factors actively instruct the lineage choice of hematopoietic stem and progenitor cells or are only selectively allowing survival and proliferation of already intrinsically lineage-committed cells has been debated over decades. Recent results demonstrated that cytokines can instruct lineage choice. However, the precise function of individual cytokine-triggered signaling molecules in inducing cellular events like proliferation, lineage choice, and differentiation remains largely elusive. Signal transduction pathways activated by different cytokine receptors are highly overlapping, but support themore » production of distinct hematopoietic lineages. Cellular context, signaling dynamics, and the crosstalk of different signaling pathways determine the cellular response of a given extrinsic signal. New tools to manipulate and continuously quantify signaling events at the single cell level are therefore required to thoroughly interrogate how dynamic signaling networks yield a specific cellular response. - Highlights: • Recent studies provided definite proof for lineage-instructive action of cytokines. • Signaling pathways involved in hematopoietic lineage instruction remain elusive. • New tools are emerging to quantitatively study dynamic signaling networks over time.« less

Binding affinity of amyloid oligomers to cellular membranes is a generic indicator of cellular dysfunction in protein misfolding diseases

PubMed Central

Evangelisti, Elisa; Cascella, Roberta; Becatti, Matteo; Marrazza, Giovanna; Dobson, Christopher M.; Chiti, Fabrizio; Stefani, Massimo; Cecchi, Cristina

2016-01-01

The conversion of peptides or proteins from their soluble native states into intractable amyloid deposits is associated with a wide range of human disorders. Misfolded protein oligomers formed during the process of aggregation have been identified as the primary pathogenic agents in many such conditions. Here, we show the existence of a quantitative relationship between the degree of binding to neuronal cells of different types of oligomers formed from a model protein, HypF-N, and the GM1 content of the plasma membranes. In addition, remarkably similar behavior is observed for oligomers of the Aβ42 peptide associated with Alzheimer’s disease. Further analysis has revealed the existence of a linear correlation between the level of the influx of Ca2+ across neuronal membranes that triggers cellular damage, and the fraction of oligomeric species bound to the membrane. Our findings indicate that the susceptibility of neuronal cells to different types of misfolded oligomeric assemblies is directly related to the extent of binding of such oligomers to the cellular membrane. PMID:27619987

[Computer simulation of thyroid regulatory mechanisms in health and malignancy].

PubMed

Abduvaliev, A A; Gil'dieva, M S; Khidirov, B N; Saĭdalieva, M; Saatov, T S

2010-07-01

The paper describes a computer model for regulation of the number of thyroid follicular cells in health and malignancy. The authors'computer program for mathematical simulation of the regulatory mechanisms of a thyroid follicular cellular community cannot be now referred to as good commercial products. For commercialization of this product, it is necessary to draw up a direct relation of the introduced corrected values from the actually existing normal values, such as the peripheral blood concentrations of thyroid hormones or the mean values of endocrine tissue mitotic activity. However, the described computer program has been also used in researches by our scientific group in the study of thyroid cancer. The available biological experimental data and theoretical provisions on thyroid structural and functional organization at the cellular level allow one to construct mathematical models for quantitative analysis of the regulation of the size of a cellular community of a thyroid follicle in health and abnormalities, by using the method for simulation of the regulatory mechanisms of living systems and the equations of cellular community regulatory communities.

Effect of alternate energy substrates on mammalian brain metabolism during ischemic events.

PubMed

Koppaka, S S; Puchowicz; LaManna, J C; Gatica, J E

2008-01-01

Regulation of brain metabolism and cerebral blood flow involves complex control systems with several interacting variables at both cellular and organ levels. Quantitative understanding of the spatially and temporally heterogeneous brain control mechanisms during internal and external stimuli requires the development and validation of a computational (mathematical) model of metabolic processes in brain. This paper describes a computational model of cellular metabolism in blood-perfused brain tissue, which considers the astrocyte-neuron lactate-shuttle (ANLS) hypothesis. The model structure consists of neurons, astrocytes, extra-cellular space, and a surrounding capillary network. Each cell is further compartmentalized into cytosol and mitochondria. Inter-compartment interaction is accounted in the form of passive and carrier-mediated transport. Our model was validated against experimental data reported by Crumrine and LaManna, who studied the effect of ischemia and its recovery on various intra-cellular tissue substrates under standard diet conditions. The effect of ketone bodies on brain metabolism was also examined under ischemic conditions following cardiac resuscitation through our model simulations. The influence of ketone bodies on lactate dynamics on mammalian brain following ischemia is studied incorporating experimental data.

Quantitative Systems Pharmacology Modeling of Acid Sphingomyelinase Deficiency and the Enzyme Replacement Therapy Olipudase Alfa Is an Innovative Tool for Linking Pathophysiology and Pharmacology.

PubMed

Kaddi, Chanchala D; Niesner, Bradley; Baek, Rena; Jasper, Paul; Pappas, John; Tolsma, John; Li, Jing; van Rijn, Zachary; Tao, Mengdi; Ortemann-Renon, Catherine; Easton, Rachael; Tan, Sharon; Puga, Ana Cristina; Schuchman, Edward H; Barrett, Jeffrey S; Azer, Karim

2018-06-19

Acid sphingomyelinase deficiency (ASMD) is a rare lysosomal storage disorder with heterogeneous clinical manifestations, including hepatosplenomegaly and infiltrative pulmonary disease, and is associated with significant morbidity and mortality. Olipudase alfa (recombinant human acid sphingomyelinase) is an enzyme replacement therapy under development for the non-neurological manifestations of ASMD. We present a quantitative systems pharmacology (QSP) model supporting the clinical development of olipudase alfa. The model is multiscale and mechanistic, linking the enzymatic deficiency driving the disease to molecular-level, cellular-level, and organ-level effects. Model development was informed by natural history, and preclinical and clinical studies. By considering patient-specific pharmacokinetic (PK) profiles and indicators of disease severity, the model describes pharmacodynamic (PD) and clinical end points for individual patients. The ASMD QSP model provides a platform for quantitatively assessing systemic pharmacological effects in adult and pediatric patients, and explaining variability within and across these patient populations, thereby supporting the extrapolation of treatment response from adults to pediatrics. © 2018 The Authors CPT: Pharmacometrics & Systems Pharmacology published by Wiley Periodicals, Inc. on behalf of American Society for Clinical Pharmacology and Therapeutics.

SPECHT - single-stage phosphopeptide enrichment and stable-isotope chemical tagging: quantitative phosphoproteomics of insulin action in muscle.

PubMed

Kettenbach, Arminja N; Sano, Hiroyuki; Keller, Susanna R; Lienhard, Gustav E; Gerber, Scott A

2015-01-30

The study of cellular signaling remains a significant challenge for translational and clinical research. In particular, robust and accurate methods for quantitative phosphoproteomics in tissues and tumors represent significant hurdles for such efforts. In the present work, we design, implement and validate a method for single-stage phosphopeptide enrichment and stable isotope chemical tagging, or SPECHT, that enables the use of iTRAQ, TMT and/or reductive dimethyl-labeling strategies to be applied to phosphoproteomics experiments performed on primary tissue. We develop and validate our approach using reductive dimethyl-labeling and HeLa cells in culture, and find these results indistinguishable from data generated from more traditional SILAC-labeled HeLa cells mixed at the cell level. We apply the SPECHT approach to the quantitative analysis of insulin signaling in a murine myotube cell line and muscle tissue, identify known as well as new phosphorylation events, and validate these phosphorylation sites using phospho-specific antibodies. Taken together, our work validates chemical tagging post-single-stage phosphoenrichment as a general strategy for studying cellular signaling in primary tissues. Through the use of a quantitatively reproducible, proteome-wide phosphopeptide enrichment strategy, we demonstrated the feasibility of post-phosphopeptide purification chemical labeling and tagging as an enabling approach for quantitative phosphoproteomics of primary tissues. Using reductive dimethyl labeling as a generalized chemical tagging strategy, we compared the performance of post-phosphopeptide purification chemical tagging to the well established community standard, SILAC, in insulin-stimulated tissue culture cells. We then extended our method to the analysis of low-dose insulin signaling in murine muscle tissue, and report on the analytical and biological significance of our results. Copyright © 2014 Elsevier B.V. All rights reserved.

Measurement of the traction force of biological cells by digital holography

PubMed Central

Yu, Xiao; Cross, Michael; Liu, Changgeng; Clark, David C.; Haynie, Donald T.; Kim, Myung K.

2011-01-01

The traction force produced by biological cells has been visualized as distortions in flexible substrata. We have utilized quantitative phase microscopy by digital holography (DH-QPM) to study the wrinkling of a silicone rubber film by motile fibroblasts. Surface deformation and the cellular traction force have been measured from phase profiles in a direct and straightforward manner. DH-QPM is shown to provide highly efficient and versatile means for quantitatively analyzing cellular motility. PMID:22254175

Purification of infectious human herpesvirus 6A virions and association of host cell proteins

PubMed Central

Hammarstedt, Maria; Ahlqvist, Jenny; Jacobson, Steven; Garoff, Henrik; Fogdell-Hahn, Anna

2007-01-01

Background Viruses that are incorporating host cell proteins might trigger autoimmune diseases. It is therefore of interest to identify possible host proteins associated with viruses, especially for enveloped viruses that have been suggested to play a role in autoimmune diseases, like human herpesvirus 6A (HHV-6A) in multiple sclerosis (MS). Results We have established a method for rapid and morphology preserving purification of HHV-6A virions, which in combination with parallel analyses with background control material released from mock-infected cells facilitates qualitative and quantitative investigations of the protein content of HHV-6A virions. In our iodixanol gradient purified preparation, we detected high levels of viral DNA by real-time PCR and viral proteins by metabolic labelling, silver staining and western blots. In contrast, the background level of cellular contamination was low in the purified samples as demonstrated by the silver staining and metabolic labelling analyses. Western blot analyses showed that the cellular complement protein CD46, the receptor for HHV-6A, is associated with the purified and infectious virions. Also, the cellular proteins clathrin, ezrin and Tsg101 are associated with intact HHV-6A virions. Conclusion Cellular proteins are associated with HHV-6A virions. The relevance of the association in disease and especially in autoimmunity will be further investigated. PMID:17949490

Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster

NASA Astrophysics Data System (ADS)

Kim, Miju; Kim, Tae-Jin; Kim, Hye Mi; Doh, Junsang; Lee, Kyung-Mi

2017-01-01

Multi-cellular cluster formation of natural killer (NK) cells occurs during in vivo priming and potentiates their activation to IL-2. However, the precise mechanism underlying this synergy within NK cell clusters remains unclear. We employed lymphocyte-laden microwell technologies to modulate contact-mediated multi-cellular interactions among activating NK cells and to quantitatively assess the molecular events occurring in multi-cellular clusters of NK cells. NK cells in social microwells, which allow cell-to-cell contact, exhibited significantly higher levels of IL-2 receptor (IL-2R) signaling compared with those in lonesome microwells, which prevent intercellular contact. Further, CD25, an IL-2R α chain, and lytic granules of NK cells in social microwells were polarized toward MTOC. Live cell imaging of lytic granules revealed their dynamic and prolonged polarization toward neighboring NK cells without degranulation. These results suggest that IL-2 bound on CD25 of one NK cells triggered IL-2 signaling of neighboring NK cells. These results were further corroborated by findings that CD25-KO NK cells exhibited lower proliferation than WT NK cells, and when mixed with WT NK cells, underwent significantly higher level of proliferation. These data highlights the existence of IL-2 trans-presentation between NK cells in the local microenvironment where the availability of IL-2 is limited.

Proteasome inhibitors alter levels of intracellular peptides in HEK293T and SH-SY5Y cells.

PubMed

Dasgupta, Sayani; Castro, Leandro M; Dulman, Russell; Yang, Ciyu; Schmidt, Marion; Ferro, Emer S; Fricker, Lloyd D

2014-01-01

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell.

Proteasome Inhibitors Alter Levels of Intracellular Peptides in HEK293T and SH-SY5Y Cells

PubMed Central

Dasgupta, Sayani; Castro, Leandro M.; Dulman, Russell; Yang, Ciyu; Schmidt, Marion; Ferro, Emer S.; Fricker, Lloyd D.

2014-01-01

The proteasome cleaves intracellular proteins into peptides. Earlier studies found that treatment of human embryonic kidney 293T (HEK293T) cells with epoxomicin (an irreversible proteasome inhibitor) generally caused a decrease in levels of intracellular peptides. However, bortezomib (an antitumor drug and proteasome inhibitor) caused an unexpected increase in the levels of most intracellular peptides in HEK293T and SH-SY5Y cells. To address this apparent paradox, quantitative peptidomics was used to study the effect of a variety of other proteasome inhibitors on peptide levels in HEK293T and SH-SY5Y cells. Inhibitors tested included carfilzomib, MG132, MG262, MLN2238, AM114, and clasto-Lactacystin β-lactone. Only MG262 caused a substantial elevation in peptide levels that was comparable to the effect of bortezomib, although carfilzomib and MLN2238 elevated the levels of some peptides. To explore off-target effects, the proteosome inhibitors were tested with various cellular peptidases. Bortezomib did not inhibit tripeptidyl peptidase 2 and only weakly inhibited cellular aminopeptidase activity, as did some of the other proteasome inhibitors. However, potent inhibitors of tripeptidyl peptidase 2 (butabindide) and cellular aminopeptidases (bestatin) did not substantially alter the peptidome, indicating that the increase in peptide levels due to proteasome inhibitors is not a result of peptidase inhibition. Although we cannot exclude other possibilities, we presume that the paradoxical increase in peptide levels upon treatment with bortezomib and other inhibitors is the result of allosteric effects of these compounds on the proteasome. Because intracellular peptides are likely to be functional, it is possible that some of the physiologic effects of bortezomib and carfilzomib arise from the perturbation of peptide levels inside the cell. PMID:25079948

Quantitative phase-contrast digital holographic microscopy for cell dynamic evaluation

NASA Astrophysics Data System (ADS)

Yu, Lingfeng; Mohanty, Samarendra; Berns, Michael W.; Chen, Zhongping

2009-02-01

The laser microbeam uses lasers to alter and/or to ablate intracellular organelles and cellular and tissue samples, and, today, has become an important tool for cell biologists to study the molecular mechanism of complex biological systems by removing individual cells or sub-cellular organelles. However, absolute quantitation of the localized alteration/damage to transparent phase objects, such as the cell membrane or chromosomes, was not possible using conventional phase-contrast or differential interference contrast microscopy. We report the development of phase-contrast digital holographic microscopy for quantitative evaluation of cell dynamic changes in real time during laser microsurgery. Quantitative phase images are recorded during the process of laser microsurgery and thus, the dynamic change in phase can be continuously evaluated. Out-of-focus organelles are re-focused by numerical reconstruction algorithms.

Intra-Articular Cellular Therapy for Osteoarthritis and Focal Cartilage Defects of the Knee: A Systematic Review of the Literature and Study Quality Analysis.

PubMed

Chahla, Jorge; Piuzzi, Nicolas S; Mitchell, Justin J; Dean, Chase S; Pascual-Garrido, Cecilia; LaPrade, Robert F; Muschler, George F

2016-09-21

Intra-articular cellular therapy injections constitute an appealing strategy that may modify the intra-articular milieu or regenerate cartilage in the settings of osteoarthritis and focal cartilage defects. However, little consensus exists regarding the indications for cellular therapies, optimal cell sources, methods of preparation and delivery, or means by which outcomes should be reported. We present a systematic review of the current literature regarding the safety and efficacy of cellular therapy delivered by intra-articular injection in the knee that provided a Level of Evidence of III or higher. A total of 420 papers were screened. Methodological quality was assessed using a modified Coleman methodology score. Only 6 studies (4 Level II and 2 Level III) met the criteria to be included in this review; 3 studies were on treatment of osteoarthritis and 3 were on treatment of focal cartilage defects. These included 4 randomized controlled studies without blinding, 1 prospective cohort study, and 1 retrospective therapeutic case-control study. The studies varied widely with respect to cell sources, cell characterization, adjuvant therapies, and assessment of outcomes. Outcome was reported in a total of 300 knees (124 in the osteoarthritis studies and 176 in the cartilage defect studies). Mean follow-up was 21.0 months (range, 12 to 36 months). All studies reported improved outcomes with intra-articular cellular therapy and no major adverse events. The mean modified Coleman methodology score was 59.1 ± 16 (range, 32 to 82). The studies of intra-articular cellular therapy injections for osteoarthritis and focal cartilage defects in the human knee suggested positive results with respect to clinical improvement and safety. However, the improvement was modest and a placebo effect cannot be disregarded. The overall quality of the literature was poor, and the methodological quality was fair, even among Level-II and III studies. Effective clinical assessment and optimization of injection therapies will demand greater attention to study methodology, including blinding; standardized quantitative methods for cell harvesting, processing, characterization, and delivery; and standardized reporting of clinical and structural outcomes. Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence. Copyright © 2016 by The Journal of Bone and Joint Surgery, Incorporated.

Digital encoding of cellular mRNAs enabling precise and absolute gene expression measurement by single-molecule counting.

PubMed

Fu, Glenn K; Wilhelmy, Julie; Stern, David; Fan, H Christina; Fodor, Stephen P A

2014-03-18

We present a new approach for the sensitive detection and accurate quantitation of messenger ribonucleic acid (mRNA) gene transcripts in single cells. First, the entire population of mRNAs is encoded with molecular barcodes during reverse transcription. After amplification of the gene targets of interest, molecular barcodes are counted by sequencing or scored on a simple hybridization detector to reveal the number of molecules in the starting sample. Since absolute quantities are measured, calibration to standards is unnecessary, and many of the relative quantitation challenges such as polymerase chain reaction (PCR) bias are avoided. We apply the method to gene expression analysis of minute sample quantities and demonstrate precise measurements with sensitivity down to sub single-cell levels. The method is an easy, single-tube, end point assay utilizing standard thermal cyclers and PCR reagents. Accurate and precise measurements are obtained without any need for cycle-to-cycle intensity-based real-time monitoring or physical partitioning into multiple reactions (e.g., digital PCR). Further, since all mRNA molecules are encoded with molecular barcodes, amplification can be used to generate more material for multiple measurements and technical replicates can be carried out on limited samples. The method is particularly useful for small sample quantities, such as single-cell experiments. Digital encoding of cellular content preserves true abundance levels and overcomes distortions introduced by amplification.

Network representations of immune system complexity

PubMed Central

Subramanian, Naeha; Torabi-Parizi, Parizad; Gottschalk, Rachel A.; Germain, Ronald N.; Dutta, Bhaskar

2015-01-01

The mammalian immune system is a dynamic multi-scale system composed of a hierarchically organized set of molecular, cellular and organismal networks that act in concert to promote effective host defense. These networks range from those involving gene regulatory and protein-protein interactions underlying intracellular signaling pathways and single cell responses to increasingly complex networks of in vivo cellular interaction, positioning and migration that determine the overall immune response of an organism. Immunity is thus not the product of simple signaling events but rather non-linear behaviors arising from dynamic, feedback-regulated interactions among many components. One of the major goals of systems immunology is to quantitatively measure these complex multi-scale spatial and temporal interactions, permitting development of computational models that can be used to predict responses to perturbation. Recent technological advances permit collection of comprehensive datasets at multiple molecular and cellular levels while advances in network biology support representation of the relationships of components at each level as physical or functional interaction networks. The latter facilitate effective visualization of patterns and recognition of emergent properties arising from the many interactions of genes, molecules, and cells of the immune system. We illustrate the power of integrating ‘omics’ and network modeling approaches for unbiased reconstruction of signaling and transcriptional networks with a focus on applications involving the innate immune system. We further discuss future possibilities for reconstruction of increasingly complex cellular and organism-level networks and development of sophisticated computational tools for prediction of emergent immune behavior arising from the concerted action of these networks. PMID:25625853

Quantitative chemoproteomics for site-specific analysis of protein alkylation by 4-hydroxy-2-nonenal in cells.

PubMed

Yang, Jing; Tallman, Keri A; Porter, Ned A; Liebler, Daniel C

2015-03-03

Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly in situ. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner.

Comparison of Mucin Levels at the Ocular Surface of Postmenopausal Women With and Without a History of Dry Eye

PubMed Central

Gipson, Ilene K.; Spurr-Michaud, Sandra J.; Senchyna, Michelle; Ritter, Robert; Schaumberg, Debra

2011-01-01

Purpose Determine 1) if levels of the glycocalyx membrane mucins, MUC1 and MUC16, and the secreted goblet cell mucin MUC5AC are altered in conjunctival cells and tears of postmenopausal women presenting with a history of non-Sjögren's dry eye, and 2) if mucin levels correlate with dry eye clinical diagnostic data. Methods Eighty-four postmenopausal women with a history of non-Sjögren's dry eye and 30 normal subjects were recruited for this study. Impression cytology samples were collected for mucin mRNA and protein analysis. Tears were collected for mucin protein assay. qPCR, western blot, and ELISA assays were used to quantitate MUC1, MUC16 and MUC5AC levels. Results Postmenopausal women with a history of dry eye displayed significantly increased MUC1 mRNA expression and cellular protein compared to normal subjects (P<0.001 and P<0.0l, respectively). Similarly, cellular MUC16 protein levels were significantly higher (P<0.001). Mucin levels were found to be correlated with the clinical characterization of the subjects, including staining and symptoms. Although cellular MUC5AC protein levels were increased in symptomatic subjects, the increase did not reach statistical significance. Conclusion Elevation in MUC1 and MUC16 mRNA and/or protein levels in postmenopausal non-Sjögren's dry eye patients with a history of dry eye may be a compensatory response to irritation and inflammation associated with the disease. Understanding the pattern of mucin expression associated with dry eye pathology may clarify factors involved in the progression of the disease and enhance the development of targeted therapies. PMID:22089171

The relationship between in vitro cellular aging and in vivo human age.

PubMed Central

Schneider, E L; Mitsui, Y

1976-01-01

Differences between early and late passage cell cultures on the organelle and macromolecular levels have been attributed to cellular "aging". However, concern has been expressed over whether changes in diploid cell populations after serial passage in vitro accurately reflect human cellular aging in vivo. Studies were therefore undertaken to determine if significant differences would be observed in the in vitro lifespans of skin fibroblast cultures from old and young normal, non-hospitalized volunteers and to examine if parameters that change with in vitro "aging" are altered as a function of age in vivo. Statistically signigificant (P less than 0.05) decreases were found in the rate of fibroblast migration, onset of cell culture senescence, in vitro lifespan, cell population replication rate, and cell number at confluency of fibroblast cultures derived from the old donor group when compared to parallel cultures from young donors. No significant differences were observed in modal cell volumes and cellular macromolecular contents. The differences observed in cell cultures from old and young donors were quantitatively and qualitatively distinct from those cellular alterations observed in early and late passage WI-38 cells (in vitro "aging"). Therefore, although early and late passage cultures of human diploid cells may provide an important cell system for examining loss of replicative potential, fibroblast cultures derived from old and young human donors may be a more appropriate model system for studying human cellular aging. PMID:1068470

Whole-body and Whole-Organ Clearing and Imaging Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals.

PubMed

Susaki, Etsuo A; Ueda, Hiroki R

2016-01-21

Organism-level systems biology aims to identify, analyze, control and design cellular circuits in organisms. Many experimental and computational approaches have been developed over the years to allow us to conduct these studies. Some of the most powerful methods are based on using optical imaging in combination with fluorescent labeling, and for those one of the long-standing stumbling blocks has been tissue opacity. Recently, the solutions to this problem have started to emerge based on whole-body and whole-organ clearing techniques that employ innovative tissue-clearing chemistry. Here, we review these advancements and discuss how combining new clearing techniques with high-performing fluorescent proteins or small molecule tags, rapid volume imaging and efficient image informatics is resulting in comprehensive and quantitative organ-wide, single-cell resolution experimental data. These technologies are starting to yield information on connectivity and dynamics in cellular circuits at unprecedented resolution, and bring us closer to system-level understanding of physiology and diseases of complex mammalian systems. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

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

PubMed

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

2014-10-01

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.

Tumor Heterogenity Research Interactive Visualization Environment (THRIVE) | Informatics Technology for Cancer Research (ITCR)

Cancer.gov

A platform for quantitative evaluation of intratumoral spatial heterogeneity in multiplexed immunofluorescence images, via characterization of the spatial interactions between different cellular phenotypes and non-cellular constituents in the tumor microenvironment.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

NASA Astrophysics Data System (ADS)

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-03-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent.

Early spatiotemporal-specific changes in intermediate signals are predictive of cytotoxic sensitivity to TNFα and co-treatments

PubMed Central

Loo, Lit-Hsin; Bougen-Zhukov, Nicola Michelle; Tan, Wei-Ling Cecilia

2017-01-01

Signaling pathways can generate different cellular responses to the same cytotoxic agents. Current quantitative models for predicting these differential responses are usually based on large numbers of intracellular gene products or signals at different levels of signaling cascades. Here, we report a study to predict cellular sensitivity to tumor necrosis factor alpha (TNFα) using high-throughput cellular imaging and machine-learning methods. We measured and compared 1170 protein phosphorylation events in a panel of human lung cancer cell lines based on different signals, subcellular regions, and time points within one hour of TNFα treatment. We found that two spatiotemporal-specific changes in an intermediate signaling protein, p90 ribosomal S6 kinase (RSK), are sufficient to predict the TNFα sensitivity of these cell lines. Our models could also predict the combined effects of TNFα and other kinase inhibitors, many of which are not known to target RSK directly. Therefore, early spatiotemporal-specific changes in intermediate signals are sufficient to represent the complex cellular responses to these perturbations. Our study provides a general framework for the development of rapid, signaling-based cytotoxicity screens that may be used to predict cellular sensitivity to a cytotoxic agent, or identify co-treatments that may sensitize or desensitize cells to the agent. PMID:28272488

Multiplexed 3D FRET imaging in deep tissue of live embryos

PubMed Central

Zhao, Ming; Wan, Xiaoyang; Li, Yu; Zhou, Weibin; Peng, Leilei

2015-01-01

Current deep tissue microscopy techniques are mostly restricted to intensity mapping of fluorophores, which significantly limit their applications in investigating biochemical processes in vivo. We present a deep tissue multiplexed functional imaging method that probes multiple Förster resonant energy transfer (FRET) sensors in live embryos with high spatial resolution. The method simultaneously images fluorescence lifetimes in 3D with multiple excitation lasers. Through quantitative analysis of triple-channel intensity and lifetime images, we demonstrated that Ca2+ and cAMP levels of live embryos expressing dual FRET sensors can be monitored simultaneously at microscopic resolution. The method is compatible with a broad range of FRET sensors currently available for probing various cellular biochemical functions. It opens the door to imaging complex cellular circuitries in whole live organisms. PMID:26387920

Bypass transition and spot nucleation in boundary layers

NASA Astrophysics Data System (ADS)

Kreilos, Tobias; Khapko, Taras; Schlatter, Philipp; Duguet, Yohann; Henningson, Dan S.; Eckhardt, Bruno

2016-08-01

The spatiotemporal aspects of the transition to turbulence are considered in the case of a boundary-layer flow developing above a flat plate exposed to free-stream turbulence. Combining results on the receptivity to free-stream turbulence with the nonlinear concept of a transition threshold, a physically motivated model suggests a spatial distribution of spot nucleation events. To describe the evolution of turbulent spots a probabilistic cellular automaton is introduced, with all parameters directly obtained from numerical simulations of the boundary layer. The nucleation rates are then combined with the cellular automaton model, yielding excellent quantitative agreement with the statistical characteristics for different free-stream turbulence levels. We thus show how the recent theoretical progress on transitional wall-bounded flows can be extended to the much wider class of spatially developing boundary-layer flows.

Cell Motility Dynamics: A Novel Segmentation Algorithm to Quantify Multi-Cellular Bright Field Microscopy Images

PubMed Central

Zaritsky, Assaf; Natan, Sari; Horev, Judith; Hecht, Inbal; Wolf, Lior; Ben-Jacob, Eshel; Tsarfaty, Ilan

2011-01-01

Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single-cell processing to perform objective, accurate quantitative analyses for various biological applications. PMID:22096600

Cell motility dynamics: a novel segmentation algorithm to quantify multi-cellular bright field microscopy images.

PubMed

Zaritsky, Assaf; Natan, Sari; Horev, Judith; Hecht, Inbal; Wolf, Lior; Ben-Jacob, Eshel; Tsarfaty, Ilan

2011-01-01

Confocal microscopy analysis of fluorescence and morphology is becoming the standard tool in cell biology and molecular imaging. Accurate quantification algorithms are required to enhance the understanding of different biological phenomena. We present a novel approach based on image-segmentation of multi-cellular regions in bright field images demonstrating enhanced quantitative analyses and better understanding of cell motility. We present MultiCellSeg, a segmentation algorithm to separate between multi-cellular and background regions for bright field images, which is based on classification of local patches within an image: a cascade of Support Vector Machines (SVMs) is applied using basic image features. Post processing includes additional classification and graph-cut segmentation to reclassify erroneous regions and refine the segmentation. This approach leads to a parameter-free and robust algorithm. Comparison to an alternative algorithm on wound healing assay images demonstrates its superiority. The proposed approach was used to evaluate common cell migration models such as wound healing and scatter assay. It was applied to quantify the acceleration effect of Hepatocyte growth factor/scatter factor (HGF/SF) on healing rate in a time lapse confocal microscopy wound healing assay and demonstrated that the healing rate is linear in both treated and untreated cells, and that HGF/SF accelerates the healing rate by approximately two-fold. A novel fully automated, accurate, zero-parameters method to classify and score scatter-assay images was developed and demonstrated that multi-cellular texture is an excellent descriptor to measure HGF/SF-induced cell scattering. We show that exploitation of textural information from differential interference contrast (DIC) images on the multi-cellular level can prove beneficial for the analyses of wound healing and scatter assays. The proposed approach is generic and can be used alone or alongside traditional fluorescence single-cell processing to perform objective, accurate quantitative analyses for various biological applications.

Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

PubMed Central

Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

2011-01-01

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID:21048197

Insulin-producing cells could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells

PubMed Central

2013-01-01

Objective The aim of this study was to compare the difference between insulin-producing cells (IPCs) and normal human pancreatic beta cells both in physiological function and morphological features in cellular level. Methods The levels of insulin secretion were measured by enzyme-linked immunosorbent assay. The insulin gene expression was determined by real-time quantitative polymerase chain reaction. The morphological features were detected by atomic force microscopy (AFM) and laser confocal scanning microscopy. Results IPCs and normal human pancreatic beta cells were similar to each other under the observation in AFM with the porous structure features in the cytoplasm. Both number of membrane particle size and average roughness of normal human beta cells were higher than those of IPCs. Conclusions Our results firstly revealed that the cellular ultrastructure of IPCs was closer to that of normal human pancreatic beta cells, but they still could not mimic the physiological regulation of insulin secretion performed by pancreatic beta cells. PMID:23421382

Identification of Glutathione S-Transferase Pi as a Protein Involved in Parkinson Disease Progression

PubMed Central

Shi, Min; Bradner, Joshua; Bammler, Theo K.; Eaton, David L.; Zhang, JianPeng; Ye, ZuCheng; Wilson, Angela M.; Montine, Thomas J.; Pan, Catherine; Zhang, Jing

2009-01-01

Parkinson disease (PD) typically affects the cortical regions during the later stages of disease, with neuronal loss, gliosis, and formation of diffuse cortical Lewy bodies in a significant portion of patients with dementia. To identify novel proteins involved in PD progression, we prepared synaptosomal fractions from the frontal cortices of pathologically verified PD patients at different stages along with age-matched controls. Protein expression profiles were compared using a robust quantitative proteomic technique. Approximately 100 proteins displayed significant differences in their relative abundances between PD patients at various stages and controls; three of these proteins were validated using independent techniques. One of the confirmed proteins, glutathione S-transferase Pi, was further investigated in cellular models of PD, demonstrating that its level was intimately associated with several critical cellular processes that are directly related to neurodegeneration in PD. These results have, for the first time, suggested that the levels of glutathione S-transferase Pi may play an important role in modulating the progression of PD. PMID:19498008

A Comparison of Raman Spectral Features of Frozen and Deparaffinized Tissues in Neuroblastoma and Ganglioneuroma

NASA Astrophysics Data System (ADS)

Devpura, Suneetha; Thakur, Jagdish S.; Poulik, Janet M.; Rabah, Raja; Naik, Vaman M.; Naik, Ratna

2012-02-01

We have investigated the cellular regions in neuroblastoma and ganglioneuroma using Raman spectroscopy and compared their spectral characteristics with those of normal adrenal gland. Thin sections from both frozen and deparaffinized tissues, obtained from the same tissue specimen, were studied in conjunction with the pathological examination of the tissues. We found a significant difference in the spectral features of frozen sections of normal adrenal gland, neuroblastoma, and ganglioneuroma when compared to deparaffinized tissues. The quantitative analysis of the Raman data using chemometric methods of principal component analysis and discriminant function analysis obtained from the frozen tissues show a sensitivity and specificity of 100% each. The biochemical identification based on the spectral differences shows that the normal adrenal gland tissues have higher levels of carotenoids, lipids, and cholesterol compared to the neuroblastoma and ganglioneuroma frozen tissues. However, deparaffinized tissues show complete removal of these biochemicals in adrenal tissues. This study demonstrates that Raman spectroscopy combined with chemometric methods can successfully distinguish neuroblastoma and ganglioneuroma at cellular level.

Multiplex, quantitative cellular analysis in large tissue volumes with clearing-enhanced 3D microscopy (Ce3D)

PubMed Central

Li, Weizhe; Germain, Ronald N.

2017-01-01

Organ homeostasis, cellular differentiation, signal relay, and in situ function all depend on the spatial organization of cells in complex tissues. For this reason, comprehensive, high-resolution mapping of cell positioning, phenotypic identity, and functional state in the context of macroscale tissue structure is critical to a deeper understanding of diverse biological processes. Here we report an easy to use method, clearing-enhanced 3D (Ce3D), which generates excellent tissue transparency for most organs, preserves cellular morphology and protein fluorescence, and is robustly compatible with antibody-based immunolabeling. This enhanced signal quality and capacity for extensive probe multiplexing permits quantitative analysis of distinct, highly intermixed cell populations in intact Ce3D-treated tissues via 3D histo-cytometry. We use this technology to demonstrate large-volume, high-resolution microscopy of diverse cell types in lymphoid and nonlymphoid organs, as well as to perform quantitative analysis of the composition and tissue distribution of multiple cell populations in lymphoid tissues. Combined with histo-cytometry, Ce3D provides a comprehensive strategy for volumetric quantitative imaging and analysis that bridges the gap between conventional section imaging and disassociation-based techniques. PMID:28808033

Effects of Space Radiation on Humoral and Cellular Immunity in Rhesus Monkeys.

DTIC Science & Technology

1992-12-01

55318-1084) immunoplates that are routinely used for quantitative assays of human Ig levels. It seemed justified to use the human system to test the...of Ig between the irradiated and control monkeys of different ages. The tests were set up and read at 18 and 72 h by the same operator, taking careful...note of the lot number and the standard reference curves for each test kit. The samples were suitably diluted to obtain clear-cut reactions (i.e

Segmentation and Quantitative Analysis of Epithelial Tissues.

PubMed

Aigouy, Benoit; Umetsu, Daiki; Eaton, Suzanne

2016-01-01

Epithelia are tissues that regulate exchanges with the environment. They are very dynamic and can acquire virtually any shape; at the cellular level, they are composed of cells tightly connected by junctions. Most often epithelia are amenable to live imaging; however, the large number of cells composing an epithelium and the absence of informatics tools dedicated to epithelial analysis largely prevented tissue scale studies. Here we present Tissue Analyzer, a free tool that can be used to segment and analyze epithelial cells and monitor tissue dynamics.

SuperSegger: robust image segmentation, analysis and lineage tracking of bacterial cells.

PubMed

Stylianidou, Stella; Brennan, Connor; Nissen, Silas B; Kuwada, Nathan J; Wiggins, Paul A

2016-11-01

Many quantitative cell biology questions require fast yet reliable automated image segmentation to identify and link cells from frame-to-frame, and characterize the cell morphology and fluorescence. We present SuperSegger, an automated MATLAB-based image processing package well-suited to quantitative analysis of high-throughput live-cell fluorescence microscopy of bacterial cells. SuperSegger incorporates machine-learning algorithms to optimize cellular boundaries and automated error resolution to reliably link cells from frame-to-frame. Unlike existing packages, it can reliably segment microcolonies with many cells, facilitating the analysis of cell-cycle dynamics in bacteria as well as cell-contact mediated phenomena. This package has a range of built-in capabilities for characterizing bacterial cells, including the identification of cell division events, mother, daughter and neighbouring cells, and computing statistics on cellular fluorescence, the location and intensity of fluorescent foci. SuperSegger provides a variety of postprocessing data visualization tools for single cell and population level analysis, such as histograms, kymographs, frame mosaics, movies and consensus images. Finally, we demonstrate the power of the package by analyzing lag phase growth with single cell resolution. © 2016 John Wiley & Sons Ltd.

Diffusion MRI in early cancer therapeutic response assessment

PubMed Central

Galbán, C. J.; Hoff, B. A.; Chenevert, T. L.; Ross, B. D.

2016-01-01

Imaging biomarkers for the predictive assessment of treatment response in patients with cancer earlier than standard tumor volumetric metrics would provide new opportunities to individualize therapy. Diffusion-weighted MRI (DW-MRI), highly sensitive to microenvironmental alterations at the cellular level, has been evaluated extensively as a technique for the generation of quantitative and early imaging biomarkers of therapeutic response and clinical outcome. First demonstrated in a rodent tumor model, subsequent studies have shown that DW-MRI can be applied to many different solid tumors for the detection of changes in cellularity as measured indirectly by an increase in the apparent diffusion coefficient (ADC) of water molecules within the lesion. The introduction of quantitative DW-MRI into the treatment management of patients with cancer may aid physicians to individualize therapy, thereby minimizing unnecessary systemic toxicity associated with ineffective therapies, saving valuable time, reducing patient care costs and ultimately improving clinical outcome. This review covers the theoretical basis behind the application of DW-MRI to monitor therapeutic response in cancer, the analytical techniques used and the results obtained from various clinical studies that have demonstrated the efficacy of DW-MRI for the prediction of cancer treatment response. PMID:26773848

Optical Phase Measurements of Disorder Strength Link Microstructure to Cell Stiffness.

PubMed

Eldridge, Will J; Steelman, Zachary A; Loomis, Brianna; Wax, Adam

2017-02-28

There have been sustained efforts on the part of cell biologists to understand the mechanisms by which cells respond to mechanical stimuli. To this end, many rheological tools have been developed to characterize cellular stiffness. However, measurement of cellular viscoelastic properties has been limited in scope by the nature of most microrheological methods, which require direct mechanical contact, applied at the single-cell level. In this article, we describe, to our knowledge, a new analysis approach for quantitative phase imaging that relates refractive index variance to disorder strength, a parameter that is linked to cell stiffness. Significantly, both disorder strength and cell stiffness are measured with the same phase imaging system, presenting a unique alternative for label-free, noncontact, single-shot imaging of cellular rheologic properties. To demonstrate the potential applicability of the technique, we measure phase disorder strength and shear stiffness across five cellular populations with varying mechanical properties and demonstrate an inverse relationship between these two parameters. The existence of this relationship suggests that predictions of cell mechanical properties can be obtained from examining the disorder strength of cell structure using this, to our knowledge, novel, noncontact technique. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Integrating Genomic Analysis with the Genetic Basis of Gene Expression: Preliminary Evidence of the Identification of Causal Genes for Cardiovascular and Metabolic Traits Related to Nutrition in Mexicans123

PubMed Central

Bastarrachea, Raúl A.; Gallegos-Cabriales, Esther C.; Nava-González, Edna J.; Haack, Karin; Voruganti, V. Saroja; Charlesworth, Jac; Laviada-Molina, Hugo A.; Veloz-Garza, Rosa A.; Cardenas-Villarreal, Velia Margarita; Valdovinos-Chavez, Salvador B.; Gomez-Aguilar, Patricia; Meléndez, Guillermo; López-Alvarenga, Juan Carlos; Göring, Harald H. H.; Cole, Shelley A.; Blangero, John; Comuzzie, Anthony G.; Kent, Jack W.

2012-01-01

Whole-transcriptome expression profiling provides novel phenotypes for analysis of complex traits. Gene expression measurements reflect quantitative variation in transcript-specific messenger RNA levels and represent phenotypes lying close to the action of genes. Understanding the genetic basis of gene expression will provide insight into the processes that connect genotype to clinically significant traits representing a central tenet of system biology. Synchronous in vivo expression profiles of lymphocytes, muscle, and subcutaneous fat were obtained from healthy Mexican men. Most genes were expressed at detectable levels in multiple tissues, and RNA levels were correlated between tissue types. A subset of transcripts with high reliability of expression across tissues (estimated by intraclass correlation coefficients) was enriched for cis-regulated genes, suggesting that proximal sequence variants may influence expression similarly in different cellular environments. This integrative global gene expression profiling approach is proving extremely useful for identifying genes and pathways that contribute to complex clinical traits. Clearly, the coincidence of clinical trait quantitative trait loci and expression quantitative trait loci can help in the prioritization of positional candidate genes. Such data will be crucial for the formal integration of positional and transcriptomic information characterized as genetical genomics. PMID:22797999

Phenotype classification of single cells using SRS microscopy, RNA sequencing, and microfluidics (Conference Presentation)

NASA Astrophysics Data System (ADS)

Streets, Aaron M.; Cao, Chen; Zhang, Xiannian; Huang, Yanyi

2016-03-01

Phenotype classification of single cells reveals biological variation that is masked in ensemble measurement. This heterogeneity is found in gene and protein expression as well as in cell morphology. Many techniques are available to probe phenotypic heterogeneity at the single cell level, for example quantitative imaging and single-cell RNA sequencing, but it is difficult to perform multiple assays on the same single cell. In order to directly track correlation between morphology and gene expression at the single cell level, we developed a microfluidic platform for quantitative coherent Raman imaging and immediate RNA sequencing (RNA-Seq) of single cells. With this device we actively sort and trap cells for analysis with stimulated Raman scattering microscopy (SRS). The cells are then processed in parallel pipelines for lysis, and preparation of cDNA for high-throughput transcriptome sequencing. SRS microscopy offers three-dimensional imaging with chemical specificity for quantitative analysis of protein and lipid distribution in single cells. Meanwhile, the microfluidic platform facilitates single-cell manipulation, minimizes contamination, and furthermore, provides improved RNA-Seq detection sensitivity and measurement precision, which is necessary for differentiating biological variability from technical noise. By combining coherent Raman microscopy with RNA sequencing, we can better understand the relationship between cellular morphology and gene expression at the single-cell level.

Scaling of number, size, and metabolic rate of cells with body size in mammals.

PubMed

Savage, Van M; Allen, Andrew P; Brown, James H; Gillooly, James F; Herman, Alexander B; Woodruff, William H; West, Geoffrey B

2007-03-13

The size and metabolic rate of cells affect processes from the molecular to the organismal level. We present a quantitative, theoretical framework for studying relationships among cell volume, cellular metabolic rate, body size, and whole-organism metabolic rate that helps reveal the feedback between these levels of organization. We use this framework to show that average cell volume and average cellular metabolic rate cannot both remain constant with changes in body size because of the well known body-size dependence of whole-organism metabolic rate. Based on empirical data compiled for 18 cell types in mammals, we find that many cell types, including erythrocytes, hepatocytes, fibroblasts, and epithelial cells, follow a strategy in which cellular metabolic rate is body size dependent and cell volume is body size invariant. We suggest that this scaling holds for all quickly dividing cells, and conversely, that slowly dividing cells are expected to follow a strategy in which cell volume is body size dependent and cellular metabolic rate is roughly invariant with body size. Data for slowly dividing neurons and adipocytes show that cell volume does indeed scale with body size. From these results, we argue that the particular strategy followed depends on the structural and functional properties of the cell type. We also discuss consequences of these two strategies for cell number and capillary densities. Our results and conceptual framework emphasize fundamental constraints that link the structure and function of cells to that of whole organisms.

Algorithm for cellular reprogramming.

PubMed

Ronquist, Scott; Patterson, Geoff; Muir, Lindsey A; Lindsly, Stephen; Chen, Haiming; Brown, Markus; Wicha, Max S; Bloch, Anthony; Brockett, Roger; Rajapakse, Indika

2017-11-07

The day we understand the time evolution of subcellular events at a level of detail comparable to physical systems governed by Newton's laws of motion seems far away. Even so, quantitative approaches to cellular dynamics add to our understanding of cell biology. With data-guided frameworks we can develop better predictions about, and methods for, control over specific biological processes and system-wide cell behavior. Here we describe an approach for optimizing the use of transcription factors (TFs) in cellular reprogramming, based on a device commonly used in optimal control. We construct an approximate model for the natural evolution of a cell-cycle-synchronized population of human fibroblasts, based on data obtained by sampling the expression of 22,083 genes at several time points during the cell cycle. To arrive at a model of moderate complexity, we cluster gene expression based on division of the genome into topologically associating domains (TADs) and then model the dynamics of TAD expression levels. Based on this dynamical model and additional data, such as known TF binding sites and activity, we develop a methodology for identifying the top TF candidates for a specific cellular reprogramming task. Our data-guided methodology identifies a number of TFs previously validated for reprogramming and/or natural differentiation and predicts some potentially useful combinations of TFs. Our findings highlight the immense potential of dynamical models, mathematics, and data-guided methodologies for improving strategies for control over biological processes. Copyright © 2017 the Author(s). Published by PNAS.

High-Performance Liquid Chromatography (HPLC)-Based Detection and Quantitation of Cellular c-di-GMP.

PubMed

Petrova, Olga E; Sauer, Karin

2017-01-01

The modulation of c-di-GMP levels plays a vital role in the regulation of various processes in a wide array of bacterial species. Thus, investigation of c-di-GMP regulation requires reliable methods for the assessment of c-di-GMP levels and turnover. Reversed-phase high-performance liquid chromatography (RP-HPLC) analysis has become a commonly used approach to accomplish these goals. The following describes the extraction and HPLC-based detection and quantification of c-di-GMP from Pseudomonas aeruginosa samples, a procedure that is amenable to modifications for the analysis of c-di-GMP in other bacterial species.

A high-throughput cellular assay to quantify the p53-degradation activity of E6 from different human papillomavirus types.

PubMed

Gagnon, David; Archambault, Jacques

2015-01-01

A subset of human papillomaviruses (HPVs), known as the high-risk types, are the causative agents of cervical cancer and other malignancies of the anogenital region and oral mucosa. The capacity of these viruses to induce cancer and to immortalize cells in culture relies in part on a critical function of their E6 oncoprotein, that of promoting the poly-ubiquitination of the cellular tumor suppressor protein p53 and its subsequent degradation by the proteasome. Here, we describe a cellular assay to measure the p53-degradation activity of E6 from different HPV types. This assay is based on a translational fusion of p53 to Renilla luciferase (Rluc-p53) that remains sensitive to degradation by high-risk E6 and whose steady-state levels can be accurately measured in standard luciferase assays. The p53-degradation activity of any E6 protein can be tested and quantified in transiently transfected cells by determining the amount of E6-expression vector required to reduce by half the levels of RLuc-p53 luciferase activity (50 % effective concentration [EC50]). The high-throughput and quantitative nature of this assay makes it particularly useful to compare the p53-degradation activities of E6 from several HPV types in parallel.

Exploring the Spatial and Temporal Organization of a Cell’s Proteome

PubMed Central

Beck, Martin; Topf, Maya; Frazier, Zachary; Tjong, Harianto; Xu, Min; Zhang, Shihua; Alber, Frank

2013-01-01

To increase our current understanding of cellular processes, such as cell signaling and division, knowledge is needed about the spatial and temporal organization of the proteome at different organizational levels. These levels cover a wide range of length and time scales: from the atomic structures of macromolecules for inferring their molecular function, to the quantitative description of their abundance, and distribution in the cell. Emerging new experimental technologies are greatly increasing the availability of such spatial information on the molecular organization in living cells. This review addresses three fields that have significantly contributed to our understanding of the proteome’s spatial and temporal organization: first, methods for the structure determination of individual macromolecular assemblies, specifically the fitting of atomic structures into density maps generated from electron microscopy techniques; second, research that visualizes the spatial distributions of these complexes within the cellular context using cryo electron tomography techniques combined with computational image processing; and third, methods for the spatial modeling of the dynamic organization of the proteome, specifically those methods for simulating reaction and diffusion of proteins and complexes in crowded intracellular fluids. The long-term goal is to integrate the varied data about a proteome’s organization into a spatially explicit, predictive model of cellular processes. PMID:21094684

Multiscale modeling of the dynamics of multicellular systems

NASA Astrophysics Data System (ADS)

Kosztin, Ioan

2011-03-01

Describing the biomechanical properties of cellular systems, regarded as complex highly viscoelastic materials, is a difficult problem of great conceptual and practical value. Here we present a novel approach, referred to as the Cellular Particle Dynamics (CPD) method, for: (i) quantitatively relating biomechanical properties at the cell level to those at the multicellular and tissue level, and (ii) describing and predicting the time evolution of multicellular systems that undergo biomechanical relaxations. In CPD cells are modeled as an ensemble of cellular particles (CPs) that interact via short range contact interactions, characterized by an attractive (adhesive interaction) and a repulsive (excluded volume interaction) component. The time evolution of the spatial conformation of the multicellular system is determined by following the trajectories of all CPs through integration of their equations of motion. Cell and multicellular level biomechanical properties (e.g., viscosity, surface tension and shear modulus) are determined through the combined use of experiments and theory of continuum viscoelastic media. The same biomechanical properties are also ``measured'' computationally by employing the CPD method, the results being expressed in terms of CPD parameters. Once these parameters have been calibrated experimentally, the formalism provides a systematic framework to predict the time evolution of complex multicellular systems during shape-changing biomechanical transformations. By design, the CPD method is rather flexible and most suitable for multiscale modeling of multicellular system. The spatial level of detail of the system can be easily tuned by changing the number of CPs in a cell. Thus, CPD can be used equally well to describe both cell level processes (e.g., the adhesion of two cells) and tissue level processes (e.g., the formation of 3D constructs of millions of cells through bioprinting). Work supported by NSF [FIBR-0526854 and PHY-0957914]. Computer time provided by the University of Missouri Bioinformatics Consortium.

Quantitative interactome reveals that porcine reproductive and respiratory syndrome virus nonstructural protein 2 forms a complex with viral nucleocapsid protein and cellular vimentin.

PubMed

Song, Tao; Fang, Liurong; Wang, Dang; Zhang, Ruoxi; Zeng, Songlin; An, Kang; Chen, Huanchun; Xiao, Shaobo

2016-06-16

Porcine reproductive and respiratory syndrome virus (PRRSV) is an Arterivirus that has heavily impacted the global swine industry. The PRRSV nonstructural protein 2 (nsp2) plays crucial roles in viral replication and host immune regulation, most likely by interacting with viral or cellular proteins that have not yet been identified. In this study, a quantitative interactome approach based on immunoprecipitation and stable isotope labeling with amino acids in cell culture (SILAC) was performed to identify nsp2-interacting proteins in PRRSV-infected cells with an nsp2-specific monoclonal antibody. Nine viral proteins and 62 cellular proteins were identified as potential nsp2-interacting partners. Our data demonstrate that the PRRSV nsp1α, nsp1β, and nucleocapsid proteins all interact directly with nsp2. Nsp2-interacting cellular proteins were classified into different functional groups and an interactome network of nsp2 was generated. Interestingly, cellular vimentin, a known receptor for PRRSV, forms a complex with nsp2 by using viral nucleocapsid protein as an intermediate. Taken together, the nsp2 interactome under the condition of virus infection clarifies a role of nsp2 in PRRSV replication and immune evasion. Viral proteins must interact with other virus-encoded proteins and/or host cellular proteins to function, and interactome analysis is an ideal approach for identifying such interacting proteins. In this study, we used the quantitative interactome methodology to identify the viral and cellular proteins that potentially interact with the nonstructural protein 2 (nsp2) of porcine reproductive and respiratory syndrome virus (PRRSV) under virus infection conditions, thus providing a rich source of potential viral and cellular interaction partners for PRRSV nsp2. Based on the interactome data, we further demonstrated that PRRSV nsp2 and nucleocapsid protein together with cellular vimentin, form a complex that may be essential for viral attachment and replication, which partly explains the role of nsp2 in PRRSV replication and immune evasion. Copyright © 2016 Elsevier B.V. All rights reserved.

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...

Photosynthesis and Respiration in a Jar.

ERIC Educational Resources Information Center

Buttner, Joseph K.

2000-01-01

Describes an activity that reduces the biosphere to a water-filled jar to simulate the relationship between cellular respiration, photosynthesis, and energy. Allows students in high school biology and related courses to explore quantitatively cellular respiration and photosynthesis in almost any laboratory setting. (ASK)

Cellular kinetics of CTL019 in relapsed/refractory B-cell acute lymphoblastic leukemia and chronic lymphocytic leukemia

PubMed Central

Maude, Shannon L.; Porter, David L.; Frey, Noelle; Wood, Patricia; Han, Xia; Waldron, Edward; Chakraborty, Abhijit; Awasthi, Rakesh; Levine, Bruce L.; Melenhorst, J. Joseph; Grupp, Stephan A.; June, Carl H.; Lacey, Simon F.

2017-01-01

Tisagenlecleucel (CTL019) is an investigational immunotherapy that involves reprogramming a patient’s own T cells with a transgene encoding a chimeric antigen receptor to identify and eliminate CD19-expressing cells. We previously reported that CTL019 achieved impressive clinical efficacy in patients with relapsed/refractory B-cell acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL), including the expansion and persistence of CTL019 cells, which correlates with response to therapy. Here, we performed formal cellular kinetic analyses of CTL019 in a larger cohort of 103 patients treated with CTL019 in 2 different diseases (ALL and CLL). CTL019 was measured in peripheral blood and bone marrow, using quantitative polymerase chain reaction and flow cytometry. CTL019 levels in peripheral blood typically peaked at 10 to 14 days postinfusion and then declined slowly over time. Patients with complete response (CR)/CR with incomplete count recovery had higher levels of CTL019 in peripheral blood, with greater maximal concentration and area under the curve values compared with nonresponding patients (P < .0001 for each). CTL019 transgene levels were measurable up to 780 days in peripheral blood. CTL019 trafficking and persistence were observed in bone marrow and cerebrospinal fluid. CTL019 expansion correlated with severity of cytokine release syndrome (CRS) and preinfusion tumor burden in pediatric ALL. The results described here are the first detailed formal presentation of cellular kinetics across 2 diseases and highlight the importance of the application of in vivo cellular kinetic analyses to characterize clinical efficacy and CRS severity associated with CTL019 therapy. PMID:28935694

Evaluation of cellular immunological responses in mono- and polymorphic clinical forms of post-kala-azar dermal leishmaniasis in India.

PubMed

Kaushal, H; Bras-Gonçalves, R; Avishek, K; Kumar Deep, D; Petitdidier, E; Lemesre, J-L; Papierok, G; Kumar, S; Ramesh, V; Salotra, P

2016-07-01

Post-kala-azar dermal leishmaniasis (PKDL) is a chronic dermal complication that occurs usually after recovery from visceral leishmaniasis (VL). The disease manifests into macular, papular and/or nodular clinical types with mono- or polymorphic presentations. Here, we investigated differences in immunological response between these two distinct clinical forms in Indian PKDL patients. Peripheral blood mononuclear cells of PKDL and naive individuals were exposed in vitro to total soluble Leishmania antigen (TSLA). The proliferation index was evaluated using an enzyme-linked immunosorbent assay (ELISA)-based lymphoproliferative assay. Cytokines and granzyme B levels were determined by cytometric bead array. Parasite load in tissue biopsy samples of PKDL was quantified by quantitative polymerase chain reaction (qPCR). The proportion of different lymphoid subsets in peripheral blood and the activated T cell population were estimated using flow cytometry. The study demonstrated heightened cellular immune responses in the polymorphic PKDL group compared to the naive group. The polymorphic group showed significantly higher lymphoproliferation, increased cytokines and granzyme B levels upon TSLA stimulation, and a raised proportion of circulating natural killer (NK) T cells against naive controls. Furthermore, the polymorphic group showed a significantly elevated proportion of activated CD4(+) and CD8(+) T cells upon in-vitro TSLA stimulation. Thus, the polymorphic variants showed pronounced cellular immunity while the monomorphic form demonstrated a comparatively lower cellular response. Additionally, the elevated level of both activated CD4(+) and CD8(+) T cells, coupled with high granzyme B secretion upon in-vitro TSLA stimulation, indicated the role of cytotoxic cells in resistance to L. donovani infection in polymorphic PKDL. © 2016 British Society for Immunology.

Angle-resolved light scattering of individual rod-shaped bacteria based on Fourier transform light scattering

NASA Astrophysics Data System (ADS)

Jo, Youngju; Jung, Jaehwang; Lee, Jee Woong; Shin, Della; Park, Hyunjoo; Nam, Ki Tae; Park, Ji-Ho; Park, Yongkeun

2014-05-01

Two-dimensional angle-resolved light scattering maps of individual rod-shaped bacteria are measured at the single-cell level. Using quantitative phase imaging and Fourier transform light scattering techniques, the light scattering patterns of individual bacteria in four rod-shaped species (Bacillus subtilis, Lactobacillus casei, Synechococcus elongatus, and Escherichia coli) are measured with unprecedented sensitivity in a broad angular range from -70° to 70°. The measured light scattering patterns are analyzed along the two principal axes of rod-shaped bacteria in order to systematically investigate the species-specific characteristics of anisotropic light scattering. In addition, the cellular dry mass of individual bacteria is calculated and used to demonstrate that the cell-to-cell variations in light scattering within bacterial species is related to the cellular dry mass and growth.

A mathematical model of cortical bone remodeling at cellular level under mechanical stimulus

NASA Astrophysics Data System (ADS)

Qin, Qing-Hua; Wang, Ya-Nan

2012-12-01

A bone cell population dynamics model for cortical bone remodeling under mechanical stimulus is developed in this paper. The external experiments extracted from the literature which have not been used in the creation of the model are used to test the validity of the model. Not only can the model compare reasonably well with these experimental results such as the increase percentage of final values of bone mineral content (BMC) and bone fracture energy (BFE) among different loading schemes (which proves the validity of the model), but also predict the realtime development pattern of BMC and BFE, as well as the dynamics of osteoblasts (OBA), osteoclasts (OCA), nitric oxide (NO) and prostaglandin E2 (PGE2) for each loading scheme, which can hardly be monitored through experiment. In conclusion, the model is the first of its kind that is able to provide an insight into the quantitative mechanism of bone remodeling at cellular level by which bone cells are activated by mechanical stimulus in order to start resorption/formation of bone mass. More importantly, this model has laid a solid foundation based on which future work such as systemic control theory analysis of bone remodeling under mechanical stimulus can be investigated. The to-be identified control mechanism will help to develop effective drugs and combined nonpharmacological therapies to combat bone loss pathologies. Also this deeper understanding of how mechanical forces quantitatively interact with skeletal tissue is essential for the generation of bone tissue for tissue replacement purposes in tissue engineering.

Putative regulatory sites unraveled by network-embedded thermodynamic analysis of metabolome data

PubMed Central

Kümmel, Anne; Panke, Sven; Heinemann, Matthias

2006-01-01

As one of the most recent members of the omics family, large-scale quantitative metabolomics data are currently complementing our systems biology data pool and offer the chance to integrate the metabolite level into the functional analysis of cellular networks. Network-embedded thermodynamic analysis (NET analysis) is presented as a framework for mechanistic and model-based analysis of these data. By coupling the data to an operating metabolic network via the second law of thermodynamics and the metabolites' Gibbs energies of formation, NET analysis allows inferring functional principles from quantitative metabolite data; for example it identifies reactions that are subject to active allosteric or genetic regulation as exemplified with quantitative metabolite data from Escherichia coli and Saccharomyces cerevisiae. Moreover, the optimization framework of NET analysis was demonstrated to be a valuable tool to systematically investigate data sets for consistency, for the extension of sub-omic metabolome data sets and for resolving intracompartmental concentrations from cell-averaged metabolome data. Without requiring any kind of kinetic modeling, NET analysis represents a perfectly scalable and unbiased approach to uncover insights from quantitative metabolome data. PMID:16788595

Avoiding Misannotation of In-Source Fragmentation Products as Cellular Metabolites in Liquid Chromatography–Mass Spectrometry-Based Metabolomics

DOE PAGES

Xu, Yi-Fan; Lu, Wenyun; Rabinowitz, Joshua D.

2015-01-15

Liquid chromatography–mass spectrometry (LC-MS) technology allows for rapid quantitation of cellular metabolites, with metabolites identified by mass spectrometry and chromatographic retention time. Recently, with the development of rapid scanning high-resolution high accuracy mass spectrometers and the desire for high throughput screening, minimal or no chromatographic separation has become increasingly popular. Furthermore, when analyzing complex cellular extracts, however, the lack of chromatographic separation could potentially result in misannotation of structurally related metabolites. Here, we show that, even using electrospray ionization, a soft ionization method, in-source fragmentation generates unwanted byproducts of identical mass to common metabolites. For example, nucleotide-triphosphates generate nucleotide-diphosphates, andmore » hexose-phosphates generate triose-phosphates. We also evaluated yeast intracellular metabolite extracts and found more than 20 cases of in-source fragments that mimic common metabolites. Finally and accordingly, chromatographic separation is required for accurate quantitation of many common cellular metabolites.« less

Isolating specific cell and tissue compartments from 3D images for quantitative regional distribution analysis using novel computer algorithms.

PubMed

Fenrich, Keith K; Zhao, Ethan Y; Wei, Yuan; Garg, Anirudh; Rose, P Ken

2014-04-15

Isolating specific cellular and tissue compartments from 3D image stacks for quantitative distribution analysis is crucial for understanding cellular and tissue physiology under normal and pathological conditions. Current approaches are limited because they are designed to map the distributions of synapses onto the dendrites of stained neurons and/or require specific proprietary software packages for their implementation. To overcome these obstacles, we developed algorithms to Grow and Shrink Volumes of Interest (GSVI) to isolate specific cellular and tissue compartments from 3D image stacks for quantitative analysis and incorporated these algorithms into a user-friendly computer program that is open source and downloadable at no cost. The GSVI algorithm was used to isolate perivascular regions in the cortex of live animals and cell membrane regions of stained spinal motoneurons in histological sections. We tracked the real-time, intravital biodistribution of injected fluorophores with sub-cellular resolution from the vascular lumen to the perivascular and parenchymal space following a vascular microlesion, and mapped the precise distributions of membrane-associated KCC2 and gephyrin immunolabeling in dendritic and somatic regions of spinal motoneurons. Compared to existing approaches, the GSVI approach is specifically designed for isolating perivascular regions and membrane-associated regions for quantitative analysis, is user-friendly, and free. The GSVI algorithm is useful to quantify regional differences of stained biomarkers (e.g., cell membrane-associated channels) in relation to cell functions, and the effects of therapeutic strategies on the redistributions of biomolecules, drugs, and cells in diseased or injured tissues. Copyright © 2014 Elsevier B.V. All rights reserved.

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.

Interleukin-7 in Patients With Chronic Hepatitis B May Have Effect on T Follicular Helper Cells and Specific Cellular Immunity.

PubMed

Zhong, Hua; Xibing, Gu; Yaping, Dai; Zheng, Wang; Decai, Fu; Xiaoye, Guo; Hangyuan, Wu; Dong, Wang; Zhonghua, Lu

2016-09-01

In patients with chronic hepatitis B (CHB), the relation of interkeukin-7 (IL-7) to either the T follicular helper cells (Tfh cells) or to a specific cellular immune response is not clear. The present study aims to explore the possible relationship of IL-7 to Tfh cells and to hepatitis B virus (HBV)-specific cellular immune response in patients with CHB. Ninety-one adult patients with CHB were divided into groups A, B, and C, according to the patients' IL-7 levels (low, medium, and high). Tfh cells and HBV-specific cytotoxic T lymphocytes (CTLs) were detected with flow cytometry; IL-7 and IL-21 were determined with a double antibody sandwich enzyme-linked immunosorbent assay; and HBV DNA was determined by using a real-time fluorescent quantitative polymerase chain reaction. The results showed that the levels of IL-7, Tfh cells, IL-21, and HBV-specific CTLs of patients in group C were significantly higher than those of patients in group B, (P < 0.01 for each comparison) and that the levels of these four parameters of patients in group B were significantly higher than those of the patients in group A (P < 0.01 for each comparison). Meanwhile, the level of HBV DNA of the patients in group C was significantly lower than that of the patients in group B (P < 0.01), and that of the patients in group B was significantly lower than that of the patients in group A (P < 0.05). Multiple linear regression analyses showed that IL-7, Tfh cells, IL-21, and HBV-specific CTL might have effects on HBV DNA and that only the HBV-specific CTL had an independent effect on HBV DNA (P < 0.01). IL-7, Tfh cells, and IL-21 showed independent effects on HBV-specific CTL (P < 0.05, P < 0.01, and P < 0.01). This study suggests that the IL-7 level of CHB patients may be related to Tfh cells. In CHB patients, IL-7 possibly increases the level of Tfh cells and HBV-specific cellular immune responses and thereby reduces the HBV DNA level.

Design and validation of a new ratiometric intracellular pH imaging probe using lanthanide-doped upconverting nanoparticles.

PubMed

Du, Shuoren; Hernández-Gil, Javier; Dong, Hao; Zheng, Xiaoyu; Lyu, Guangming; Bañobre-López, Manuel; Gallo, Juan; Sun, Ling-Dong; Yan, Chun-Hua; Long, Nicholas J

2017-10-17

pH homeostasis is strictly controlled at a subcellular level. A deregulation of the intra/extra/subcellular pH environment is associated with a number of diseases and as such, the monitoring of the pH state of cells and tissues is a valuable diagnostic tool. To date, only a few tools have been developed to measure the pH in living cells with the spatial resolution needed for intracellular imaging. Among the techniques available, only optical imaging offers enough resolution and biocompatibility to be proposed for subcellular pH monitoring. We present herein a ratiometric probe based on upconversion nanoparticles modified with a pH sensitive moiety for the quantitative imaging of pH at the subcellular level in living cells. This system provides the properties required for live cell quantitative imaging i.e. positive cellular uptake, biocompatibility, long wavelength excitation, sensitive response to pH within a biologically relevant range, and self-referenced signal.

Gene Transfer into Rat Brain Using Adenoviral Vectors

PubMed Central

Puntel, Mariana; Kroeger, Kurt M.; Sanderson, Nicholas S.R.; Thomas, Clare E.; Castro, Maria G.; Lowenstein, Pedro R.

2010-01-01

Viral vector–mediated gene delivery is an attractive procedure for introducing genes into the brain, both for purposes of basic neuroscience research and to develop gene therapy for neurological diseases. Replication-defective adenoviruses possess many features which make them ideal vectors for this purpose—efficiently transducing terminally differentiated cells such as neurons and glial cells, resulting in high levels of transgene expression in vivo. Also, in the absence of anti-adenovirus immunity, these vectors can sustain very long-term transgene expression within the brain parenchyma. This unit provides protocols for the stereotactic injection of adenoviral vectors into the brain, followed by protocols to detect transgene expression or infiltrates of immune cells by immunocytochemistry or immunofluorescence. ELISPOT and neutralizing antibody assay methodologies are provided to quantitate the levels of cellular and humoral immune responses against adenoviruses. Quantitation of adenoviral vector genomes within the rat brain using qPCR is also described. Curr. Protoc. Neurosci. 50:4.24.1–4.24.49. © 2010 by John Wiley & Sons, Inc. PMID:20066657

Quantitative image analysis of cellular heterogeneity in breast tumors complements genomic profiling.

PubMed

Yuan, Yinyin; Failmezger, Henrik; Rueda, Oscar M; Ali, H Raza; Gräf, Stefan; Chin, Suet-Feung; Schwarz, Roland F; Curtis, Christina; Dunning, Mark J; Bardwell, Helen; Johnson, Nicola; Doyle, Sarah; Turashvili, Gulisa; Provenzano, Elena; Aparicio, Sam; Caldas, Carlos; Markowetz, Florian

2012-10-24

Solid tumors are heterogeneous tissues composed of a mixture of cancer and normal cells, which complicates the interpretation of their molecular profiles. Furthermore, tissue architecture is generally not reflected in molecular assays, rendering this rich information underused. To address these challenges, we developed a computational approach based on standard hematoxylin and eosin-stained tissue sections and demonstrated its power in a discovery and validation cohort of 323 and 241 breast tumors, respectively. To deconvolute cellular heterogeneity and detect subtle genomic aberrations, we introduced an algorithm based on tumor cellularity to increase the comparability of copy number profiles between samples. We next devised a predictor for survival in estrogen receptor-negative breast cancer that integrated both image-based and gene expression analyses and significantly outperformed classifiers that use single data types, such as microarray expression signatures. Image processing also allowed us to describe and validate an independent prognostic factor based on quantitative analysis of spatial patterns between stromal cells, which are not detectable by molecular assays. Our quantitative, image-based method could benefit any large-scale cancer study by refining and complementing molecular assays of tumor samples.

Comparative transcript profiling by SuperSAGE identifies novel candidate genes for controlling potato quantitative resistance to late blight not compromised by late maturity.

PubMed

Draffehn, Astrid M; Li, Li; Krezdorn, Nicolas; Ding, Jia; Lübeck, Jens; Strahwald, Josef; Muktar, Meki S; Walkemeier, Birgit; Rotter, Björn; Gebhardt, Christiane

2013-01-01

Resistance to pathogens is essential for survival of wild and cultivated plants. Pathogen susceptibility causes major losses of crop yield and quality. Durable field resistance combined with high yield and other superior agronomic characters are therefore, important objectives in every crop breeding program. Precision and efficacy of resistance breeding can be enhanced by molecular diagnostic tools, which result from knowledge of the molecular basis of resistance and susceptibility. Breeding uses resistance conferred by single R genes and polygenic quantitative resistance. The latter is partial but considered more durable. Molecular mechanisms of plant pathogen interactions are elucidated mainly in experimental systems involving single R genes, whereas most genes important for quantitative resistance in crops like potato are unknown. Quantitative resistance of potato to Phytophthora infestans causing late blight is often compromised by late plant maturity, a negative agronomic character. Our objective was to identify candidate genes for quantitative resistance to late blight not compromised by late plant maturity. We used diagnostic DNA-markers to select plants with different field levels of maturity corrected resistance (MCR) to late blight and compared their leaf transcriptomes before and after infection with P. infestans using SuperSAGE (serial analysis of gene expression) technology and next generation sequencing. We identified 2034 transcripts up or down regulated upon infection, including a homolog of the kiwi fruit allergen kiwellin. 806 transcripts showed differential expression between groups of genotypes with contrasting MCR levels. The observed expression patterns suggest that MCR is in part controlled by differential transcript levels in uninfected plants. Functional annotation suggests that, besides biotic and abiotic stress responses, general cellular processes such as photosynthesis, protein biosynthesis, and degradation play a role in MCR.

Ionizing Radiation: The issue of radiation quality

NASA Astrophysics Data System (ADS)

Prise, Kevin; Schettino, Giuseppe

Types of Ionising radiations are differentiated from each other by fundamental characteristics of their energy deposition patterns when they interact with biological materials. At the level of the DNA these non-random patterns drive differences in the yields and distributions of DNA damage patterns and specifically the production of clustered damage or complex lesions. The complex radiation fields found in space bring significant challenges for developing a mechanistic understanding of radiation effects from the perspective of radiation quality as these consist of a diverse range of particle and energy types unique to the space environment. Linear energy transfer, energy deposited per unit track length in units of keV per micron, has long been used as a comparator for different types of radiation but has limitations in that it is an average value. Difference in primary core ionizations relative to secondary delta ray ranges vary significantly with particle mass and energy leading to complex interrelationships with damage production at the cellular level. At the cellular level a greater mechanistic understanding is necessary, linking energy deposition patterns to DNA damage patterns and cellular response, to build appropriate biophysical models that are predictive for different radiation qualities and mixed field exposures. Defined studies using monoenergetic beams delivered under controlled conditions are building quantitative data sets of both initial and long term changes in cells as a basis for a great mechanistic understanding of radiation quality effects of relevance to not only space exposures but clinical application of ion-beams.

Development and application of a quantitative PCR assay to study equine herpesvirus 5 invasion and replication in equine tissues in vitro and in vivo.

PubMed

Zarski, Lila M; High, Emily A; Nelli, Rahul K; Bolin, Steven R; Williams, Kurt J; Hussey, Gisela

2017-10-01

Equine herpesvirus 5 (EHV-5) infection is associated with pulmonary fibrosis in horses, but further studies on EHV-5 persistence in equine cells are needed to fully understand viral and host contributions to disease pathogenesis. Our aim was to develop a quantitative PCR (qPCR) assay to measure EHV-5 viral copy number in equine cell cultures, blood lymphocytes, and nasal swabs of horses. Furthermore, we used a recently developed equine primary respiratory cell culture system to study EHV-5 pathogenesis at the respiratory tract. PCR primers and a probe were designed to target gene E11 of the EHV-5 genome. Sensitivity and repeatability were established, and specificity was verified by testing multiple isolates of EHV-5, as well as DNA from other equine herpesviruses. Four-week old fully differentiated (mature), newly seeded (immature) primary equine respiratory epithelial cell (ERECs), and equine dermal cell cultures were inoculated with EHV-5 and the cells and supernatants collected daily for 14days. Blood lymphocytes and nasal swabs were collected from horses experimentally infected with equine herpesvirus 1 (EHV-1). The qPCR assay detected EHV-5 at stable concentrations throughout 14days in inoculated mature EREC and equine dermal cell cultures (peaking at 202 and 5861 viral genomes per 10 6 cellular β actin, respectively). EHV-5 copies detected in the immature EREC cultures increased over 14days and reached levels greater than 10,000 viral genomes per 10 6 cellular β actin. Moreover, EHV-5 was detected in the lymphocytes of 76% of horses and in the nasal swabs of 84% of horses experimentally infected with EHV-1 pre-inoculation with EHV-1. Post-inoculation with EHV-1, EHV-5 was detected in lymphocytes of 52% of horses while EHV-5 levels in nasal swabs were not significantly different from pre-inoculation levels. In conclusion, qPCR was a reliable technique to investigate viral load in in vivo and in vitro samples, and EHV-5 replication in equine epithelial cells may be influenced by cellular stages of differentiation. Copyright © 2017 Elsevier B.V. All rights reserved.

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...

Quantitative Detection of Low-Abundance Transcripts at Single-Cell Level in Human Epidermal Keratinocytes by Digital Droplet Reverse Transcription-Polymerase Chain Reaction.

PubMed

Auvré, Frédéric; Coutier, Julien; Martin, Michèle T; Fortunel, Nicolas O

2018-05-08

Genetic and epigenetic characterization of the large cellular diversity observed within tissues is essential to understanding the molecular networks that ensure the regulation of homeostasis, repair, and regeneration, but also pathophysiological processes. Skin is composed of multiple cell lineages and is therefore fully concerned by this complexity. Even within one particular lineage, such as epidermal keratinocytes, different immaturity statuses or differentiation stages are represented, which are still incompletely characterized. Accordingly, there is presently great demand for methods and technologies enabling molecular investigation at single-cell level. Also, most current methods used to analyze gene expression at RNA level, such as RT-qPCR, do not directly provide quantitative data, but rather comparative ratios between two conditions. A second important need in skin biology is thus to determine the number of RNA molecules in a given cell sample. Here, we describe a workflow that we have set up to meet these specific needs, by means of transcript quantification in cellular micro-samples using flow cytometry sorting and reverse transcription-digital droplet polymerase chain reaction. As a proof-of-principle, the workflow was tested for the detection of transcription factor transcripts expressed at low levels in keratinocyte precursor cells. A linear correlation was found between quantification values and keratinocyte input numbers in a low quantity range from 40 cells to 1 cell. Interpretable signals were repeatedly obtained from single-cell samples corresponding to estimated expression levels as low as 10-20 transcript copies per keratinocyte or less. The present workflow may have broad applications for the detection and quantification of low-abundance nucleic acid species in single cells, opening up perspectives for the study of cell-to-cell genetic and molecular heterogeneity. Interestingly, the process described here does not require internal references such as house-keeping gene expression, as it is initiated with defined cell numbers, precisely sorted by flow cytometry.

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

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

Leichner, P.K.

This report summarizes research in beta-particle dosimetry, quantitative single-photon emission computed tomography (SPECT), the clinical implementation of these two areas of research in radioimmunotherapy (RIT), and postgraduate training provided since the inception of this grant on July 15, 1989. To improve beta-particle dosimetry, a point source function was developed that is valid for a wide range of beta emitters. Analytical solutions for beta-particle dose rates within out outside slabs of finite thickness were validated in experimental tumors and are now being used in clinical RIT. Quantitative SPECT based on the circular harmonic transform (CHT) algorithm was validated in phantom, experimental,more » and clinical studies. This has led to improved macrodosimetry in clinical RIT. In dosimetry at the multi-cellular level studies were made of the HepG2 human hepatoblastoma grown subcutaneously in nude mice. Histologic sections and autoradiographs were prepared to quantitate activity distributions of radiolabeled antibodies. Absorbed-dose calculations are being carried out for {sup 131}I and {sup 90}Y beta particles for these antibody distributions.« less

Multichannel series piezoelectric quartz crystal cell sensor for real time and quantitative monitoring of the living cell and assessment of cytotoxicity.

PubMed

Tong, Feifei; Lian, Yan; Zhou, Huang; Shi, Xiaohong; He, Fengjiao

2014-10-21

A new multichannel series piezoelectric quartz crystal (MSPQC) cell sensor for real time monitoring of living cells in vitro was reported in this paper. The constructed sensor was used successfully to monitor adhesion, spreading, proliferation, and apoptosis of MG63 osteosarcoma cells and investigate the effects of different concentrations of cobalt chloride on MG63 cells. Quantitative real time and dynamic cell analyses data were conducted using the MSPQC cell sensor. Compared with methods such as fluorescence staining and morphology observation by microscopy, the MSPQC cell sensor is noninvasive, label free, simple, cheap, and capable of online monitoring. It can automatically record the growth status of cells and quantitatively evaluate cell proliferation and the apoptotic response to drugs. It will be a valuable detection and analysis tool for the acquisition of cellular level information and is anticipated to have application in the field of cell biology research or cytotoxicity testing in the future.

Advancing Cell Biology Through Proteomics in Space and Time (PROSPECTS)*

PubMed Central

Lamond, Angus I.; Uhlen, Mathias; Horning, Stevan; Makarov, Alexander; Robinson, Carol V.; Serrano, Luis; Hartl, F. Ulrich; Baumeister, Wolfgang; Werenskiold, Anne Katrin; Andersen, Jens S.; Vorm, Ole; Linial, Michal; Aebersold, Ruedi; Mann, Matthias

2012-01-01

The term “proteomics” encompasses the large-scale detection and analysis of proteins and their post-translational modifications. Driven by major improvements in mass spectrometric instrumentation, methodology, and data analysis, the proteomics field has burgeoned in recent years. It now provides a range of sensitive and quantitative approaches for measuring protein structures and dynamics that promise to revolutionize our understanding of cell biology and molecular mechanisms in both human cells and model organisms. The Proteomics Specification in Time and Space (PROSPECTS) Network is a unique EU-funded project that brings together leading European research groups, spanning from instrumentation to biomedicine, in a collaborative five year initiative to develop new methods and applications for the functional analysis of cellular proteins. This special issue of Molecular and Cellular Proteomics presents 16 research papers reporting major recent progress by the PROSPECTS groups, including improvements to the resolution and sensitivity of the Orbitrap family of mass spectrometers, systematic detection of proteins using highly characterized antibody collections, and new methods for absolute as well as relative quantification of protein levels. Manuscripts in this issue exemplify approaches for performing quantitative measurements of cell proteomes and for studying their dynamic responses to perturbation, both during normal cellular responses and in disease mechanisms. Here we present a perspective on how the proteomics field is moving beyond simply identifying proteins with high sensitivity toward providing a powerful and versatile set of assay systems for characterizing proteome dynamics and thereby creating a new “third generation” proteomics strategy that offers an indispensible tool for cell biology and molecular medicine. PMID:22311636

The Role of Extracellular Vesicles in Metastasis

DTIC Science & Technology

2017-10-01

quantitative characterization of each cancerous ESV subpopulation’s role in cargo transfer. Specifically, we aim to (1) optimize an existing...the first quantitative data on which ESV subpopulations (exosomes, MVs, or oncosomes) manipulate the tumor microenvironment, the ESV cargo transferred...without cellular contaminants or without damaging the cargo. A second hindrance has been the lack of quantitative methods for measuring very small

Engineering challenges of BioNEMS: the integration of microfluidics, micro- and nanodevices, models and external control for systems biology.

PubMed

Wikswo, J P; Prokop, A; Baudenbacher, F; Cliffel, D; Csukas, B; Velkovsky, M

2006-08-01

Systems biology, i.e. quantitative, postgenomic, postproteomic, dynamic, multiscale physiology, addresses in an integrative, quantitative manner the shockwave of genetic and proteomic information using computer models that may eventually have 10(6) dynamic variables with non-linear interactions. Historically, single biological measurements are made over minutes, suggesting the challenge of specifying 10(6) model parameters. Except for fluorescence and micro-electrode recordings, most cellular measurements have inadequate bandwidth to discern the time course of critical intracellular biochemical events. Micro-array expression profiles of thousands of genes cannot determine quantitative dynamic cellular signalling and metabolic variables. Major gaps must be bridged between the computational vision and experimental reality. The analysis of cellular signalling dynamics and control requires, first, micro- and nano-instruments that measure simultaneously multiple extracellular and intracellular variables with sufficient bandwidth; secondly, the ability to open existing internal control and signalling loops; thirdly, external BioMEMS micro-actuators that provide high bandwidth feedback and externally addressable intracellular nano-actuators; and, fourthly, real-time, closed-loop, single-cell control algorithms. The unravelling of the nested and coupled nature of cellular control loops requires simultaneous recording of multiple single-cell signatures. Externally controlled nano-actuators, needed to effect changes in the biochemical, mechanical and electrical environment both outside and inside the cell, will provide a major impetus for nanoscience.

Network signatures of nuclear and cytoplasmic density alterations in a model of pre and postmetastatic colorectal cancer

NASA Astrophysics Data System (ADS)

Damania, Dhwanil; Subramanian, Hariharan; Backman, Vadim; Anderson, Eric C.; Wong, Melissa H.; McCarty, Owen J. T.; Phillips, Kevin G.

2014-01-01

Cells contributing to the pathogenesis of cancer possess cytoplasmic and nuclear structural alterations that accompany their aberrant genetic, epigenetic, and molecular perturbations. Although it is known that architectural changes in primary and metastatic tumor cells can be quantified through variations in cellular density at the nanometer and micrometer spatial scales, the interdependent relationships among nuclear and cytoplasmic density as a function of tumorigenic potential has not been thoroughly investigated. We present a combined optical approach utilizing quantitative phase microscopy and partial wave spectroscopic microscopy to perform parallel structural characterizations of cellular architecture. Using the isogenic SW480 and SW620 cell lines as a model of pre and postmetastatic transition in colorectal cancer, we demonstrate that nuclear and cytoplasmic nanoscale disorder, micron-scale dry mass content, mean dry mass density, and shape metrics of the dry mass density histogram are uniquely correlated within and across different cellular compartments for a given cell type. The correlations of these physical parameters can be interpreted as networks whose nodal importance and level of connection independence differ according to disease stage. This work demonstrates how optically derived biophysical parameters are linked within and across different cellular compartments during the architectural orchestration of the metastatic phenotype.

Macromolecular Expression and Function: A New Paradigm for NASA Risk Assessment

NASA Technical Reports Server (NTRS)

Richmond, Robert

2003-01-01

Predicting risks in humans of either acute effects such as bone loss or muscle wasting, or late effects such as cancer, is challenging. To an approximation, this is because uncertainties of exposure to stress factors or toxic agents and the uniformity of processing subsequent damage at the cellular level within a complex set of biological variables degrade the confidence of predicting pathologic outcome. A cellular biodosimeter that simultaneously reports 1) the type of damage due to that exposure, 2) the quantity of damage incurred by that exposure, and 3) the dataset used to assess risk of developing pathologic outcome caused by that exposure would therefore be useful for predicting ultimate risks faced by an individual, such as an astronaut. It is suggested that such a biodosimeter can be based upon analyses of gene-expression and protein expression whereby large datasets of cellular response to damage are obtained and analyzed for expression-profiles correlated with established end points and molecular markers predictive for risks being assessed. The usefulness of multiparametric cellular biodosimeters could be realized by quantitatively profiling these datasets using techniques of bioinformatics. Such an approach contributes to the foundation of molecular epidemiology as a new scientific discipline, and represents a new paradigm of risk assessment.

What determines organ size differences between species? A meta-analysis of the cellular basis.

PubMed

Gázquez, Ayelén; Beemster, Gerrit T S

2017-07-01

Little is known about how the characteristic differences in organ size between species are regulated. At the cellular level, the size of an organ is strictly regulated by cell division and expansion during its development. We performed a meta-analysis of the growth parameters of roots, and Graminae and eudicotyledonous leaves, to address the question of how quantitative variation in these two processes contributes to size differences across a range of species. We extracted or derived cellular parameters from published kinematic growth analyses. These data were subjected to linear regression analyses to identify the parameters that determine differences in organ growth. Our results demonstrate that, across all species and organs, similar conclusions can be made: cell number rather than cell size determines the final size of plant organs; cell number is determined by meristem size rather than the rate at which cells divide; cells that are small when leaving the meristem compensate by expanding for longer; mature cell size is primarily determined by the duration of cell expansion. These results identify the regulation of the transition from cell division to expansion as the key cellular mechanism targeted by the evolution of organ size. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Mass spectrometry-based proteomics: from cancer biology to protein biomarkers, drug targets, and clinical applications.

PubMed

Jimenez, Connie R; Verheul, Henk M W

2014-01-01

Proteomics is optimally suited to bridge the gap between genomic information on the one hand and biologic functions and disease phenotypes at the other, since it studies the expression and/or post-translational modification (especially phosphorylation) of proteins--the major cellular players bringing about cellular functions--at a global level in biologic specimens. Mass spectrometry technology and (bio)informatic tools have matured to the extent that they can provide high-throughput, comprehensive, and quantitative protein inventories of cells, tissues, and biofluids in clinical samples at low level. In this article, we focus on next-generation proteomics employing nanoliquid chromatography coupled to high-resolution tandem mass spectrometry for in-depth (phospho)protein profiling of tumor tissues and (proximal) biofluids, with a focus on studies employing clinical material. In addition, we highlight emerging proteogenomic approaches for the identification of tumor-specific protein variants, and targeted multiplex mass spectrometry strategies for large-scale biomarker validation. Below we provide a discussion of recent progress, some research highlights, and challenges that remain for clinical translation of proteomic discoveries.

First cellular approach of the effects of global warming on groundwater organisms: a study of the HSP70 gene expression.

PubMed

Colson-Proch, Céline; Morales, Anne; Hervant, Frédéric; Konecny, Lara; Moulin, Colette; Douady, Christophe J

2010-05-01

Whereas the consequences of global warming at population or community levels are well documented, studies at the cellular level are still scarce. The study of the physiological or metabolic effects of such small increases in temperature (between +2 degrees C and +6 degrees C) is difficult because they are below the amplitude of the daily or seasonal thermal variations occurring in most environments. In contrast, subterranean biotopes are highly thermally buffered (+/-1 degrees C within a year), and underground water organisms could thus be particularly well suited to characterise cellular responses of global warming. To this purpose, we studied genes encoding chaperone proteins of the HSP70 family in amphipod crustaceans belonging to the ubiquitous subterranean genus Niphargus. An HSP70 sequence was identified in eight populations of two complexes of species of the Niphargus genus (Niphargus rhenorhodanensis and Niphargus virei complexes). Expression profiles were determined for one of these by reverse transcription and quantitative polymerase chain reaction, confirming the inducible nature of this gene. An increase in temperature of 2 degrees C seemed to be without effect on N. rhenorhodanensis physiology, whereas a heat shock of +6 degrees C represented an important thermal stress for these individuals. Thus, this study shows that although Niphargus individuals do not undergo any daily or seasonal thermal variations in underground water, they display an inducible HSP70 heat shock response. This controlled laboratory-based physiological experiment constitutes a first step towards field investigations of the cellular consequences of global warming on subterranean organisms.

Fluorescein-methotrexate transport in dogfish shark (Squalus acanthias) choroid plexus.

PubMed

Baehr, Carsten H; Fricker, Gert; Miller, David S

2006-08-01

The vertebrate choroid plexus removes potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We used confocal microscopy and quantitative image analysis to characterize the mechanisms driving transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact lateral choroid plexus from dogfish shark, Squalus acanthias, an evolutionarily ancient vertebrate. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium/vascular space exceeded bath levels by 5- to 10-fold, and fluorescence in the epithelial cells was slightly below bath levels. FL-MTX accumulation in both tissue compartments was reduced by NaCN, Na removal, and ouabain, but not by a 10-fold increase in medium K. Certain organic anions, e.g., probenecid, MTX, and taurocholate, reduced FL-MTX accumulation in both tissue compartments; p-aminohippurate and estrone sulfate reduced subepithelial/vascular accumulation, but not cellular accumulation. At low concentrations, digoxin, leukotriene C4, and MK-571 reduced fluorescence in the subepithelium/vascular space while increasing cellular fluorescence, indicating preferential inhibition of efflux over uptake. In the presence of 10 microM digoxin (reduced efflux, enhanced cellular accumulation), cellular FL-MTX accumulation was specific, concentrative, and Na dependent. Thus transepithelial FL-MTX transport involved the following two carrier-mediated steps: electroneutral, Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane. Finally, FL-MTX accumulation in both tissue compartments was reduced by phorbol ester and increased by forskolin, indicating antagonistic modulation by protein kinase C and protein kinase A.

Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease

PubMed Central

ALAPAN, YUNUS; KIM, CEONNE; ADHIKARI, ANIMA; GRAY, KAYLA E.; GURKAN-CAVUSOGLU, EVREN; LITTLE, JANE A.; GURKAN, UMUT A.

2016-01-01

Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

A systems approach to integrative biology: an overview of statistical methods to elucidate association and architecture.

PubMed

Ciaccio, Mark F; Finkle, Justin D; Xue, Albert Y; Bagheri, Neda

2014-07-01

An organism's ability to maintain a desired physiological response relies extensively on how cellular and molecular signaling networks interpret and react to environmental cues. The capacity to quantitatively predict how networks respond to a changing environment by modifying signaling regulation and phenotypic responses will help inform and predict the impact of a changing global enivronment on organisms and ecosystems. Many computational strategies have been developed to resolve cue-signal-response networks. However, selecting a strategy that answers a specific biological question requires knowledge both of the type of data being collected, and of the strengths and weaknesses of different computational regimes. We broadly explore several computational approaches, and we evaluate their accuracy in predicting a given response. Specifically, we describe how statistical algorithms can be used in the context of integrative and comparative biology to elucidate the genomic, proteomic, and/or cellular networks responsible for robust physiological response. As a case study, we apply this strategy to a dataset of quantitative levels of protein abundance from the mussel, Mytilus galloprovincialis, to uncover the temperature-dependent signaling network. © The Author 2014. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.

An FD-LC-MS/MS Proteomic Strategy for Revealing Cellular Protein Networks: A Conditional Superoxide Dismutase 1 Knockout Cells

PubMed Central

Ichibangase, Tomoko; Sugawara, Yasuhiro; Yamabe, Akio; Koshiyama, Akiyo; Yoshimura, Akari; Enomoto, Takemi; Imai, Kazuhiro

2012-01-01

Systems biology aims to understand biological phenomena in terms of complex biological and molecular interactions, and thus proteomics plays an important role in elucidating protein networks. However, many proteomic methods have suffered from their high variability, resulting in only showing altered protein names. Here, we propose a strategy for elucidating cellular protein networks based on an FD-LC-MS/MS proteomic method. The strategy permits reproducible relative quantitation of differences in protein levels between different cell populations and allows for integration of the data with those obtained through other methods. We demonstrate the validity of the approach through a comparison of differential protein expression in normal and conditional superoxide dismutase 1 gene knockout cells and believe that beginning with an FD-LC-MS/MS proteomic approach will enable researchers to elucidate protein networks more easily and comprehensively. PMID:23029042

Semi-Automated Curation Allows Causal Network Model Building for the Quantification of Age-Dependent Plaque Progression in ApoE-/- Mouse.

PubMed

Szostak, Justyna; Martin, Florian; Talikka, Marja; Peitsch, Manuel C; Hoeng, Julia

2016-01-01

The cellular and molecular mechanisms behind the process of atherosclerotic plaque destabilization are complex, and molecular data from aortic plaques are difficult to interpret. Biological network models may overcome these difficulties and precisely quantify the molecular mechanisms impacted during disease progression. The atherosclerosis plaque destabilization biological network model was constructed with the semiautomated curation pipeline, BELIEF. Cellular and molecular mechanisms promoting plaque destabilization or rupture were captured in the network model. Public transcriptomic data sets were used to demonstrate the specificity of the network model and to capture the different mechanisms that were impacted in ApoE -/- mouse aorta at 6 and 32 weeks. We concluded that network models combined with the network perturbation amplitude algorithm provide a sensitive, quantitative method to follow disease progression at the molecular level. This approach can be used to investigate and quantify molecular mechanisms during plaque progression.

Tunable Single-Cell Extraction for Molecular Analyses.

PubMed

Guillaume-Gentil, Orane; Grindberg, Rashel V; Kooger, Romain; Dorwling-Carter, Livie; Martinez, Vincent; Ossola, Dario; Pilhofer, Martin; Zambelli, Tomaso; Vorholt, Julia A

2016-07-14

Because of cellular heterogeneity, the analysis of endogenous molecules from single cells is of significant interest and has major implications. While micromanipulation or cell sorting followed by cell lysis is already used for subsequent molecular examinations, approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Here, we demonstrate the quantitative extraction from single cells with spatiotemporal control using fluidic force microscopy. We further present a comprehensive analysis of the soluble molecules withdrawn from the cytoplasm or the nucleus, including the detection of enzyme activities and transcript abundances. This approach has uncovered the ability of cells to withstand extraction of up to several picoliters and opens opportunities to study cellular dynamics and cell-cell communication under physiological conditions at the single-cell level. Copyright © 2016 Elsevier Inc. All rights reserved.

Multiscale Systems Analysis of Root Growth and Development: Modeling Beyond the Network and Cellular Scales

PubMed Central

Band, Leah R.; Fozard, John A.; Godin, Christophe; Jensen, Oliver E.; Pridmore, Tony; Bennett, Malcolm J.; King, John R.

2012-01-01

Over recent decades, we have gained detailed knowledge of many processes involved in root growth and development. However, with this knowledge come increasing complexity and an increasing need for mechanistic modeling to understand how those individual processes interact. One major challenge is in relating genotypes to phenotypes, requiring us to move beyond the network and cellular scales, to use multiscale modeling to predict emergent dynamics at the tissue and organ levels. In this review, we highlight recent developments in multiscale modeling, illustrating how these are generating new mechanistic insights into the regulation of root growth and development. We consider how these models are motivating new biological data analysis and explore directions for future research. This modeling progress will be crucial as we move from a qualitative to an increasingly quantitative understanding of root biology, generating predictive tools that accelerate the development of improved crop varieties. PMID:23110897

Quantitative and Qualitative Analysis of Biomarkers in Fusarium verticillioides

USDA-ARS?s Scientific Manuscript database

In this study, a combination HPLC-DART-TOF-MS system was utilized to identify and quantitatively analyze carbohydrates in wild type and mutant strains of Fusarium verticillioides. Carbohydrate fractions were isolated from F. verticillioides cellular extracts by HPLC using a cation-exchange size-excl...

Multiscale image analysis reveals structural heterogeneity of the cell microenvironment in homotypic spheroids.

PubMed

Schmitz, Alexander; Fischer, Sabine C; Mattheyer, Christian; Pampaloni, Francesco; Stelzer, Ernst H K

2017-03-03

Three-dimensional multicellular aggregates such as spheroids provide reliable in vitro substitutes for tissues. Quantitative characterization of spheroids at the cellular level is fundamental. We present the first pipeline that provides three-dimensional, high-quality images of intact spheroids at cellular resolution and a comprehensive image analysis that completes traditional image segmentation by algorithms from other fields. The pipeline combines light sheet-based fluorescence microscopy of optically cleared spheroids with automated nuclei segmentation (F score: 0.88) and concepts from graph analysis and computational topology. Incorporating cell graphs and alpha shapes provided more than 30 features of individual nuclei, the cellular neighborhood and the spheroid morphology. The application of our pipeline to a set of breast carcinoma spheroids revealed two concentric layers of different cell density for more than 30,000 cells. The thickness of the outer cell layer depends on a spheroid's size and varies between 50% and 75% of its radius. In differently-sized spheroids, we detected patches of different cell densities ranging from 5 × 10 5 to 1 × 10 6  cells/mm 3 . Since cell density affects cell behavior in tissues, structural heterogeneities need to be incorporated into existing models. Our image analysis pipeline provides a multiscale approach to obtain the relevant data for a system-level understanding of tissue architecture.

A Quantitative Three-Dimensional Image Analysis Tool for Maximal Acquisition of Spatial Heterogeneity Data.

PubMed

Allenby, Mark C; Misener, Ruth; Panoskaltsis, Nicki; Mantalaris, Athanasios

2017-02-01

Three-dimensional (3D) imaging techniques provide spatial insight into environmental and cellular interactions and are implemented in various fields, including tissue engineering, but have been restricted by limited quantification tools that misrepresent or underutilize the cellular phenomena captured. This study develops image postprocessing algorithms pairing complex Euclidean metrics with Monte Carlo simulations to quantitatively assess cell and microenvironment spatial distributions while utilizing, for the first time, the entire 3D image captured. Although current methods only analyze a central fraction of presented confocal microscopy images, the proposed algorithms can utilize 210% more cells to calculate 3D spatial distributions that can span a 23-fold longer distance. These algorithms seek to leverage the high sample cost of 3D tissue imaging techniques by extracting maximal quantitative data throughout the captured image.

Fluorescence-based detection and quantification of features of cellular senescence.

PubMed

Cho, Sohee; Hwang, Eun Seong

2011-01-01

Cellular senescence is a spontaneous organismal defense mechanism against tumor progression which is raised upon the activation of oncoproteins or other cellular environmental stresses that must be circumvented for tumorigenesis to occur. It involves growth-arrest state of normal cells after a number of active divisions. There are multiple experimental routes that can drive cells into a state of senescence. Normal somatic cells and cancer cells enter a state of senescence upon overexpression of oncogenic Ras or Raf protein or by imposing certain kinds of stress such as cellular tumor suppressor function. Both flow cytometry and confocal imaging analysis techniques are very useful in quantitative analysis of cellular senescence phenomenon. They allow quantitative estimates of multiple different phenotypes expressed in multiple cell populations simultaneously. Here we review the various types of fluorescence methodologies including confocal imaging and flow cytometry that are frequently utilized to study a variety of senescence. First, we discuss key cell biological changes occurring during senescence and review the current understanding on the mechanisms of these changes with the goal of improving existing protocols and further developing new ones. Next, we list specific senescence phenotypes associated with each cellular trait along with the principles of their assay methods and the significance of the assay outcomes. We conclude by selecting appropriate references that demonstrate a typical example of each method. Copyright © 2011 Elsevier Inc. All rights reserved.

A quantitative evaluation of cell migration by the phagokinetic track motility assay.

PubMed

Nogalski, Maciej T; Chan, Gary C T; Stevenson, Emily V; Collins-McMillen, Donna K; Yurochko, Andrew D

2012-12-04

Cellular motility is an important biological process for both unicellular and multicellular organisms. It is essential for movement of unicellular organisms towards a source of nutrients or away from unsuitable conditions, as well as in multicellular organisms for tissue development, immune surveillance and wound healing, just to mention a few roles(1,2,3). Deregulation of this process can lead to serious neurological, cardiovascular and immunological diseases, as well as exacerbated tumor formation and spread(4,5). Molecularly, actin polymerization and receptor recycling have been shown to play important roles in creating cellular extensions (lamellipodia), that drive the forward movement of the cell(6,7,8). However, many biological questions about cell migration remain unanswered. The central role for cellular motility in human health and disease underlines the importance of understanding the specific mechanisms involved in this process and makes accurate methods for evaluating cell motility particularly important. Microscopes are usually used to visualize the movement of cells. However, cells move rather slowly, making the quantitative measurement of cell migration a resource-consuming process requiring expensive cameras and software to create quantitative time-lapsed movies of motile cells. Therefore, the ability to perform a quantitative measurement of cell migration that is cost-effective, non-laborious, and that utilizes common laboratory equipment is a great need for many researchers. The phagokinetic track motility assay utilizes the ability of a moving cell to clear gold particles from its path to create a measurable track on a colloidal gold-coated glass coverslip(9,10). With the use of freely available software, multiple tracks can be evaluated for each treatment to accomplish statistical requirements. The assay can be utilized to assess motility of many cell types, such as cancer cells(11,12), fibroblasts(9), neutrophils(13), skeletal muscle cells(14), keratinocytes(15), trophoblasts(16), endothelial cells(17), and monocytes(10,18-22). The protocol involves the creation of slides coated with gold nanoparticles (Au°) that are generated by a reduction of chloroauric acid (Au(3+)) by sodium citrate. This method was developed by Turkevich et al. in 1951(23) and then improved in the 1970s by Frens et al.(24,25). As a result of this chemical reduction step, gold particles (10-20 nm in diameter) precipitate from the reaction mixture and can be applied to glass coverslips, which are then ready for use in cellular migration analyses(9,26,27). In general, the phagokinetic track motility assay is a quick, quantitative and easy measure of cellular motility. In addition, it can be utilized as a simple high-throughput assay, for use with cell types that are not amenable to time-lapsed imaging, as well as other uses depending on the needs of the researcher. Together, the ability to quantitatively measure cellular motility of multiple cell types without the need for expensive microscopes and software, along with the use of common laboratory equipment and chemicals, make the phagokinetic track motility assay a solid choice for scientists with an interest in understanding cellular motility.

In silico method for modelling metabolism and gene product expression at genome scale

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

Lerman, Joshua A.; Hyduke, Daniel R.; Latif, Haythem

2012-07-03

Transcription and translation use raw materials and energy generated metabolically to create the macromolecular machinery responsible for all cellular functions, including metabolism. A biochemically accurate model of molecular biology and metabolism will facilitate comprehensive and quantitative computations of an organism's molecular constitution as a function of genetic and environmental parameters. Here we formulate a model of metabolism and macromolecular expression. Prototyping it using the simple microorganism Thermotoga maritima, we show our model accurately simulates variations in cellular composition and gene expression. Moreover, through in silico comparative transcriptomics, the model allows the discovery of new regulons and improving the genome andmore » transcription unit annotations. Our method presents a framework for investigating molecular biology and cellular physiology in silico and may allow quantitative interpretation of multi-omics data sets in the context of an integrated biochemical description of an organism.« less

Regulation of Glycan Structures in Animal Tissues

PubMed Central

Nairn, Alison V.; York, William S.; Harris, Kyle; Hall, Erica M.; Pierce, J. Michael; Moremen, Kelley W.

2008-01-01

Glycan structures covalently attached to proteins and lipids play numerous roles in mammalian cells, including protein folding, targeting, recognition, and adhesion at the molecular or cellular level. Regulating the abundance of glycan structures on cellular glycoproteins and glycolipids is a complex process that depends on numerous factors. Most models for glycan regulation hypothesize that transcriptional control of the enzymes involved in glycan synthesis, modification, and catabolism determines glycan abundance and diversity. However, few broad-based studies have examined correlations between glycan structures and transcripts encoding the relevant biosynthetic and catabolic enzymes. Low transcript abundance for many glycan-related genes has hampered broad-based transcript profiling for comparison with glycan structural data. In an effort to facilitate comparison with glycan structural data and to identify the molecular basis of alterations in glycan structures, we have developed a medium-throughput quantitative real time reverse transcriptase-PCR platform for the analysis of transcripts encoding glycan-related enzymes and proteins in mouse tissues and cells. The method employs a comprehensive list of >700 genes, including enzymes involved in sugar-nucleotide biosynthesis, transporters, glycan extension, modification, recognition, catabolism, and numerous glycosylated core proteins. Comparison with parallel microarray analyses indicates a significantly greater sensitivity and dynamic range for our quantitative real time reverse transcriptase-PCR approach, particularly for the numerous low abundance glycan-related enzymes. Mapping of the genes and transcript levels to their respective biosynthetic pathway steps allowed a comparison with glycan structural data and provides support for a model where many, but not all, changes in glycan abundance result from alterations in transcript expression of corresponding biosynthetic enzymes. PMID:18411279

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

2017-04-01

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 D 3 (25(OH)D 3 ) 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)D 3 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)D 3 directly in aqueous samples, the cellular integrity was maintained in our application. We show the time-dependent uptake of 25(OH)D 3 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.

Exposure of Daphnia magna to trichloroethylene (TCE) and vinyl chloride (VC): evaluation of gene transcription, cellular activity, and life-history parameters.

PubMed

Houde, Magali; Douville, Mélanie; Gagnon, Pierre; Sproull, Jim; Cloutier, François

2015-06-01

Trichloroethylene (TCE) is a ubiquitous contaminant classified as a human carcinogen. Vinyl chloride (VC) is primarily used to manufacture polyvinyl chloride and can also be a degradation product of TCE. Very few data exist on the toxicity of TCE and VC in aquatic organisms particularly at environmentally relevant concentrations. The aim of this study was to evaluate the sub-lethal effects (10 day exposure; 0.1; 1; 10 µg/L) of TCE and VC in Daphnia magna at the gene, cellular, and life-history levels. Results indicated impacts of VC on the regulation of genes related to glutathione-S-transferase (GST), juvenile hormone esterase (JHE), and the vitelline outer layer membrane protein (VMO1). On the cellular level, exposure to 0.1, 1, and 10 µg/L of VC significantly increased the activity of JHE in D. magna and TCE increased the activity of chitinase (at 1 and 10 µg/L). Results for life-history parameters indicated a possible tendency of TCE to affect the number of molts at the individual level in D. magna (p=0.051). Measurement of VG-like proteins using the alkali-labile phosphates (ALP) assay did not show differences between TCE treated organisms and controls. However, semi-quantitative measurement using gradient gel electrophoresis (213-218 kDa) indicated significant decrease in VG-like protein levels following exposure to TCE at all three concentrations. Overall, results indicate effects of TCE and VC on genes and proteins related to metabolism, reproduction, and growth in D. magna. Crown Copyright © 2015. Published by Elsevier Inc. All rights reserved.

Reduction of In-Stent Restenosis Risk on Nickel-Free Stainless Steel by Regulating Cell Apoptosis and Cell Cycle

PubMed Central

Li, Liming; Pan, Shuang; Zhou, Xiaohang; Meng, Xin; Han, Xiaoxi; Ren, Yibin; Yang, Ke; Guan, Yifu

2013-01-01

High nitrogen nickel-free austenitic stainless steel (HNNF SS) is one of the biomaterials developed recently for circumventing the in-stent restenosis (ISR) in coronary stent applications. To understand the ISR-resistance mechanism, we have conducted a comparative study of cellular and molecular responses of human umbilical vein endothelial cells (HUVECs) to HNNF SS and 316L SS (nickel-containing austenitic 316L stainless steel) which is the stent material used currently. CCK-8 analysis and flow cytometric analysis were used to assess the cellular responses (proliferation, apoptosis, and cell cycle), and quantitative real-time PCR (qRT-PCR) was used to analyze the gene expression profile of HUVECs exposed to HNNF SS and 316L SS, respectively. Flow cytometry analysis revealed that 316L SS could activate the cellular apoptosis more efficiently and initiate an earlier entry into the S-phase of cell cycle than HNNF SS. At the molecular level, qRT-PCR results showed that the genes regulating cell apoptosis and autophagy were overexpressed on 316L SS. Further examination indicated that nickel released from 316L SS triggered the cell apoptosis via Fas-Caspase8-Caspase3 exogenous pathway. These molecular mechanisms of HUVECs present a good model for elucidating the observed cellular responses. The findings in this study furnish valuable information for understanding the mechanism of ISR-resistance on the cellular and molecular basis as well as for developing new biomedical materials for stent applications. PMID:23638002

Investigating the role of melanin in UVA/UVB- and hydrogen peroxide-induced cellular and mitochondrial ROS production and mitochondrial DNA damage in human melanoma cells.

PubMed

Swalwell, Helen; Latimer, Jennifer; Haywood, Rachel M; Birch-Machin, Mark A

2012-02-01

Skin cancer incidence is dramatically increasing worldwide, with exposure to ultraviolet radiation (UVR) a predominant factor. The UVA component initiates oxidative stress in human skin, although its exact role in the initiation of skin cancer, particularly malignant melanoma, remains unclear and is controversial because there is evidence for a melanin-dependent mechanism in UVA-linked melanoma studies. Nonpigmented (CHL-1, A375), moderately pigmented (FM55, SKmel23), and highly pigmented (FM94, hyperpigmented FM55) human melanoma cell lines have been used to investigate UVA-induced production of reactive oxygen species using FACS analysis, at both the cellular (dihydrorhodamine-123) and the mitochondrial (MitoSOX) level, where most cellular stress is generated. For the first time, downstream mtDNA damage (utilizing a quantitative long-PCR assay) has been investigated. Using UVA, UVB, and H(2)O(2) as cellular stressors, we have explored the dual roles of melanin as a photoprotector and photosensitizer. The presence of melanin has no influence over cellular oxidative stress generation, whereas, in contrast, melanin protects against mitochondrial superoxide generation and mtDNA damage (one-way ANOVA with post hoc Tukey's analysis, P<0.001). We show that if melanin binds directly to DNA, it acts as a direct photosensitizer of mtDNA damage during UVA irradiation (P<0.001), providing evidence for the dual roles of melanin. Copyright © 2011 Elsevier Inc. All rights reserved.

Quantitative interaction screen of telomeric repeat-containing RNA reveals novel TERRA regulators

PubMed Central

Scheibe, Marion; Arnoult, Nausica; Kappei, Dennis; Buchholz, Frank; Decottignies, Anabelle; Butter, Falk; Mann, Matthias

2013-01-01

Telomeres are actively transcribed into telomeric repeat-containing RNA (TERRA), which has been implicated in the regulation of telomere length and heterochromatin formation. Here, we applied quantitative mass spectrometry (MS)–based proteomics to obtain a high-confidence interactome of TERRA. Using SILAC-labeled nuclear cell lysates in an RNA pull-down experiment and two different salt conditions, we distinguished 115 proteins binding specifically to TERRA out of a large set of background binders. While TERRA binders identified in two previous studies showed little overlap, using quantitative mass spectrometry we obtained many candidates reported in these two studies. To test whether novel candidates found here are involved in TERRA regulation, we performed an esiRNA-based interference analysis for 15 of them. Knockdown of 10 genes encoding candidate proteins significantly affected total cellular levels of TERRA, and RNAi of five candidates perturbed TERRA recruitment to telomeres. Notably, depletion of SRRT/ARS2, involved in miRNA processing, up-regulated both total and telomere-bound TERRA. Conversely, knockdown of MORF4L2, a component of the NuA4 histone acetyltransferase complex, reduced TERRA levels both globally and for telomere-bound TERRA. We thus identified new proteins involved in the homeostasis and telomeric abundance of TERRA, extending our knowledge of TERRA regulation. PMID:23921659

Prospects and challenges of quantitative phase imaging in tumor cell biology

NASA Astrophysics Data System (ADS)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Understanding metal homeostasis in primary cultured neurons. Studies using single neuron subcellular and quantitative metallomics.

PubMed

Colvin, Robert A; Lai, Barry; Holmes, William R; Lee, Daewoo

2015-07-01

The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be associated with ferritin cages or transferrin receptor endosomes. The iron content and its distribution in puncta were similar in all neuron types studied including primary dopaminergic neurons. In summary, quantitative measurements of steady state metal levels in single primary cultured neurons made possible by SRXRF analyses provide unique information on the relative levels of each metal in neuronal soma and processes, subcellular location of zinc loads, and have confirmed and extended the characterization of heretofore poorly understood cytoplasmic iron puncta.

Hemochromatosis Enhances Tumor Progression via Upregulation of Intracellular Iron in Head and Neck Cancer

PubMed Central

Lenarduzzi, Michelle; Hui, Angela B. Y.; Yue, Shijun; Ito, Emma; Shi, Wei; Williams, Justin; Bruce, Jeff; Sakemura-Nakatsugawa, Noriko; Xu, Wei; Schimmer, Aaron; Liu, Fei-Fei

2013-01-01

Introduction Despite improvements in treatment strategies for head and neck squamous cell carcinoma (HNSCC), outcomes have not significantly improved; highlighting the importance of identifying novel therapeutic approaches to target this disease. To address this challenge, we proceeded to evaluate the role of iron in HNSCC. Experimental Design Expression levels of iron-related genes were evaluated in HNSCC cell lines using quantitative RT-PCR. Cellular phenotypic effects were assessed using viability (MTS), clonogenic survival, BrdU, and tumor formation assays. The prognostic significance of iron-related proteins was determined using immunohistochemistry. Results In a panel of HNSCC cell lines, hemochromatosis (HFE) was one of the most overexpressed genes involved in iron regulation. In vitro knockdown of HFE in HNSCC cell lines significantly decreased hepcidin (HAMP) expression and intracellular iron level. This in turn, resulted in a significant decrease in HNSCC cell viability, clonogenicity, DNA synthesis, and Wnt signalling. These cellular changes were reversed by re-introducing iron back into HNSCC cells after HFE knockdown, indicating that iron was mediating this phenotype. Concordantly, treating HNSCC cells with an iron chelator, ciclopirox olamine (CPX), significantly reduced viability and clonogenic survival. Finally, patients with high HFE expression experienced a reduced survival compared to patients with low HFE expression. Conclusions Our data identify HFE as potentially novel prognostic marker in HNSCC that promotes tumour progression via HAMP and elevated intracellular iron levels, leading to increased cellular proliferation and tumour formation. Hence, these findings suggest that iron chelators might have a therapeutic role in HNSCC management. PMID:23991213

Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.

PubMed

Lee, Wooram; Lazar, Iulia M

2014-07-01

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.

Detecting the Extent of Cellular Decomposition after Sub-Eutectoid Annealing in Rolled UMo Foils

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

Kautz, Elizabeth J.; Jana, Saumyadeep; Devaraj, Arun

2017-07-31

This report presents an automated image processing approach to quantifying microstructure image data, specifically the extent of eutectoid (cellular) decomposition in rolled U-10Mo foils. An image processing approach is used here to be able to quantitatively describe microstructure image data in order to relate microstructure to processing parameters (time, temperature, deformation).

Cellular Phone Use in Class: Implications for Teaching and Learning a Pilot Study

ERIC Educational Resources Information Center

Burns, Shari M.; Lohenry, Kevin

2010-01-01

Students equipped with the cell phones enter college classrooms daily. Realizing the impact of technology on fellow learners and faculty represents an area of concern. A pilot study was conducted to determine student and faculty perception regarding cellular phone use in the classroom. A quantitative descriptive study examined the perception of…

Cellular Motility--Experiments on Contractile and Motile Mechanisms in the Slime Mould, Physarum Polycephalum

ERIC Educational Resources Information Center

Holmes, R. P.; Stewart, P. R.

1977-01-01

Actin and myosin have now been demonstrated to be important constituents of many eukaryotic cells. Their role is primarily that of a contractile system underlying all aspects of cellular motility. Described here is a simple experimental system to demonstrate quantitatively aspects of motility and its regulation in a slime mold. (Author/MA)

Modeling of Receptor Tyrosine Kinase Signaling: Computational and Experimental Protocols.

PubMed

Fey, Dirk; Aksamitiene, Edita; Kiyatkin, Anatoly; Kholodenko, Boris N

2017-01-01

The advent of systems biology has convincingly demonstrated that the integration of experiments and dynamic modelling is a powerful approach to understand the cellular network biology. Here we present experimental and computational protocols that are necessary for applying this integrative approach to the quantitative studies of receptor tyrosine kinase (RTK) signaling networks. Signaling by RTKs controls multiple cellular processes, including the regulation of cell survival, motility, proliferation, differentiation, glucose metabolism, and apoptosis. We describe methods of model building and training on experimentally obtained quantitative datasets, as well as experimental methods of obtaining quantitative dose-response and temporal dependencies of protein phosphorylation and activities. The presented methods make possible (1) both the fine-grained modeling of complex signaling dynamics and identification of salient, course-grained network structures (such as feedback loops) that bring about intricate dynamics, and (2) experimental validation of dynamic models.

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome*

PubMed Central

Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K.; Moore, Dirk F.; Sleat, David E.; Lobel, Peter

2017-01-01

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene. PMID:27923875

Accounting for Protein Subcellular Localization: A Compartmental Map of the Rat Liver Proteome.

PubMed

Jadot, Michel; Boonen, Marielle; Thirion, Jaqueline; Wang, Nan; Xing, Jinchuan; Zhao, Caifeng; Tannous, Abla; Qian, Meiqian; Zheng, Haiyan; Everett, John K; Moore, Dirk F; Sleat, David E; Lobel, Peter

2017-02-01

Accurate knowledge of the intracellular location of proteins is important for numerous areas of biomedical research including assessing fidelity of putative protein-protein interactions, modeling cellular processes at a system-wide level and investigating metabolic and disease pathways. Many proteins have not been localized, or have been incompletely localized, partly because most studies do not account for entire subcellular distribution. Thus, proteins are frequently assigned to one organelle whereas a significant fraction may reside elsewhere. As a step toward a comprehensive cellular map, we used subcellular fractionation with classic balance sheet analysis and isobaric labeling/quantitative mass spectrometry to assign locations to >6000 rat liver proteins. We provide quantitative data and error estimates describing the distribution of each protein among the eight major cellular compartments: nucleus, mitochondria, lysosomes, peroxisomes, endoplasmic reticulum, Golgi, plasma membrane and cytosol. Accounting for total intracellular distribution improves quality of organelle assignments and assigns proteins with multiple locations. Protein assignments and supporting data are available online through the Prolocate website (http://prolocate.cabm.rutgers.edu). As an example of the utility of this data set, we have used organelle assignments to help analyze whole exome sequencing data from an infant dying at 6 months of age from a suspected neurodegenerative lysosomal storage disorder of unknown etiology. Sequencing data was prioritized using lists of lysosomal proteins comprising well-established residents of this organelle as well as novel candidates identified in this study. The latter included copper transporter 1, encoded by SLC31A1, which we localized to both the plasma membrane and lysosome. The patient harbors two predicted loss of function mutations in SLC31A1, suggesting that this may represent a heretofore undescribed recessive lysosomal storage disease gene. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Guo, Lei; Xiao, Yongsheng; Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu

Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 humanmore » skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA(III) perturbs Nrf2 pathway and selenoprotein synthesis.« less

Increased fibulin-1 plasma levels in polycystic ovary syndrome (PCOS) patients: possible contribution to the link between PCOS and cardiovascular risk.

PubMed

Scarinci, E; Tropea, A; Russo, G; Notaristefano, G; Messana, C; Alesiani, O; Fabozzi, S M; Lanzone, A; Apa, R

2018-04-21

To investigate a possible relation between fibulin-1 plasma levels and PCOS. ELISA quantitative determination of human fibulin-1. 50 women with PCOS and 40 control patients who attended the Unit of Human Reproductive Pathophysiology, Università Cattolica del Sacro Cuore, Rome, were enrolled. Ultrasonographic pelvic examinations, hormonal profile assays, oral tolerance test OGTT, lipid profile and ELISA quantitative determination of human fibulin-1 were performed. Fibulin-1 levels were found to be statistically significantly higher in PCOS patients than in matched control women. No statistically significant positive correlation was found between fibulin-1 and AUCi, HOMA-IR, total cholesterol, LDL, AMH, androstenedione and FAI, whereas a statistically significant positive correlation was found between fibulin-1 and 17OHP (p = 0.016) in the PCOS group. However, multivariable linear regression analysis showed that 17 OH P did not independently predict fibulin-1 levels (p = 0.089). Our data could contribute to explain the hypothesized increased cardiovascular risk and vascular damage in patients with PCOS. A better understanding of the cellular and molecular mechanisms involved in cardiometabolic disorders associated with PCOS is mandatory to identify new therapeutic strategies to eventually prevent the progression of cardiovascular diseases in these patients.

Identification of suitable reference genes for hepatic microRNA quantitation.

PubMed

Lamba, Vishal; Ghodke-Puranik, Yogita; Guan, Weihua; Lamba, Jatinder K

2014-03-07

MicroRNAs (miRNAs) are short (~22 nt) endogenous RNAs that play important roles in regulating expression of a wide variety of genes involved in different cellular processes. Alterations in microRNA expression patterns have been associated with a number of human diseases. Accurate quantitation of microRNA levels is important for their use as biomarkers and in determining their functions. Real time PCR is the gold standard and the most frequently used technique for miRNA quantitation. Real time PCR data analysis includes normalizing the amplification data to suitable endogenous control/s to ensure that microRNA quantitation is not affected by the variability that is potentially introduced at different experimental steps. U6 (RNU6A) and RNU6B are two commonly used endogenous controls in microRNA quantitation. The present study was designed to investigate inter-individual variability and gender differences in hepatic microRNA expression as well as to identify the best endogenous control/s that could be used for normalization of real-time expression data in liver samples. We used Taqman based real time PCR to quantitate hepatic expression levels of 22 microRNAs along with U6 and RNU6B in 50 human livers samples (25 M, 25 F). To identify the best endogenous controls for use in data analysis, we evaluated the amplified candidates for their stability (least variability) in expression using two commonly used software programs: Normfinder and GeNormplus, Both Normfinder and GeNormplus identified U6 to be among the least stable of all the candidates analyzed, and RNU6B was also not among the top genes in stability. mir-152 and mir-23b were identified to be the two most stable candidates by both Normfinder and GeNormplus in our analysis, and were used as endogenous controls for normalization of hepatic miRNA levels. Measurements of microRNA stability indicate that U6 and RNU6B are not suitable for use as endogenous controls for normalizing microRNA relative quantitation data in hepatic tissue, and their use can led to possibly erroneous conclusions.

From Single-Cell Dynamics to Scaling Laws in Oncology

NASA Astrophysics Data System (ADS)

Chignola, Roberto; Sega, Michela; Stella, Sabrina; Vyshemirsky, Vladislav; Milotti, Edoardo

We are developing a biophysical model of tumor biology. We follow a strictly quantitative approach where each step of model development is validated by comparing simulation outputs with experimental data. While this strategy may slow down our advancements, at the same time it provides an invaluable reward: we can trust simulation outputs and use the model to explore territories of cancer biology where current experimental techniques fail. Here, we review our multi-scale biophysical modeling approach and show how a description of cancer at the cellular level has led us to general laws obeyed by both in vitro and in vivo tumors.

Switch-like Arp2/3 activation upon WASP and WIP recruitment to an apparent threshold level by multivalent linker proteins in vivo.

PubMed

Sun, Yidi; Leong, Nicole T; Jiang, Tommy; Tangara, Astou; Darzacq, Xavier; Drubin, David G

2017-08-16

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.

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

PubMed Central

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

2015-01-01

Abstract. 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 w, Δt, z, α, μ, α1, and α2, 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. PMID:26158101

Fungicidal efficiency of electrolyzed oxidizing water on Candida albicans and its biochemical mechanism.

PubMed

Zeng, Xinping; Ye, Guoqing; Tang, Wenwei; Ouyang, Ting; Tian, Lin; Ni, Yaming; Li, Ping

2011-07-01

The fungicidal influencing factors of electrolyzed oxidizing water (EOW) on Candida albicans were investigated by suspension quantitative germicidal tests. Results showed that EOW possessed predominant fungicidal rate on C. albican, as high as consumately 100% after 0.5min duration of 65.5mg/L active available chlorine concentration (ACC). The fungicidal effect was promoted proportionally along with ACC but was inhibited by organic interferential bovine serum albumin (BSA). The fungicidal mechanism was also investigated at a biological molecular level by detecting series of biochemical indices. Fluorescent microscopy showed that almost all C. albicans cells were stained red in 1min, suggesting that cell membrane was one of EOW's action targets. Transmission electron microscopy (TEM) showed that EOW destroyed the cellular protective barriers and imposed some damage upon the nucleus area, which verified EOW's effects on microbial ultra-structures. EOW improved membrane permeabilities with the result that the leakages of cellular inclusions (K(+), proteins and DNA) and the conductivity increased rapidly. The dehydrogenase relative activities of C. albicans decreased by 44.0% after 10min, indicating that EOW also had a destructive effect on cellular dehydrogenase. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Metabolic Adaptation to Muscle Ischemia

NASA Technical Reports Server (NTRS)

Cabrera, Marco E.; Coon, Jennifer E.; Kalhan, Satish C.; Radhakrishnan, Krishnan; Saidel, Gerald M.; Stanley, William C.

2000-01-01

Although all tissues in the body can adapt to varying physiological/pathological conditions, muscle is the most adaptable. To understand the significance of cellular events and their role in controlling metabolic adaptations in complex physiological systems, it is necessary to link cellular and system levels by means of mechanistic computational models. The main objective of this work is to improve understanding of the regulation of energy metabolism during skeletal/cardiac muscle ischemia by combining in vivo experiments and quantitative models of metabolism. Our main focus is to investigate factors affecting lactate metabolism (e.g., NADH/NAD) and the inter-regulation between carbohydrate and fatty acid metabolism during a reduction in regional blood flow. A mechanistic mathematical model of energy metabolism has been developed to link cellular metabolic processes and their control mechanisms to tissue (skeletal muscle) and organ (heart) physiological responses. We applied this model to simulate the relationship between tissue oxygenation, redox state, and lactate metabolism in skeletal muscle. The model was validated using human data from published occlusion studies. Currently, we are investigating the difference in the responses to sudden vs. gradual onset ischemia in swine by combining in vivo experimental studies with computational models of myocardial energy metabolism during normal and ischemic conditions.

Peptidomic analysis of human cell lines

PubMed Central

Gelman, Julia S.; Sironi, Juan; Castro, Leandro M.; Ferro, Emer S.; Fricker, Lloyd D.

2011-01-01

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells. PMID:21204522

Multiplexed Nanoplasmonic Temporal Profiling of T-Cell Response under Immunomodulatory Agent Exposure

PubMed Central

2016-01-01

Immunomodulatory drugs—agents regulating the immune response—are commonly used for treating immune system disorders and minimizing graft versus host disease in persons receiving organ transplants. At the cellular level, immunosuppressant drugs are used to inhibit pro-inflammatory or tissue-damaging responses of cells. However, few studies have so far precisely characterized the cellular-level effect of immunomodulatory treatment. The primary challenge arises due to the rapid and transient nature of T-cell immune responses to such treatment. T-cell responses involve a highly interactive network of different types of cytokines, which makes precise monitoring of drug-modulated T-cell response difficult. Here, we present a nanoplasmonic biosensing approach to quantitatively characterize cytokine secretion behaviors of T cells with a fine time-resolution (every 10 min) that are altered by an immunosuppressive drug used in the treatment of T-cell-mediated diseases. With a microfluidic platform integrating antibody-conjugated gold nanorod (AuNR) arrays, the technique enables simultaneous multi-time-point measurements of pro-inflammatory (IL-2, IFN-γ, and TNF-α) and anti-inflammatory (IL-10) cytokines secreted by T cells. The integrated nanoplasmonic biosensors achieve precise measurements with low operating sample volume (1 μL), short assay time (∼30 min), heightened sensitivity (∼20–30 pg/mL), and negligible sensor crosstalk. Data obtained from the multicytokine secretion profiles with high practicality resulting from all of these sensing capabilities provide a comprehensive picture of the time-varying cellular functional state during pharmacologic immunosuppression. The capability to monitor cellular functional response demonstrated in this study has great potential to ultimately permit personalized immunomodulatory treatment. PMID:27478873

Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression.

PubMed

Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

2016-08-01

Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP‑1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP‑1 cells were differentiated to macrophages by phorbol 12‑myristate 13‑acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon‑γ (IFN‑γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription‑quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme‑linked immunosorbent assay. IRF5 protein and nuclei co‑localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN‑γ stimulation‑induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels.

Mangiferin inhibits macrophage classical activation via downregulating interferon regulatory factor 5 expression

PubMed Central

Wei, Zhiquan; Yan, Li; Chen, Yixin; Bao, Chuanhong; Deng, Jing; Deng, Jiagang

2016-01-01

Mangiferin is a natural polyphenol and the predominant effective component of Mangifera indica Linn. leaves. For hundreds of years, Mangifera indica Linn. leaf has been used as an ingredient in numerous traditional Chinese medicine preparations for the treatment of bronchitis. However, the pharmacological mechanism of mangiferin in the treatment of bronchitis remains to be elucidated. Macrophage classical activation is important role in the process of bronchial airway inflammation, and interferon regulatory factor 5 (IRF5) has been identified as a key regulatory factor for macrophage classical activation. The present study used the THP-1 human monocyte cell line to investigate whether mangiferin inhibits macrophage classical activation via suppressing IRF5 expression in vitro. THP-1 cells were differentiated to macrophages by phorbol 12-myristate 13-acetate. Macrophages were polarized to M1 macrophages following stimulation with lipopolysaccharide (LPS)/interferon-γ (IFN-γ). Flow cytometric analysis was conducted to detect the M1 macrophages. Reverse transcription-quantitative polymerase chain reaction was used to investigate cellular IRF5 gene expression. Levels of proinflammatory cytokines and IRF5 were assessed following cell culture and cellular homogenization using enzyme-linked immunosorbent assay. IRF5 protein and nuclei co-localization was performed in macrophages with laser scanning confocal microscope immunofluorescence analysis. The results of the present study demonstrated that mangiferin significantly inhibits LPS/IFN-γ stimulation-induced classical activation of macrophages in vitro and markedly decreases proinflammatory cytokine release. In addition, cellular IRF5 expression was markedly downregulated. These results suggest that the inhibitory effect of mangiferin on classical activation of macrophages may be exerted via downregulation of cellular IRF5 expression levels. PMID:27277156

A cellular and molecular basis for the selective desmopressin-induced ACTH release in Cushing disease patients: key role of AVPR1b receptor and potential therapeutic implications.

PubMed

Luque, R M; Ibáñez-Costa, A; López-Sánchez, L M; Jiménez-Reina, L; Venegas-Moreno, E; Gálvez, M A; Villa-Osaba, A; Madrazo-Atutxa, A M; Japón, M A; de la Riva, A; Cano, D A; Benito-López, P; Soto-Moreno, A; Gahete, M D; Leal-Cerro, A; Castaño, J P

2013-10-01

Desmopressin is a synthetic agonist of vasopressin receptors (AVPRs). The desmopressin stimulation test is used in the diagnosis and postsurgery prognosis of Cushing disease (CD). However, the cellular and molecular mechanisms underlying the desmopressin-induced ACTH increase in patients with CD are poorly understood. The objectives of this study were to determine, for the first time, whether desmopressin acts directly and exclusively on pituitary corticotropinoma cells to stimulate ACTH expression/release and to elucidate the cellular and molecular mechanisms involved in desmopressin-induced ACTH increase in CD. A total of 8 normal pituitaries (NPs), 23 corticotropinomas, 14 nonfunctioning pituitary adenomas, 17 somatotropinomas, and 3 prolactinomas were analyzed for AVPR expression by quantitative real-time RT-PCR. Primary cultures derived from corticotropinomas, nonfunctioning pituitary adenomas, somatotropinomas, prolactinomas, and NPs were treated with desmopressin, and ACTH secretion/expression, [Ca(2+)]i kinetics, and AVPR expression and/or proliferative response were evaluated. The relationship between AVPR expression and plasma adrenocorticotropin/cortisol levels obtained from desmopressin tests was assessed. Desmopressin affects all functional parameters evaluated in corticotropinoma cells but not in NPs or other pituitary adenomas cells. These effects might be due to the dramatic elevation of AVPR1b expression levels found in corticotropinomas. In line with this notion, the use of an AVPR1b antagonist completely blocked desmopressin stimulatory effects. Remarkably, only AVPR1b expression was positively correlated with elevated plasma adrenocorticotropin levels in corticotropinomas. The present results provide a cellular and molecular basis to support the desmopressin stimulation test as a reliable, specific test for the diagnosis and postsurgery prognosis of CD. Furthermore, our data indicate that AVPR1b is responsible for the direct/exclusive desmopressin stimulatory pituitary effects observed in CD, thus opening the possibility of exploring AVPR1b antagonists as potential therapeutic tools for CD treatment.

Repression of anti-proliferative factor Tob1 in osteoarthritic cartilage

PubMed Central

Gebauer, Mathias; Saas, Joachim; Haag, Jochen; Dietz, Uwe; Takigawa, Masaharu; Bartnik, Eckart; Aigner, Thomas

2005-01-01

Osteoarthritis is the most common degenerative disorder of the modern world. However, many basic cellular features and molecular processes of the disease are poorly understood. In the present study we used oligonucleotide-based microarray analysis of genes of known or assumed relevance to the cellular phenotype to screen for relevant differences in gene expression between normal and osteoarthritic chondrocytes. Custom made oligonucleotide DNA arrays were used to screen for differentially expressed genes in normal (n = 9) and osteoarthritic (n = 10) cartilage samples. Real-time polymerase chain reaction (PCR) with gene-specific primers was used for quantification. Primary human adult articular chondrocytes and chondrosarcoma cell line HCS-2/8 were used to study changes in gene expression levels after stimulation with interleukin-1β and bone morphogenetic protein, as well as the dependence on cell differentiation. In situ hybridization with a gene-specific probe was applied to detect mRNA expression levels in fetal growth plate cartilage. Overall, more than 200 significantly regulated genes were detected between normal and osteoarthritic cartilage (P < 0.01). One of the significantly repressed genes, Tob1, encodes a protein belonging to a family involved in silencing cells in terms of proliferation and functional activity. The repression of Tob1 was confirmed by quantitative PCR and correlated to markers of chondrocyte activity and proliferation in vivo. Tob1 expression was also detected at a decreased level in isolated chondrocytes and in the chondrosarcoma cell line HCS-2/8. Again, in these cells it was negatively correlated with proliferative activity and positively with cellular differentiation. Altogether, the downregulation of the expression of Tob1 in osteoarthritic chondrocytes might be an important aspect of the cellular processes taking place during osteoarthritic cartilage degeneration. Activation, the reinitiation of proliferative activity and the loss of a stable phenotype are three major changes in osteoarthritic chondrocytes that are highly significantly correlated with the repression of Tob1 expression. PMID:15743474

Quantitative Microscopic Analysis of Plasma Membrane Receptor Dynamics in Living Plant Cells.

PubMed

Luo, Yu; Russinova, Eugenia

2017-01-01

Plasma membrane-localized receptors are essential for cellular communication and signal transduction. In Arabidopsis thaliana, BRASSINOSTEROID INSENSITIVE1 (BRI1) is one of the receptors that is activated by binding to its ligand, the brassinosteroid (BR) hormone, at the cell surface to regulate diverse plant developmental processes. The availability of BRI1 in the plasma membrane is related to its signaling output and is known to be controlled by the dynamic endomembrane trafficking. Advances in fluorescence labeling and confocal microscopy techniques enabled us to gain a better understanding of plasma membrane receptor dynamics in living cells. Here we describe different quantitative microscopy methods to monitor the relative steady-state levels of the BRI1 protein in the plasma membrane of root epidermal cells and its relative exocytosis and recycling rates. The methods can be applied also to analyze similar dynamics of other plasma membrane-localized receptors.

Oxidative DNA damage background estimated by a system model of base excision repair

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

Sokhansanj, B A; Wilson, III, D M

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parametersmore » from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.« less

Quantitative Large-Scale Three-Dimensional Imaging of Human Kidney Biopsies: A Bridge to Precision Medicine in Kidney Disease.

PubMed

Winfree, Seth; Dagher, Pierre C; Dunn, Kenneth W; Eadon, Michael T; Ferkowicz, Michael; Barwinska, Daria; Kelly, Katherine J; Sutton, Timothy A; El-Achkar, Tarek M

2018-06-05

Kidney biopsy remains the gold standard for uncovering the pathogenesis of acute and chronic kidney diseases. However, the ability to perform high resolution, quantitative, molecular and cellular interrogation of this precious tissue is still at a developing stage compared to other fields such as oncology. Here, we discuss recent advances in performing large-scale, three-dimensional (3D), multi-fluorescence imaging of kidney biopsies and quantitative analysis referred to as 3D tissue cytometry. This approach allows the accurate measurement of specific cell types and their spatial distribution in a thick section spanning the entire length of the biopsy. By uncovering specific disease signatures, including rare occurrences, and linking them to the biology in situ, this approach will enhance our understanding of disease pathogenesis. Furthermore, by providing accurate quantitation of cellular events, 3D cytometry may improve the accuracy of prognosticating the clinical course and response to therapy. Therefore, large-scale 3D imaging and cytometry of kidney biopsy is poised to become a bridge towards personalized medicine for patients with kidney disease. © 2018 S. Karger AG, Basel.

Quantitative measures for redox signaling.

PubMed

Pillay, Ché S; Eagling, Beatrice D; Driscoll, Scott R E; Rohwer, Johann M

2016-07-01

Redox signaling is now recognized as an important regulatory mechanism for a number of cellular processes including the antioxidant response, phosphokinase signal transduction and redox metabolism. While there has been considerable progress in identifying the cellular machinery involved in redox signaling, quantitative measures of redox signals have been lacking, limiting efforts aimed at understanding and comparing redox signaling under normoxic and pathogenic conditions. Here we have outlined some of the accepted principles for redox signaling, including the description of hydrogen peroxide as a signaling molecule and the role of kinetics in conferring specificity to these signaling events. Based on these principles, we then develop a working definition for redox signaling and review a number of quantitative methods that have been employed to describe signaling in other systems. Using computational modeling and published data, we show how time- and concentration- dependent analyses, in particular, could be used to quantitatively describe redox signaling and therefore provide important insights into the functional organization of redox networks. Finally, we consider some of the key challenges with implementing these methods. Copyright © 2016 Elsevier Inc. All rights reserved.

Inhibiting the NF-kappaB pathway to assess its function in the cellular response to space radiation

NASA Astrophysics Data System (ADS)

Koch, Kristina; Baumstark-Khan, Christa; Hellweg, Christine; Testard, Isabelle; Reitz, Guenther

2012-07-01

Radiation is regarded as one of the limiting factors for space missions. Therefore the cellular radiation response needs to be studied in order to estimate risks and to develop appropriate countermeasures. Exposure of human cells to ionizing radiation can provoke cell cycle arrest, leading to cellular senescence or premature differentiation, and different types of cell death. Previous heavy ion experiments have shown that the Nuclear Factor κB (NF-κB) pathway is activated by fluences that can be reached during long-term missions and thereby NF-κB was identified as an important modulating factor in the cellular radiation response. It could improve cellular survival after exposure to high radiation doses and influence the cancer risk of astronauts. The classical and the genotoxic stress induced NF-κB pathway result in nuclear translocation of the p65/p50 dimer. Both pathways might contribute to the cellular radiation response. Chemical inhibitors were tested to suppress the NF-κB pathway in recombinant HEK-pNF-κB-d2EGFP/Neo cells. The efficacy and cytotoxicity of the inhibitors targeting different elements of the NF-κB pathway were analyzed and found mostly inappropriate as inhibitors were partly cytotoxic or unspecific. Alternatively a functional knock-out of RelA (p65) was used to identify the contribution of the NF-κB pathway to different cellular outcomes. Small hairpin RNA constructs (shRNA) were transfected into the HEK-pNF-κB-d2EGFP/Neo cell line. Their functionality was assessed by quantitative Reverse Transcriptase real-time PCR (qRT-PCR) to verify that the RelA mRNA amount was reduced by more than 80% in the knock-down cells The original cell line had been stably transfected with a reporter system to monitor NF-κB activation by measuring destabilized Enhanced Green Fluorescent Protein (d2EGFP)-expression. It was shown that after 18 hours d2EGFP reaches its highest expression level after activation of NF-κB and can be measured by FACS analysis. Results of measuring d2EGFP showed a suppressed level of EGFP(+) cells in the knock-down cell line, indicating a decreased NF-κB level. Growth behavior of the original and the knock-down cell line was investigated, showing that the decreased RelA level leads to an elongated lag phase while the doubling time during the exponential growth phase remained unaltered. Further the colony forming ability of both cell lines was compared. Both cell lines were irradiated with X-Rays. The RelA-knock-down cell line showed an increased radiosensitivity towards X-Rays, proving that NF-κB plays an important role in the survival ability of the cell. The knock-down cell line will now be used to study the involvement of NF-κB pathway in the cellular response to heavy ion exposure and other space relevant radiation qualities.

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment.

PubMed

Santos, Fátima Milhano; Gaspar, Leonor Mesquita; Ciordia, Sergio; Rocha, Ana Sílvia; Castro E Sousa, João Paulo; Paradela, Alberto; Passarinha, Luís António; Tomaz, Cândida Teixeira

2018-04-11

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

PubMed Central

Gaspar, Leonor Mesquita; Ciordia, Sergio; Rocha, Ana Sílvia; Castro e Sousa, João Paulo; Paradela, Alberto

2018-01-01

Rhegmatogenous retinal detachment (RRD) is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), growth factors (metalloproteinase inhibitor 1), and serine protease inhibitors (plasminogen activator inhibitor 1) are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses. PMID:29641463

Real time blood testing using quantitative phase imaging.

PubMed

Pham, Hoa V; Bhaduri, Basanta; Tangella, Krishnarao; Best-Popescu, Catherine; Popescu, Gabriel

2013-01-01

We demonstrate a real-time blood testing system that can provide remote diagnosis with minimal human intervention in economically challenged areas. Our instrument combines novel advances in label-free optical imaging with parallel computing. Specifically, we use quantitative phase imaging for extracting red blood cell morphology with nanoscale sensitivity and NVIDIA's CUDA programming language to perform real time cellular-level analysis. While the blood smear is translated through focus, our system is able to segment and analyze all the cells in the one megapixel field of view, at a rate of 40 frames/s. The variety of diagnostic parameters measured from each cell (e.g., surface area, sphericity, and minimum cylindrical diameter) are currently not available with current state of the art clinical instruments. In addition, we show that our instrument correctly recovers the red blood cell volume distribution, as evidenced by the excellent agreement with the cell counter results obtained on normal patients and those with microcytic and macrocytic anemia. The final data outputted by our instrument represent arrays of numbers associated with these morphological parameters and not images. Thus, the memory necessary to store these data is of the order of kilobytes, which allows for their remote transmission via, for example, the cellular network. We envision that such a system will dramatically increase access for blood testing and furthermore, may pave the way to digital hematology.

Quantitative implementation of the endogenous molecular-cellular network hypothesis in hepatocellular carcinoma.

PubMed

Wang, Gaowei; Zhu, Xiaomei; Gu, Jianren; Ao, Ping

2014-06-06

A quantitative hypothesis for cancer genesis and progression-the endogenous molecular-cellular network hypothesis, intended to include both genetic and epigenetic causes of cancer-has been proposed recently. Using this hypothesis, here we address the molecular basis for maintaining normal liver and hepatocellular carcinoma (HCC), and the potential strategy to cure or relieve HCC. First, we elaborate the basic assumptions of the hypothesis and establish a core working network of HCC according to the hypothesis. Second, we quantify the working network by a nonlinear dynamical system. We show that the working network reproduces the main known features of normal liver and HCC at both the modular and molecular levels. Lastly, the validated working network reveals that (i) specific positive feedback loops are responsible for the maintenance of normal liver and HCC; (ii) inhibiting proliferation and inflammation-related positive feedback loops and simultaneously inducing a liver-specific positive feedback loop is predicated as a potential strategy to cure or relieve HCC; and (iii) the genesis and regression of HCC are asymmetric. In light of the characteristic properties of the nonlinear dynamical system, we demonstrate that positive feedback loops must exist as a simple and general molecular basis for the maintenance of heritable phenotypes, such as normal liver and HCC, and regulating the positive feedback loops directly or indirectly provides potential strategies to cure or relieve HCC.

Studying the relationship between redox and cell growth using quantitative phase imaging (Conference Presentation)

NASA Astrophysics Data System (ADS)

Sridharan, Shamira; Leslie, Matthew T.; Bapst, Natalya; Smith, John; Gaskins, H. Rex; Popescu, Gabriel

2016-03-01

Quantitative phase imaging has been used in the past to study the dry mass of cells and study cell growth under various treatment conditions. However, the relationship between cellular redox and growth rates has not yet been studied in this context. This study employed the recombinant Glrx-roGFP2 redox biosensor targeted to the mitochondrial matrix or cytosolic compartments of A549 lung epithelial carcinoma cells. The Glrx-roGFP2s biosensor consists of a modified GFP protein containing internal cysteine residues sensitive to the local redox environment. The formation/dissolution of sulfide bridges contorts the internal chromophore, dictating corresponding changes in florescence emission that provide direct measures of the local redox potential. Combining 2-channel florescent imaging of the redox sensor with quantitative phase imaging allowed observation of redox homeostasis alongside measurements of cellular mass during full cycles of cellular division. The results indicate that mitochondrial redox showed a stronger inverse correlation with cell growth than cytoplasmic redox states; although redox changes are restricted to a 5% range. We are now studying the relationship between mitochondrial redox and cell growth in an isogenic series of breast cell lines built upon the MCF-10A genetic background that vary both in malignancy and metastatic potential.

Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference.

PubMed

Presley, Tennille; Kuppusamy, Periannan; Zweier, Jay L; Ilangovan, Govindasamy

2006-12-15

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO(2)-independent respiration at higher pO(2) ranges, pO(2)-dependent respiration at low pO(2) ranges, and a static equilibrium with no change in pO(2) at very low pO(2) values. The approach here enables one to comprehensively analyze all of the three zones together-where the progression of O(2) diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO(2) data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry.

Yellow Fever Virus Modulates the Expression of Key Proteins Related to the microRNA Pathway in the Human Hepatocarcinoma Cell Line HepG2.

PubMed

Holanda, Gustavo Moraes; Casseb, Samir Mansour Moraes; Mello, Karla Fabiane Lopes; Vasconcelos, Pedro Fernando Costa; Cruz, Ana Cecília Ribeiro

2017-06-01

Yellow fever is a zoonotic disease caused by the yellow fever virus (YFV) and transmitted by mosquitoes of the family Culicidae. It is well known that cellular and viral microRNAs (miRNAs) are involved in modulation of viral and cellular gene expression, as well as immune response, and are considered by the scientific community as possible targets for an effective therapy against viral infections. This regulation may be involved in different levels of infection and clinical symptomatology. We used viral titration techniques, viral kinetics from 24 to 96 hours postinfection (hpi), and analyzed the expression of key proteins related to the miRNA pathway by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The expression of Dicer was different when compared over the course of infection by the distinct YFV genotypes. Drosha expression was similar during infection by YFV genotype 1 or 2, with a decrease in their expression over time and a slight increase in 96 hpi. Ago1, Ago2, and Ago4 showed different levels of expression between the viral genotypes: for YFV genotype 1 infection, Ago1 presented a positive expression, while for YFV genotype 2, it showed a negative expression, when compared with negative controls. We conclude that YFV infection modulates the proteins involved in miRNA biogenesis, which can regulate both viral replication and cellular immune response.

Impact of miR-155 and miR-126 as novel biomarkers on the assessment of disease progression and prognosis in adult T-cell leukemia.

PubMed

Ishihara, Kaori; Sasaki, Daisuke; Tsuruda, Kazuto; Inokuchi, Naoko; Nagai, Kazuhiro; Hasegawa, Hiroo; Yanagihara, Katsunori; Kamihira, Shimeru

2012-12-01

Micro RNAs (miRNAs) provide new insight in the development of cancer, but little is known about their clinical relevance as biomarkers in the assessment of diagnosis, classification, progression and prognosis of various cancers. To explore a potential novel biomarker, we examined the cellular and plasma miRNA profiles in adult T-cell leukemia (ATL) characterized by diverse clinical features. Using CD4-positive cells isolated from 2 non-infected healthy individuals, 3 chronic ATL patients and 3 acute ATL patients, cellular miRNAs were profiled by microarray. The microarray screened 5 miRNAs namely miR-155, let-7g, miR-126, miR-130a and let-7b because of the large difference in their expression in diseased vs. that of healthy controls. The expression levels of before 5 miRNAs re-quantified by reverse transcription quantifiable polymerase chain reaction (RT-qPCR) were not always accordant in cells and plasma. The high and low plasma levels of miR-155 and miR-126 changed with ATL stage. The present study revealed that there is a quantitative discrepancy between cellular and plasma miRNAs. The elevation of plasma miR-155 and the reduction in miR-126 correlated with poor prognosis, indicating their usefulness as a novel biomarker for the assessment of disease stage. Copyright © 2012 Elsevier Ltd. All rights reserved.

Bridging the gap between system and cell: The role of ultra-high field MRI in human neuroscience.

PubMed

Turner, Robert; De Haan, Daniel

2017-01-01

The volume of published research at the levels of systems and cellular neuroscience continues to increase at an accelerating rate. At the same time, progress in psychiatric medicine has stagnated and scientific confidence in cognitive psychology research is under threat due to careless analysis methods and underpowered experiments. With the advent of ultra-high field MRI, with submillimeter image voxels, imaging neuroscience holds the potential to bridge the cellular and systems levels. Use of these accurate and precisely localized quantitative measures of brain activity may go far in providing more secure foundations for psychology, and hence for more appropriate treatment and management of psychiatric illness. However, fundamental issues regarding the construction of testable mechanistic models using imaging data require careful consideration. This chapter summarizes the characteristics of acceptable models of brain function and provides concise descriptions of the relevant types of neuroimaging data that have recently become available. Approaches to data-driven experiments and analyses are described that may lead to more realistic conceptions of the competences of neural assemblages, as they vary across the brain's complex neuroanatomy. © 2017 Elsevier B.V. All rights reserved.

Time-resolved optical imaging provides a molecular snapshot of altered metabolic function in living human cancer cell models

NASA Astrophysics Data System (ADS)

Sud, Dhruv; Zhong, Wei; Beer, David G.; Mycek, Mary-Ann

2006-05-01

A fluorescence lifetime imaging microscopy (FLIM) method was developed and applied to investigate metabolic function in living human normal esophageal (HET-1) and Barrett’s adenocarcinoma (SEG-1) cells. In FLIM, image contrast is based on fluorophore excited state lifetimes, which reflect local biochemistry and molecular activity. Unique FLIM system attributes, including variable ultrafast time gating (≥ 200 ps), wide spectral tunability (337.1 - 960 nm), large temporal dynamic range (≥ 600 ps), and short data acquisition and processing times (15 s), enabled the study of two key molecules consumed at the termini of the oxidative phosphorylation pathway, NADH and oxygen, in living cells under controlled and calibrated environmental conditions. NADH is an endogenous cellular fluorophore detectable in living human tissues that has been shown to be a quantitative biomarker of dysplasia in the esophagus. Lifetime calibration of an oxygen-sensitive, ruthenium-based cellular stain enabled in vivo oxygen level measurements with a resolution of 8 μM over the entire physiological range (1 - 300 μM). Starkly higher intracellular oxygen and NADH levels in living SEG-1 vs. HET-1 cells were detected by FLIM and attributed to altered metabolic pathways in malignant cells.

Correlation of visual in vitro cytotoxicity ratings of biomaterials with quantitative in vitro cell viability measurements.

PubMed

Bhatia, Sujata K; Yetter, Ann B

2008-08-01

Medical devices and implanted biomaterials are often assessed for biological reactivity using visual scores of cell-material interactions. In such testing, biomaterials are assigned cytotoxicity ratings based on visual evidence of morphological cellular changes, including cell lysis, rounding, spreading, and proliferation. For example, ISO 10993 cytotoxicity testing of medical devices allows the use of a visual grading scale. The present study compared visual in vitro cytotoxicity ratings to quantitative in vitro cytotoxicity measurements for biomaterials to determine the level of correlation between visual scoring and a quantitative cell viability assay. Biomaterials representing a spectrum of biological reactivity levels were evaluated, including organo-tin polyvinylchloride (PVC; a known cytotoxic material), ultra-high molecular weight polyethylene (a known non-cytotoxic material), and implantable tissue adhesives. Each material was incubated in direct contact with mouse 3T3 fibroblast cell cultures for 24 h. Visual scores were assigned to the materials using a 5-point rating scale; the scorer was blinded to the material identities. Quantitative measurements of cell viability were performed using a 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay; again, the assay operator was blinded to material identities. The investigation revealed a high degree of correlation between visual cytotoxicity ratings and quantitative cell viability measurements; a Pearson's correlation gave a correlation coefficient of 0.90 between the visual cytotoxicity score and the percent viable cells. An equation relating the visual cytotoxicity score and the percent viable cells was derived. The results of this study are significant for the design and interpretation of in vitro cytotoxicity studies of novel biomaterials.

Probing the stochastic property of endoreduplication in cell size determination of Arabidopsis thaliana leaf epidermal tissue

PubMed Central

2017-01-01

Cell size distribution is highly reproducible, whereas the size of individual cells often varies greatly within a tissue. This is obvious in a population of Arabidopsis thaliana leaf epidermal cells, which ranged from 1,000 to 10,000 μm2 in size. Endoreduplication is a specialized cell cycle in which nuclear genome size (ploidy) is doubled in the absence of cell division. Although epidermal cells require endoreduplication to enhance cellular expansion, the issue of whether this mechanism is sufficient for explaining cell size distribution remains unclear due to a lack of quantitative understanding linking the occurrence of endoreduplication with cell size diversity. Here, we addressed this question by quantitatively summarizing ploidy profile and cell size distribution using a simple theoretical framework. We first found that endoreduplication dynamics is a Poisson process through cellular maturation. This finding allowed us to construct a mathematical model to predict the time evolution of a ploidy profile with a single rate constant for endoreduplication occurrence in a given time. We reproduced experimentally measured ploidy profile in both wild-type leaf tissue and endoreduplication-related mutants with this analytical solution, further demonstrating the probabilistic property of endoreduplication. We next extended the mathematical model by incorporating the element that cell size is determined according to ploidy level to examine cell size distribution. This analysis revealed that cell size is exponentially enlarged 1.5 times every endoreduplication round. Because this theoretical simulation successfully recapitulated experimentally observed cell size distributions, we concluded that Poissonian endoreduplication dynamics and exponential size-boosting are the sources of the broad cell size distribution in epidermal tissue. More generally, this study contributes to a quantitative understanding whereby stochastic dynamics generate steady-state biological heterogeneity. PMID:28926847

Sulfur isotopic constraints from a single enzyme on the cellular to global sulfur cycles

NASA Astrophysics Data System (ADS)

Sim, M. S.; Adkins, J. F.; Sessions, A. L.; Orphan, V. J.; McGlynn, S.

2017-12-01

Since first reported more than a half century ago, sulfur isotope fractionation between sulfate and sulfide has been used as a diagnostic indicator of microbial sulfate reduction, giving added dimensions to the microbial ecological and geochemical studies of the sulfur cycle. A wide range of fractionation has attracted particular attention because it may serve as a potential indicator of environmental or physiological variables such as substrate concentrations or specific respiration rates. In theory, the magnitude of isotope fractionation depends upon the sulfur isotope effect imparted by the involved enzymes and the relative rate of each enzymatic reaction. The former defines the possible range of fractionation quantitatively, while the latter responds to environmental stimuli, providing an underlying rationale for the varying fractionations. The experimental efforts so far have concentrated largely on the latter, the factors affecting the size of fractionation. Recently, however, the direct assessment of intracellular processes emerges as a promising means for the quantitative analysis of microbial sulfur isotope fractionation as a function of environmental or physiological variables. Here, we experimentally determined for the first time the sulfur isotope fractionation during APS reduction, the first reductive step in the dissimilatory sulfate reduction pathway, using the enzyme purified from Desulfovibrio vulgaris Miyazaki. APS reductase carried out the one-step, two-electron reduction of APS to sulfite, without the production of other metabolic intermediates. Nearly identical isotope effects were obtained at two different temperatures, while the rate of APS reduction more than quadrupled with a temperature increase from 20 to 32°C. When placed in context of the linear network model for microbial sulfur isotope fractionation, our finding could provide a new, semi-quantitative constraint on the sulfur cycle at levels from cellular to global.

Divergent synthesis and identification of the cellular targets of deoxyelephantopins

NASA Astrophysics Data System (ADS)

Lagoutte, Roman; Serba, Christelle; Abegg, Daniel; Hoch, Dominic G.; Adibekian, Alexander; Winssinger, Nicolas

2016-08-01

Herbal extracts containing sesquiterpene lactones have been extensively used in traditional medicine and are known to be rich in α,β-unsaturated functionalities that can covalently engage target proteins. Here we report synthetic methodologies to access analogues of deoxyelephantopin, a sesquiterpene lactone with anticancer properties. Using alkyne-tagged cellular probes and quantitative proteomics analysis, we identified several cellular targets of deoxyelephantopin. We further demonstrate that deoxyelephantopin antagonizes PPARγ activity in situ via covalent engagement of a cysteine residue in the zinc-finger motif of this nuclear receptor.

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

Harnessing cell-to-cell variations to probe bacterial structure and biophysics

NASA Astrophysics Data System (ADS)

Cass, Julie A.

Advances in microscopy and biotechnology have given us novel insights into cellular biology and physics. While bacteria were long considered to be relatively unstructured, the development of fluorescence microscopy techniques, and spatially and temporally resolved high-throughput quantitative studies, have uncovered that the bacterial cell is highly organized, and its structure rigorously maintained. In this thesis I will describe our gateTool software, designed to harness cell-to-cell variations to probe bacterial structure, and discuss two exciting aspects of structure that we have employed gateTool to investigate: (i) chromosome organization and the cellular mechanisms for controlling DNA dynamics, and (ii) the study of cell wall synthesis, and how the genes in the synthesis pathway impact cellular shape. In the first project, we develop a spatial and temporal mapping of cell-cycle-dependent chromosomal organization, and use this quantitative map to discover that chromosomal loci segregate from midcell with universal dynamics. In the second project, I describe preliminary time- lapse and snapshot imaging analysis suggesting phentoypical coherence across peptidoglycan synthesis pathways.

Astrobiological complexity with probabilistic cellular automata.

PubMed

Vukotić, Branislav; Ćirković, Milan M

2012-08-01

The search for extraterrestrial life and intelligence constitutes one of the major endeavors in science, but has yet been quantitatively modeled only rarely and in a cursory and superficial fashion. We argue that probabilistic cellular automata (PCA) represent the best quantitative framework for modeling the astrobiological history of the Milky Way and its Galactic Habitable Zone. The relevant astrobiological parameters are to be modeled as the elements of the input probability matrix for the PCA kernel. With the underlying simplicity of the cellular automata constructs, this approach enables a quick analysis of large and ambiguous space of the input parameters. We perform a simple clustering analysis of typical astrobiological histories with "Copernican" choice of input parameters and discuss the relevant boundary conditions of practical importance for planning and guiding empirical astrobiological and SETI projects. In addition to showing how the present framework is adaptable to more complex situations and updated observational databases from current and near-future space missions, we demonstrate how numerical results could offer a cautious rationale for continuation of practical SETI searches.

Elevated osteopontin and thrombospondin expression identifies malignant human breast carcinoma but is not indicative of metastatic status

PubMed Central

Wang-Rodriguez, Jessica; Urquidi, Virginia; Rivard, Amber; Goodison, Steve

2003-01-01

Background Our previous characterization of a human breast tumor metastasis model identified several candidate metastasis genes. The expression of osteopontin (OPN) correlated with the metastatic phenotype, whereas thrombospondin-1 (TSP-1) and tyrosinase-related protein-1 (TYRP-1) correlated with the nonmetastatic phenotype of independent MDA-MB-435 cell lines implanted orthotopically into athymic mice. The aim of the present study was to examine the cellular distribution of these molecules in human breast tissue and to determine whether the relative expression level of these three genes is associated with human breast tumor metastasis. Methods Sixty-eight fresh, frozen specimens including 31 primary infiltrating ductal carcinomas, 22 nodal metastases, 10 fibroadenomas, and five normal breast tissues were evaluated for OPN expression, TSP-1 expression and TYRP-1 expression. Immunohistochemistry was performed to monitor the cellular distribution and to qualitatively assess expression. Quantitative analysis was achieved by enrichment of breast epithelial cells using laser-capture microdissection and subsequent real-time, quantitative PCR. Results The epithelial components of the breast tissue were the source of OPN and TSP-1 expression, whereas TYRP-1 was present in both the epithelial and stromal components. Both OPN and TSP-1 expression were significantly higher in malignant epithelial sources over normal and benign epithelial sources, but no difference in expression levels was evident between primary tumors with or without metastases, nor between primary and metastatic carcinomas. Conclusion Elevated expression of OPN and TSP-1 may play a role in the pathogenesis of breast cancer. The multiplex analysis of these molecules may enhance our ability to diagnose and/or prognosticate human breast malignancy. PMID:12927044

Dynamic imaging of adaptive stress response pathway activation for prediction of drug induced liver injury.

PubMed

Wink, Steven; Hiemstra, Steven W; Huppelschoten, Suzanne; Klip, Janna E; van de Water, Bob

2018-05-01

Drug-induced liver injury remains a concern during drug treatment and development. There is an urgent need for improved mechanistic understanding and prediction of DILI liabilities using in vitro approaches. We have established and characterized a panel of liver cell models containing mechanism-based fluorescent protein toxicity pathway reporters to quantitatively assess the dynamics of cellular stress response pathway activation at the single cell level using automated live cell imaging. We have systematically evaluated the application of four key adaptive stress pathway reporters for the prediction of DILI liability: SRXN1-GFP (oxidative stress), CHOP-GFP (ER stress/UPR response), p21 (p53-mediated DNA damage-related response) and ICAM1 (NF-κB-mediated inflammatory signaling). 118 FDA-labeled drugs in five human exposure relevant concentrations were evaluated for reporter activation using live cell confocal imaging. Quantitative data analysis revealed activation of single or multiple reporters by most drugs in a concentration and time dependent manner. Hierarchical clustering of time course dynamics and refined single cell analysis allowed the allusion of key events in DILI liability. Concentration response modeling was performed to calculate benchmark concentrations (BMCs). Extracted temporal dynamic parameters and BMCs were used to assess the predictive power of sub-lethal adaptive stress pathway activation. Although cellular adaptive responses were activated by non-DILI and severe-DILI compounds alike, dynamic behavior and lower BMCs of pathway activation were sufficiently distinct between these compound classes. The high-level detailed temporal- and concentration-dependent evaluation of the dynamics of adaptive stress pathway activation adds to the overall understanding and prediction of drug-induced liver liabilities.

Quantitative MRI and spectroscopy of bone marrow

PubMed Central

Ruschke, Stefan; Dieckmeyer, Michael; Diefenbach, Maximilian; Franz, Daniela; Gersing, Alexandra S.; Krug, Roland; Baum, Thomas

2017-01-01

Bone marrow is one of the largest organs in the human body, enclosing adipocytes, hematopoietic stem cells, which are responsible for blood cell production, and mesenchymal stem cells, which are responsible for the production of adipocytes and bone cells. Magnetic resonance imaging (MRI) is the ideal imaging modality to monitor bone marrow changes in healthy and pathological states, thanks to its inherent rich soft‐tissue contrast. Quantitative bone marrow MRI and magnetic resonance spectroscopy (MRS) techniques have been also developed in order to quantify changes in bone marrow water–fat composition, cellularity and perfusion in different pathologies, and to assist in understanding the role of bone marrow in the pathophysiology of systemic diseases (e.g. osteoporosis). The present review summarizes a large selection of studies published until March 2017 in proton‐based quantitative MRI and MRS of bone marrow. Some basic knowledge about bone marrow anatomy and physiology is first reviewed. The most important technical aspects of quantitative MR methods measuring bone marrow water–fat composition, fatty acid composition, perfusion, and diffusion are then described. Finally, previous MR studies are reviewed on the application of quantitative MR techniques in both healthy aging and diseased bone marrow affected by osteoporosis, fractures, metabolic diseases, multiple myeloma, and bone metastases. Level of Evidence: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:332–353. PMID:28570033

Increased cortical expression of the zinc transporter SLC39A12 suggests a breakdown in zinc cellular homeostasis as part of the pathophysiology of schizophrenia

PubMed Central

Scarr, Elizabeth; Udawela, Madhara; Greenough, Mark A; Neo, Jaclyn; Suk Seo, Myoung; Money, Tammie T; Upadhyay, Aradhana; Bush, Ashley I; Everall, Ian P; Thomas, Elizabeth A; Dean, Brian

2016-01-01

Our expression microarray studies showed messenger RNA (mRNA) for solute carrier family 39 (zinc transporter), member 12 (SLC39A12) was higher in dorsolateral prefrontal cortex from subjects with schizophrenia (Sz) in comparison with controls. To better understand the significance of these data we ascertained whether SLC39A12 mRNA was altered in a number of cortical regions (Brodmann’s area (BA) 8, 9, 44) from subjects with Sz, in BA 9 from subjects with mood disorders and in rats treated with antipsychotic drugs. In addition, we determined whether inducing the expression of SLC39A12 resulted in an increased cellular zinc uptake. SLC39A12 variant 1 and 2 mRNA was measured using quantitative PCR. Zinc uptake was measured in CHO cells transfected with human SLC39A12 variant 1 and 2. In Sz, compared with controls, SLC39A12 variant 1 and 2 mRNA was higher in all cortical regions studied. The were no differences in levels of mRNA for either variant of SLC39A12 in BA 9 from subjects with mood disorders and levels of mRNA for Slc39a12 was not different in the cortex of rats treated with antipsychotic drugs. Finally, expressing both variants in CHO-K1 cells was associated with an increase in radioactive zinc uptake. As increased levels of murine Slc39a12 mRNA has been shown to correlate with increasing cellular zinc uptake, our data would be consistent with the possibility of a dysregulated zinc homeostasis in the cortex of subjects with schizophrenia due to altered expression of SLC39A12. PMID:27336053

A draft map of the mouse pluripotent stem cell spatial proteome

PubMed Central

Christoforou, Andy; Mulvey, Claire M.; Breckels, Lisa M.; Geladaki, Aikaterini; Hurrell, Tracey; Hayward, Penelope C.; Naake, Thomas; Gatto, Laurent; Viner, Rosa; Arias, Alfonso Martinez; Lilley, Kathryn S.

2016-01-01

Knowledge of the subcellular distribution of proteins is vital for understanding cellular mechanisms. Capturing the subcellular proteome in a single experiment has proven challenging, with studies focusing on specific compartments or assigning proteins to subcellular niches with low resolution and/or accuracy. Here we introduce hyperLOPIT, a method that couples extensive fractionation, quantitative high-resolution accurate mass spectrometry with multivariate data analysis. We apply hyperLOPIT to a pluripotent stem cell population whose subcellular proteome has not been extensively studied. We provide localization data on over 5,000 proteins with unprecedented spatial resolution to reveal the organization of organelles, sub-organellar compartments, protein complexes, functional networks and steady-state dynamics of proteins and unexpected subcellular locations. The method paves the way for characterizing the impact of post-transcriptional and post-translational modification on protein location and studies involving proteome-level locational changes on cellular perturbation. An interactive open-source resource is presented that enables exploration of these data. PMID:26754106

In vivo biochemistry: quantifying ion and metabolite levels in individual cells or cultures of yeast.

PubMed

Bermejo, Clara; Ewald, Jennifer C; Lanquar, Viviane; Jones, Alexander M; Frommer, Wolf B

2011-08-15

Over the past decade, we have learned that cellular processes, including signalling and metabolism, are highly compartmentalized, and that relevant changes in metabolic state can occur at sub-second timescales. Moreover, we have learned that individual cells in populations, or as part of a tissue, exist in different states. If we want to understand metabolic processes and signalling better, it will be necessary to measure biochemical and biophysical responses of individual cells with high temporal and spatial resolution. Fluorescence imaging has revolutionized all aspects of biology since it has the potential to provide information on the cellular and subcellular distribution of ions and metabolites with sub-second time resolution. In the present review we summarize recent progress in quantifying ions and metabolites in populations of yeast cells as well as in individual yeast cells with the help of quantitative fluorescent indicators, namely FRET metabolite sensors. We discuss the opportunities and potential pitfalls and the controls that help preclude misinterpretation. © The Authors Journal compilation © 2011 Biochemical Society

Scanning number and brightness yields absolute protein concentrations in live cells: a crucial parameter controlling functional bio-molecular interaction networks.

PubMed

Papini, Christina; Royer, Catherine A

2018-02-01

Biological function results from properly timed bio-molecular interactions that transduce external or internal signals, resulting in any number of cellular fates, including triggering of cell-state transitions (division, differentiation, transformation, apoptosis), metabolic homeostasis and adjustment to changing physical or nutritional environments, amongst many more. These bio-molecular interactions can be modulated by chemical modifications of proteins, nucleic acids, lipids and other small molecules. They can result in bio-molecular transport from one cellular compartment to the other and often trigger specific enzyme activities involved in bio-molecular synthesis, modification or degradation. Clearly, a mechanistic understanding of any given high level biological function requires a quantitative characterization of the principal bio-molecular interactions involved and how these may change dynamically. Such information can be obtained using fluctation analysis, in particular scanning number and brightness, and used to build and test mechanistic models of the functional network to define which characteristics are the most important for its regulation.

Cell fate determination dynamics in bacteria

NASA Astrophysics Data System (ADS)

Kuchina, Anna; Espinar, Lorena; Cagatay, Tolga; Garcia-Ojalvo, Jordi; Suel, Gurol

2010-03-01

The fitness of an organism depends on many processes that serve the purpose to adapt to changing environment in a robust and coordinated fashion. One example of such process is cellular fate determination. In the presence of a variety of alternative responses each cell adopting a particular fate represents a ``choice'' that must be tightly regulated to ensure the best survival strategy for the population taking into account the broad range of possible environmental challenges. We investigated this problem in the model organism B.Subtilis which under stress conditions differentiates terminally into highly resistant spores or initiates an alternative transient state of competence. The dynamics underlying cell fate choice remains largely unknown. We utilize quantitative fluorescent microscopy to track the activities of genes involved in these responses on a single-cell level. We explored the importance of temporal interactions between competing cell fates by re- engineering the differentiation programs. I will discuss how the precise dynamics of cellular ``decision-making'' governed by the corresponding biological circuits may enable cells to adjust to diverse environments and determine survival.

Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics

DOE PAGES

Geng, Tao; Bredeweg, Erin L.; Szymanski, Craig J.; ...

2015-11-04

Here, interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of science applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, this microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression withmore » single hyphal compartment resolution in response to carbon source starvation and exchange experiments. Although the microfluidic device is demonstrated on filamentous fungi, our technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth with applications ranging from bioenergy production to human health.« less

The impact of physiological crowding on the diffusivity of membrane bound proteins.

PubMed

Houser, Justin R; Busch, David J; Bell, David R; Li, Brian; Ren, Pengyu; Stachowiak, Jeanne C

2016-02-21

Diffusion of transmembrane and peripheral membrane-bound proteins within the crowded cellular membrane environment is essential to diverse biological processes including cellular signaling, endocytosis, and motility. Nonetheless we presently lack a detailed understanding of the influence of physiological levels of crowding on membrane protein diffusion. Utilizing quantitative in vitro measurements, here we demonstrate that the diffusivities of membrane bound proteins follow a single linearly decreasing trend with increasing membrane coverage by proteins. This trend holds for homogenous protein populations across a range of protein sizes and for heterogeneous mixtures of proteins of different sizes, such that protein diffusivity is controlled by the total coverage of the surrounding membrane. These results demonstrate that steric exclusion within the crowded membrane environment can fundamentally limit the diffusive rate of proteins, regardless of their size. In cells this "speed limit" could be modulated by changes in local membrane coverage, providing a mechanism for tuning the rate of molecular interaction and assembly.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

PubMed Central

Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

2010-01-01

DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

Correlated receptor transport processes buffer single-cell heterogeneity

PubMed Central

Kallenberger, Stefan M.; Unger, Anne L.; Legewie, Stefan; Lymperopoulos, Konstantinos; Eils, Roland

2017-01-01

Cells typically vary in their response to extracellular ligands. Receptor transport processes modulate ligand-receptor induced signal transduction and impact the variability in cellular responses. Here, we quantitatively characterized cellular variability in erythropoietin receptor (EpoR) trafficking at the single-cell level based on live-cell imaging and mathematical modeling. Using ensembles of single-cell mathematical models reduced parameter uncertainties and showed that rapid EpoR turnover, transport of internalized EpoR back to the plasma membrane, and degradation of Epo-EpoR complexes were essential for receptor trafficking. EpoR trafficking dynamics in adherent H838 lung cancer cells closely resembled the dynamics previously characterized by mathematical modeling in suspension cells, indicating that dynamic properties of the EpoR system are widely conserved. Receptor transport processes differed by one order of magnitude between individual cells. However, the concentration of activated Epo-EpoR complexes was less variable due to the correlated kinetics of opposing transport processes acting as a buffering system. PMID:28945754

Quantitative fluorescence microscopy and image deconvolution.

PubMed

Swedlow, Jason R

2013-01-01

Quantitative imaging and image deconvolution have become standard techniques for the modern cell biologist because they can form the basis of an increasing number of assays for molecular function in a cellular context. There are two major types of deconvolution approaches--deblurring and restoration algorithms. Deblurring algorithms remove blur but treat a series of optical sections as individual two-dimensional entities and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed in this chapter. Image deconvolution in fluorescence microscopy has usually been applied to high-resolution imaging to improve contrast and thus detect small, dim objects that might otherwise be obscured. Their proper use demands some consideration of the imaging hardware, the acquisition process, fundamental aspects of photon detection, and image processing. This can prove daunting for some cell biologists, but the power of these techniques has been proven many times in the works cited in the chapter and elsewhere. Their usage is now well defined, so they can be incorporated into the capabilities of most laboratories. A major application of fluorescence microscopy is the quantitative measurement of the localization, dynamics, and interactions of cellular factors. The introduction of green fluorescent protein and its spectral variants has led to a significant increase in the use of fluorescence microscopy as a quantitative assay system. For quantitative imaging assays, it is critical to consider the nature of the image-acquisition system and to validate its response to known standards. Any image-processing algorithms used before quantitative analysis should preserve the relative signal levels in different parts of the image. A very common image-processing algorithm, image deconvolution, is used to remove blurred signal from an image. There are two major types of deconvolution approaches, deblurring and restoration algorithms. Deblurring algorithms remove blur, but treat a series of optical sections as individual two-dimensional entities, and therefore sometimes mishandle blurred light. Restoration algorithms determine an object that, when convolved with the point-spread function of the microscope, could produce the image data. The advantages and disadvantages of these methods are discussed. Copyright © 1998 Elsevier Inc. All rights reserved.

Critical behavior of subcellular density organization during neutrophil activation and migration.

PubMed

Baker-Groberg, Sandra M; Phillips, Kevin G; Healy, Laura D; Itakura, Asako; Porter, Juliana E; Newton, Paul K; Nan, Xiaolin; McCarty, Owen J T

2015-12-01

Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration.

Critical behavior of subcellular density organization during neutrophil activation and migration

PubMed Central

Baker-Groberg, Sandra M.; Phillips, Kevin G.; Healy, Laura D.; Itakura, Asako; Porter, Juliana E.; Newton, Paul K.; Nan, Xiaolin; McCarty, Owen J.T.

2015-01-01

Physical theories of active matter continue to provide a quantitative understanding of dynamic cellular phenomena, including cell locomotion. Although various investigations of the rheology of cells have identified important viscoelastic and traction force parameters for use in these theoretical approaches, a key variable has remained elusive both in theoretical and experimental approaches: the spatiotemporal behavior of the subcellular density. The evolution of the subcellular density has been qualitatively observed for decades as it provides the source of image contrast in label-free imaging modalities (e.g., differential interference contrast, phase contrast) used to investigate cellular specimens. While these modalities directly visualize cell structure, they do not provide quantitative access to the structures being visualized. We present an established quantitative imaging approach, non-interferometric quantitative phase microscopy, to elucidate the subcellular density dynamics in neutrophils undergoing chemokinesis following uniform bacterial peptide stimulation. Through this approach, we identify a power law dependence of the neutrophil mean density on time with a critical point, suggesting a critical density is required for motility on 2D substrates. Next we elucidate a continuum law relating mean cell density, area, and total mass that is conserved during neutrophil polarization and migration. Together, our approach and quantitative findings will enable investigators to define the physics coupling cytoskeletal dynamics with subcellular density dynamics during cell migration. PMID:26640599

Pulmonary serotonin and histamine in experimental asbestosis

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

Keith, I.M.; Day, R.; Lemaire, S.

1986-03-01

Adult male Wistar rats were treated once with tracheal instillation of 5 mg Crysotile B asbestos fibers in 0.5 ml saline under ketamine/xylaxine anesthesia. Control rats (n = 37) received 0.5 ml saline. Test and control rats were killed at 7 and 14 d., and 1, 3 and 6 mo. post instillation. Serotonin (5-HT) was quantitated in lung tissue homogenate from all rats using HPLC and electrochemical detection. Among rats killed at 1, 3 and 6 mo., lung tissue histamine-o-phthaldialdehyde complex was quantitated using reverse phase HPLC coupled to a fluorometric detector. Furthermore, 5-HT was quantitated in the cytoplasm ofmore » grouped (NEB) and individual (NEC) neuroendocrine cells and in mast cells using formaldehyde-vapor-induced fluorescence and microspectrofluorometry, and mast cell numbers were determined. Test rats had higher pulmonary 5-HT and histamine levels than controls at 1, 3 and 6 mo. Test rats also had higher cellular 5-HT compared to controls in NEB's at 1 mo., but not in NECs, and tended to have higher 5-HT-levels in mast cells at 6 mo. Mast cell numbers were higher among tests at 1 and 3 mo. The authors results suggest that NEBs may contribute to the early asbestos induced rise in 5-HT, and that the major source of 5-HT and histamine is from the increased numbers of mast cells.« less

Statistical parametric mapping of stimuli-evoked changes in quantitative blood flow using extended-focus optical coherence microscopy (Conference Presentation)

NASA Astrophysics Data System (ADS)

Marchand, Paul J.; Bouwens, Arno; Shamaei, Vincent; Nguyen, David; Extermann, Jerome; Bolmont, Tristan; Lasser, Theo

2016-03-01

Magnetic Resonance Imaging has revolutionised our understanding of brain function through its ability to image human cerebral structures non-invasively over the entire brain. By exploiting the different magnetic properties of oxygenated and deoxygenated blood, functional MRI can indirectly map areas undergoing neural activation. Alongside the development of fMRI, powerful statistical tools have been developed in an effort to shed light on the neural pathways involved in processing of sensory and cognitive information. In spite of the major improvements made in fMRI technology, the obtained spatial resolution of hundreds of microns prevents MRI in resolving and monitoring processes occurring at the cellular level. In this regard, Optical Coherence Microscopy is an ideal instrumentation as it can image at high spatio-temporal resolution. Moreover, by measuring the mean and the width of the Doppler spectra of light scattered by moving particles, OCM allows extracting the axial and lateral velocity components of red blood cells. The ability to assess quantitatively total blood velocity, as opposed to classical axial velocity Doppler OCM, is of paramount importance in brain imaging as a large proportion of cortical vascular is oriented perpendicularly to the optical axis. We combine here quantitative blood flow imaging with extended-focus Optical Coherence Microscopy and Statistical Parametric Mapping tools to generate maps of stimuli-evoked cortical hemodynamics at the capillary level.

Epstein-Barr virus growth/latency III program alters cellular microRNA expression

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

Cameron, Jennifer E.; Tulane Cancer Center, Tulane University Health Sciences Center, 1430 Tulane Avenue, SL79, New Orleans, LA 70112; Fewell, Claire

The Epstein-Barr virus (EBV) is associated with lymphoid and epithelial cancers. Initial EBV infection alters lymphocyte gene expression, inducing cellular proliferation and differentiation as the virus transitions through consecutive latency transcription programs. Cellular microRNAs (miRNAs) are important regulators of signaling pathways and are implicated in carcinogenesis. The extent to which EBV exploits cellular miRNAs is unknown. Using micro-array analysis and quantitative PCR, we demonstrate differential expression of cellular miRNAs in type III versus type I EBV latency including elevated expression of miR-21, miR-23a, miR-24, miR-27a, miR-34a, miR-146a and b, and miR-155. In contrast, miR-28 expression was found to be lowermore » in type III latency. The EBV-mediated regulation of cellular miRNAs may contribute to EBV signaling and associated cancers.« less

Transparent metal model study of the use of a cellular growth front to form aligned monotectic composite materials

NASA Technical Reports Server (NTRS)

Kaukler, William F.

1988-01-01

The purpose of this work was to resolve a scientific controversy in the understanding of how second phase particles become aligned during unidirectional growth of a monotectic alloy. A second aspect was to make the first systematic observations of the solidification behavior of a monotectic alloy during cellular growth in-situ. This research provides the first systematic transparent model study of cellular solidification. An interface stability diagram was developed for the planar to cellular transition of the succinonitrile glycerol (SNG) system. A method was developed utilizing Fourier Transform Infrared Spectroscopy which allows quantitative compositional analysis of directionally solidified SNG along the growth axis. To determine the influence of cellular growth front on alignment for directionally solidified monotectic alloys, the planar and cellular growth morphology was observed in-situ for SNG between 8 and 17 percent glycerol and for a range of over two orders of magnitude G/R.

Dynamic systems approaches and levels of analysis in the nervous system

PubMed Central

Parker, David; Srivastava, Vipin

2013-01-01

Various analyses are applied to physiological signals. While epistemological diversity is necessary to address effects at different levels, there is often a sense of competition between analyses rather than integration. This is evidenced by the differences in the criteria needed to claim understanding in different approaches. In the nervous system, neuronal analyses that attempt to explain network outputs in cellular and synaptic terms are rightly criticized as being insufficient to explain global effects, emergent or otherwise, while higher-level statistical and mathematical analyses can provide quantitative descriptions of outputs but can only hypothesize on their underlying mechanisms. The major gap in neuroscience is arguably our inability to translate what should be seen as complementary effects between levels. We thus ultimately need approaches that allow us to bridge between different spatial and temporal levels. Analytical approaches derived from critical phenomena in the physical sciences are increasingly being applied to physiological systems, including the nervous system, and claim to provide novel insight into physiological mechanisms and opportunities for their control. Analyses of criticality have suggested several important insights that should be considered in cellular analyses. However, there is a mismatch between lower-level neurophysiological approaches and statistical phenomenological analyses that assume that lower-level effects can be abstracted away, which means that these effects are unknown or inaccessible to experimentalists. As a result experimental designs often generate data that is insufficient for analyses of criticality. This review considers the relevance of insights from analyses of criticality to neuronal network analyses, and highlights that to move the analyses forward and close the gap between the theoretical and neurobiological levels, it is necessary to consider that effects at each level are complementary rather than in competition. PMID:23386835

Crosstalk-eliminated quantitative determination of aflatoxin B1-induced hepatocellular cancer stem cells based on concurrent monitoring of CD133, CD44, and aldehyde dehydrogenase1.

PubMed

Ju, Hee; Shim, Yumi; Arumugam, Parthasarathy; Song, Joon Myong

2016-01-22

Cancer stem cells (CSCs), known as tumor initiating cells, have become a critically important issue for cancer therapy. Although much research has demonstrated the induction of hepato cellular carcinoma by aflatoxin B1, the formation of hepatocellular CSCs and their quantitative determination is hardly reported. In this work, it was found that hepatocellular CSCs were produced from HepG2 cells by aflatoxin B1-induced mutation, and their amount was quantitatively determined using crosstalk-eliminated multicolor cellular imaging based on quantum dot (Qdot) nanoprobes and an acousto-optical tunable filter (AOTF). Hepatocellular CSCs were acquired via magnetic bead-based sorting and observed using concurrent detection of three different markers: CD133, CD44, and aldehyde dehydrogenase1 (ALDH1). The DNA mutation of HepG2 cells caused by aflatoxin B1 was quantitatively observed via absorbance spectra of aflatoxin B1-8, 9-epoxide-DNA adducts. The percentages of hepatocellular CSCs formed in the entire HepG2 cells were determined to be 9.77±0.65%, 10.9±1.39%, 11.4±1.32%, and 12.8±0.7%, respectively, at 0 μM, 5 μM, 10 μM, and 20 μM of aflatoxin B1. The results matched well with those obtained utilizing flow cytometry. This study demonstrates that aflatoxin mediated mutation induced the conversion of hepatic cancer cell to hepatic CSCs by using a Qdot based constructed multicolor cellular imaging system. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Quantitative imaging assay for NF-κB nuclear translocation in primary human macrophages

PubMed Central

Noursadeghi, Mahdad; Tsang, Jhen; Haustein, Thomas; Miller, Robert F.; Chain, Benjamin M.; Katz, David R.

2008-01-01

Quantitative measurement of NF-κB nuclear translocation is an important research tool in cellular immunology. Established methodologies have a number of limitations, such as poor sensitivity, high cost or dependence on cell lines. Novel imaging methods to measure nuclear translocation of transcriptionally active components of NF-κB are being used but are also partly limited by the need for specialist imaging equipment or image analysis software. Herein we present a method for quantitative detection of NF-κB rel A nuclear translocation, using immunofluorescence microscopy and the public domain image analysis software ImageJ that can be easily adopted for cellular immunology research without the need for specialist image analysis expertise and at low cost. The method presented here is validated by demonstrating the time course and dose response of NF-κB nuclear translocation in primary human macrophages stimulated with LPS, and by comparison with a commercial NF-κB activation reporter cell line. PMID:18036607

MIiSR: Molecular Interactions in Super-Resolution Imaging Enables the Analysis of Protein Interactions, Dynamics and Formation of Multi-protein Structures.

PubMed

Caetano, Fabiana A; Dirk, Brennan S; Tam, Joshua H K; Cavanagh, P Craig; Goiko, Maria; Ferguson, Stephen S G; Pasternak, Stephen H; Dikeakos, Jimmy D; de Bruyn, John R; Heit, Bryan

2015-12-01

Our current understanding of the molecular mechanisms which regulate cellular processes such as vesicular trafficking has been enabled by conventional biochemical and microscopy techniques. However, these methods often obscure the heterogeneity of the cellular environment, thus precluding a quantitative assessment of the molecular interactions regulating these processes. Herein, we present Molecular Interactions in Super Resolution (MIiSR) software which provides quantitative analysis tools for use with super-resolution images. MIiSR combines multiple tools for analyzing intermolecular interactions, molecular clustering and image segmentation. These tools enable quantification, in the native environment of the cell, of molecular interactions and the formation of higher-order molecular complexes. The capabilities and limitations of these analytical tools are demonstrated using both modeled data and examples derived from the vesicular trafficking system, thereby providing an established and validated experimental workflow capable of quantitatively assessing molecular interactions and molecular complex formation within the heterogeneous environment of the cell.

A Review: Some biological effects of high LET radiations

NASA Technical Reports Server (NTRS)

Wiley, A., Jr.

1972-01-01

There are qualitative and quantitative differences in the biological damage observed after exposure to high LET radiation as compared to that caused by low LET radiations. This review is concerned with these differences, which are ultimately reflected at the biochemical, cellular and even whole animal levels. In general, high LET radiations seem to produce biochemical damage which is more severe and possibly less repairable. Experimental data for those effects are presented in terms of biochemical RBE's with consideration of both early and late manifestations. An LET independent process by which significant biochemical damage may result from protons, neutrons and negative pion mesons is discussed.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts.

PubMed

Milovancev, Milan; Hilgart-Martiszus, Ian; McNamara, Michael J; Goodall, Cheri P; Seguin, Bernard; Bracha, Shay; Wickramasekara, Samanthi I

2013-06-13

Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis.

System-level modeling of acetone-butanol-ethanol fermentation.

PubMed

Liao, Chen; Seo, Seung-Oh; Lu, Ting

2016-05-01

Acetone-butanol-ethanol (ABE) fermentation is a metabolic process of clostridia that produces bio-based solvents including butanol. It is enabled by an underlying metabolic reaction network and modulated by cellular gene regulation and environmental cues. Mathematical modeling has served as a valuable strategy to facilitate the understanding, characterization and optimization of this process. In this review, we highlight recent advances in system-level, quantitative modeling of ABE fermentation. We begin with an overview of integrative processes underlying the fermentation. Next we survey modeling efforts including early simple models, models with a systematic metabolic description, and those incorporating metabolism through simple gene regulation. Particular focus is given to a recent system-level model that integrates the metabolic reactions, gene regulation and environmental cues. We conclude by discussing the remaining challenges and future directions towards predictive understanding of ABE fermentation. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

High-throughput, quantitative assessment of the effects of low-dose silica nanoparticles on lung cells: grasping complex toxicity with a great depth of field.

PubMed

Pisani, Cédric; Gaillard, Jean-Charles; Nouvel, Virginie; Odorico, Michaël; Armengaud, Jean; Prat, Odette

2015-04-18

The toxicity of manufactured fumed silica nanoparticles (NPs) remains poorly investigated compared to that of crystalline silica NPs, which have been associated with lung diseases after inhalation. Amorphous silica NPs are a raw material for manufactured nanocomposites, such as cosmetics, foods, and drugs, raising concerns about their potential toxicity. The size of the NPs was determined by dynamic light scattering and their shape was visualized by atomic force microscopy (10 ± 4 nm). The pertinent toxicological concentration and dynamic ranges were determined using viability tests and cellular impedance. We combined transcriptomics and proteomics to assess the cellular and molecular effects of fumed silica in A549 human alveolar epithelial cells. The "no observed transcriptomic adverse effect level" (NOTEL) was set to 1.0 μg/cm(2), and the "lowest observed adverse transcriptional effect level" (LOTEL) was set at 1.5 μg/cm(2). We carried out genome-wide expression profiles with microarrays and identified, by shotgun proteomics, the exoproteome changes in lung cells after exposure to NP doses (0.1, 1.0, 1.5, 3.0, and 6.0 μg/cm(2)) at two time points (24 h and 72 h). The data revealed a hierarchical, dose-dependent cellular response to silica NPs. At 1.5 μg/cm(2), the Rho signaling cascade, actin cytoskeleton remodeling, and clathrin-mediated endocytosis were induced. At 3.0 μg/cm(2), many inflammatory mediators were upregulated and the coagulation system pathway was triggered. Lastly, at 6.0 μg/cm(2), oxidative stress was initiated. The proteins identified in the extracellular compartment were consistent with these findings. The alliance of two high-throughput technologies allowed the quantitative assessment of the cellular effects and molecular consequences of exposure of lung cells to low doses of NPs. These results were obtained using a pathway-driven analysis instead of isolated genes. As in photography, toxicogenomics allows, at the same time, the visualization of a wide spectrum of biological responses and a "zoom in" to the details with a great depth of field. This study illustrates how such an approach based on human cell culture models is a valuable predictive screening tool to evaluate the toxicity of many potentially harmful emerging substances, alone or in mixtures, in the framework of future regulatory reinforcements.

Estimating cellular parameters through optimization procedures: elementary principles and applications.

PubMed

Kimura, Akatsuki; Celani, Antonio; Nagao, Hiromichi; Stasevich, Timothy; Nakamura, Kazuyuki

2015-01-01

Construction of quantitative models is a primary goal of quantitative biology, which aims to understand cellular and organismal phenomena in a quantitative manner. In this article, we introduce optimization procedures to search for parameters in a quantitative model that can reproduce experimental data. The aim of optimization is to minimize the sum of squared errors (SSE) in a prediction or to maximize likelihood. A (local) maximum of likelihood or (local) minimum of the SSE can efficiently be identified using gradient approaches. Addition of a stochastic process enables us to identify the global maximum/minimum without becoming trapped in local maxima/minima. Sampling approaches take advantage of increasing computational power to test numerous sets of parameters in order to determine the optimum set. By combining Bayesian inference with gradient or sampling approaches, we can estimate both the optimum parameters and the form of the likelihood function related to the parameters. Finally, we introduce four examples of research that utilize parameter optimization to obtain biological insights from quantified data: transcriptional regulation, bacterial chemotaxis, morphogenesis, and cell cycle regulation. With practical knowledge of parameter optimization, cell and developmental biologists can develop realistic models that reproduce their observations and thus, obtain mechanistic insights into phenomena of interest.

Light scattering application for quantitative estimation of apoptosis

NASA Astrophysics Data System (ADS)

Bilyy, Rostyslav O.; Stoika, Rostyslav S.; Getman, Vasyl B.; Bilyi, Olexander I.

2004-05-01

Estimation of cell proliferation and apoptosis are in focus of instrumental methods used in modern biomedical sciences. Present study concerns monitoring of functional state of cells, specifically the development of their programmed death or apoptosis. The available methods for such purpose are either very expensive, or require time-consuming operations. Their specificity and sensitivity are frequently not sufficient for making conclusions which could be used in diagnostics or treatment monitoring. We propose a novel method for apoptosis measurement based on quantitative determination of cellular functional state taking into account their physical characteristics. This method uses the patented device -- laser microparticle analyser PRM-6 -- for analyzing light scattering by the microparticles, including cells. The method gives an opportunity for quick, quantitative, simple (without complicated preliminary cell processing) and relatively cheap measurement of apoptosis in cellular population. The elaborated method was used for studying apoptosis expression in murine leukemia cells of L1210 line and human lymphoblastic leukemia cells of K562 line. The results obtained by the proposed method permitted measuring cell number in tested sample, detecting and quantitative characterization of functional state of cells, particularly measuring the ratio of the apoptotic cells in suspension.

Transcriptional Response of Human Cells to Microbeam Irradiation with 2.1 MeV Alpha Particles

NASA Astrophysics Data System (ADS)

Hellweg, C. E.; Bogner, S.; Spitta, L.; Arenz, A.; Baumstark-Khan, C.; Greif, K. D.; Giesen, U.

Within the next decades an increasing number of human beings in space will be simultaneously exposed to different stimuli especially microgravity and radiation To assess the risks for humans during long-duration space missions the complex interplay of these parameters at the cellular level must be understood Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors Activation of the Nuclear Factor kappa B NF- kappa B pathway as a possible anti-apoptotic route represents such an important cellular stress response A screening assay for detection of NF- kappa B-dependent gene activation using the destabilized variant of Enhanced Green Fluorescent Protein d2EGFP as reporter protein had been developed It consists of Human Embryonic Kidney HEK 293 Cells stably transfected with a receptor-reporter-construct carrying d2EGFP under the control of a NF- kappa B response element Clones positive for Tumor Necrosis Factor alpha TNF- alpha inducible d2EGFP expression were selected as cellular reporters Irradiation was performed either with X-rays 150 kV 19 mA at DLR Cologne or with 2 1 MeV alpha particles LET sim 160 keV mu m at PTB Braunschweig After irradiation the following biological endpoints were determined i cell survival via the colony forming ability test ii time-dependent activation of NF- kappa B dependent d2EGFP gene expression using flow cytometry iii quantitative RT-PCR

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay.

PubMed

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M

2011-10-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin.

Nuclear protein accumulation in cellular senescence and organismal aging revealed with a novel single-cell resolution fluorescence microscopy assay

PubMed Central

De Cecco, Marco; Jeyapalan, Jessie; Zhao, Xiaoai; Tamamori-Adachi, Mimi; Sedivy, John M.

2011-01-01

Replicative cellular senescence was discovered some 50 years ago. The phenotypes of senescent cells have been investigated extensively in cell culture, and found to affect essentially all aspects of cellular physiology. The relevance of cellular senescence in the context of age-associated pathologies as well as normal aging is a topic of active and ongoing interest. Considerable effort has been devoted to biomarker discovery to enable the microscopic detection of single senescent cells in tissues. One characteristic of senescent cells documented very early in cell culture studies was an increase in cell size and total protein content, but whether this occurs in vivo is not known. A limiting factor for studies of protein content and localization has been the lack of suitable fluorescence microscopy tools. We have developed an easy and flexible method, based on the merocyanine dye known as NanoOrange, to visualize and quantitatively measure total protein levels by high resolution fluorescence microscopy. NanoOrange staining can be combined with antibody-based immunofluorescence, thus providing both specific target and total protein information in the same specimen. These methods are optimally combined with automated image analysis platforms for high throughput analysis. We document here increasing protein content and density in nuclei of senescent human and mouse fibroblasts in vitro, and in liver nuclei of aged mice in vivo. Additionally, in aged liver nuclei NanoOrange revealed protein-dense foci that colocalize with centromeric heterochromatin. PMID:22006542

Mammalian synthetic biology for studying the cell

PubMed Central

Mathur, Melina; Xiang, Joy S.

2017-01-01

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

PubMed Central

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. PMID:21731697

Quantitative regulation of bone-mimetic, oriented collagen/apatite matrix structure depends on the degree of osteoblast alignment on oriented collagen substrates.

PubMed

Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Nakano, Takayoshi

2015-02-01

Bone tissue has a specific anisotropic morphology derived from collagen fiber alignment and the related apatite crystal orientation as a bone quality index. However, the precise mechanism of cellular regulation of the crystallographic orientation of apatite has not been clarified. In this study, anisotropic construction of cell-produced mineralized matrix in vitro was established by initiating organized cellular alignment and subsequent oriented bone-like matrix (collagen/apatite) production. The oriented collagen substrates with three anisotropic levels were prepared by a hydrodynamic method. Primary osteoblasts were cultured on the fabricated substrates until mineralized matrix formation is confirmed. Osteoblast alignment was successfully regulated by the level of substrate collagen orientation, with preferential alignment along the direction of the collagen fibers. Notably, both fibrous orientation of newly synthesized collagen matrix and c-axis of produced apatite crystals showed preferential orientation along the cell direction. Because the degree of anisotropy of the deposited apatite crystals showed dependency on the directional distribution of osteoblasts cultured on the oriented collagen substrates, the cell orientation determines the crystallographic anisotropy of produced apatite crystals. To the best of our knowledge, this is the first report demonstrating that bone tissue anisotropy, even the alignment of apatite crystals, is controllable by varying the degree of osteoblast alignment via regulating the level of substrate orientation. © 2014 Wiley Periodicals, Inc.

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.

Elucidation of the Ebola virus VP24 cellular interactome and disruption of virus biology through targeted inhibition of host-cell protein function.

PubMed

García-Dorival, Isabel; Wu, Weining; Dowall, Stuart; Armstrong, Stuart; Touzelet, Olivier; Wastling, Jonathan; Barr, John N; Matthews, David; Carroll, Miles; Hewson, Roger; Hiscox, Julian A

2014-11-07

Viral pathogenesis in the infected cell is a balance between antiviral responses and subversion of host-cell processes. Many viral proteins specifically interact with host-cell proteins to promote virus biology. Understanding these interactions can lead to knowledge gains about infection and provide potential targets for antiviral therapy. One such virus is Ebola, which has profound consequences for human health and causes viral hemorrhagic fever where case fatality rates can approach 90%. The Ebola virus VP24 protein plays a critical role in the evasion of the host immune response and is likely to interact with multiple cellular proteins. To map these interactions and better understand the potential functions of VP24, label-free quantitative proteomics was used to identify cellular proteins that had a high probability of forming the VP24 cellular interactome. Several known interactions were confirmed, thus placing confidence in the technique, but new interactions were also discovered including one with ATP1A1, which is involved in osmoregulation and cell signaling. Disrupting the activity of ATP1A1 in Ebola-virus-infected cells with a small molecule inhibitor resulted in a decrease in progeny virus, thus illustrating how quantitative proteomics can be used to identify potential therapeutic targets.

Accurate Construction of Photoactivated Localization Microscopy (PALM) Images for Quantitative Measurements

PubMed Central

Coltharp, Carla; Kessler, Rene P.; Xiao, Jie

2012-01-01

Localization-based superresolution microscopy techniques such as Photoactivated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) have allowed investigations of cellular structures with unprecedented optical resolutions. One major obstacle to interpreting superresolution images, however, is the overcounting of molecule numbers caused by fluorophore photoblinking. Using both experimental and simulated images, we determined the effects of photoblinking on the accurate reconstruction of superresolution images and on quantitative measurements of structural dimension and molecule density made from those images. We found that structural dimension and relative density measurements can be made reliably from images that contain photoblinking-related overcounting, but accurate absolute density measurements, and consequently faithful representations of molecule counts and positions in cellular structures, require the application of a clustering algorithm to group localizations that originate from the same molecule. We analyzed how applying a simple algorithm with different clustering thresholds (tThresh and dThresh) affects the accuracy of reconstructed images, and developed an easy method to select optimal thresholds. We also identified an empirical criterion to evaluate whether an imaging condition is appropriate for accurate superresolution image reconstruction with the clustering algorithm. Both the threshold selection method and imaging condition criterion are easy to implement within existing PALM clustering algorithms and experimental conditions. The main advantage of our method is that it generates a superresolution image and molecule position list that faithfully represents molecule counts and positions within a cellular structure, rather than only summarizing structural properties into ensemble parameters. This feature makes it particularly useful for cellular structures of heterogeneous densities and irregular geometries, and allows a variety of quantitative measurements tailored to specific needs of different biological systems. PMID:23251611

Collective synchronization of self/non-self discrimination in T cell activation, across multiple spatio-temporal scales

NASA Astrophysics Data System (ADS)

Altan-Bonnet, Gregoire

The immune system is a collection of cells whose function is to eradicate pathogenic infections and malignant tumors while protecting healthy tissues. Recent work has delineated key molecular and cellular mechanisms associated with the ability to discriminate self from non-self agents. For example, structural studies have quantified the biophysical characteristics of antigenic molecules (those prone to trigger lymphocyte activation and a subsequent immune response). However, such molecular mechanisms were found to be highly unreliable at the individual cellular level. We will present recent efforts to build experimentally validated computational models of the immune responses at the collective cell level. Such models have become critical to delineate how higher-level integration through nonlinear amplification in signal transduction, dynamic feedback in lymphocyte differentiation and cell-to-cell communication allows the immune system to enforce reliable self/non-self discrimination at the organism level. In particular, we will present recent results demonstrating how T cells tune their antigen discrimination according to cytokine cues, and how competition for cytokine within polyclonal populations of cells shape the repertoire of responding clones. Additionally, we will present recent theoretical and experimental results demonstrating how competition between diffusion and consumption of cytokines determine the range of cell-cell communications within lymphoid organs. Finally, we will discuss how biochemically explicit models, combined with quantitative experimental validation, unravel the relevance of new feedbacks for immune regulations across multiple spatial and temporal scales.

Electron Paramagnetic Resonance Oximetry as a Quantitative Method to Measure Cellular Respiration: A Consideration of Oxygen Diffusion Interference

PubMed Central

Presley, Tennille; Kuppusamy, Periannan; Zweier, Jay L.; Ilangovan, Govindasamy

2006-01-01

Electron paramagnetic resonance (EPR) oximetry is being widely used to measure the oxygen consumption of cells, mitochondria, and submitochondrial particles. However, further improvement of this technique, in terms of data analysis, is required to use it as a quantitative tool. Here, we present a new approach for quantitative analysis of cellular respiration using EPR oximetry. The course of oxygen consumption by cells in suspension has been observed to have three distinct zones: pO2-independent respiration at higher pO2 ranges, pO2-dependent respiration at low pO2 ranges, and a static equilibrium with no change in pO2 at very low pO2 values. The approach here enables one to comprehensively analyze all of the three zones together—where the progression of O2 diffusion zones around each cell, their overlap within time, and their potential impact on the measured pO2 data are considered. The obtained results agree with previously established methods such as high-resolution respirometry measurements. Additionally, it is also demonstrated how the diffusion limitations can depend on cell density and consumption rate. In conclusion, the new approach establishes a more accurate and meaningful model to evaluate the EPR oximetry data on cellular respiration to quantify related parameters using EPR oximetry. PMID:17012319

Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

PubMed

Di Pietro, C; Piro, S; Tabbì, G; Ragusa, M; Di Pietro, V; Zimmitti, V; Cuda, F; Anello, M; Consoli, U; Salinaro, E T; Caruso, M; Vancheri, C; Crimi, N; Sabini, M G; Cirrone, G A P; Raffaele, L; Privitera, G; Pulvirenti, A; Giugno, R; Ferro, A; Cuttone, G; Lo Nigro, S; Purrello, R; Purrello, F; Purrello, M

2006-01-01

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

Matrix metalloproteinase 3 promotes cellular anti-dengue virus response via interaction with transcription factor NFκB in cell nucleus.

PubMed

Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

2014-01-01

Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation.

EmbryoMiner: A new framework for interactive knowledge discovery in large-scale cell tracking data of developing embryos.

PubMed

Schott, Benjamin; Traub, Manuel; Schlagenhauf, Cornelia; Takamiya, Masanari; Antritter, Thomas; Bartschat, Andreas; Löffler, Katharina; Blessing, Denis; Otte, Jens C; Kobitski, Andrei Y; Nienhaus, G Ulrich; Strähle, Uwe; Mikut, Ralf; Stegmaier, Johannes

2018-04-01

State-of-the-art light-sheet and confocal microscopes allow recording of entire embryos in 3D and over time (3D+t) for many hours. Fluorescently labeled structures can be segmented and tracked automatically in these terabyte-scale 3D+t images, resulting in thousands of cell migration trajectories that provide detailed insights to large-scale tissue reorganization at the cellular level. Here we present EmbryoMiner, a new interactive open-source framework suitable for in-depth analyses and comparisons of entire embryos, including an extensive set of trajectory features. Starting at the whole-embryo level, the framework can be used to iteratively focus on a region of interest within the embryo, to investigate and test specific trajectory-based hypotheses and to extract quantitative features from the isolated trajectories. Thus, the new framework provides a valuable new way to quantitatively compare corresponding anatomical regions in different embryos that were manually selected based on biological prior knowledge. As a proof of concept, we analyzed 3D+t light-sheet microscopy images of zebrafish embryos, showcasing potential user applications that can be performed using the new framework.

Spiny Nanorod and Upconversion Nanoparticle Satellite Assemblies for Ultrasensitive Detection of Messenger RNA in Living Cells.

PubMed

Gao, Rui; Hao, Changlong; Xu, Liguang; Xu, Chuanlai; Kuang, Hua

2018-04-17

Quantitation and in situ monitoring of target mRNA (mRNA) in living cells remains a significant challenge for the chemical and biomedical communities. To quantitatively detect mRNA expression levels in living cells, we have developed DNA-driven gold nanorod coated platinum-upconversion nanoparticle satellite assemblies (termed Au NR@Pt-UCNP satellites) for intracellular thymidine kinase 1 (TK1) mRNA analysis. The nanostructures were capable of recognizing target mRNA in a sequence-specific manner as luminescence of UCNPs was effectively quenched by Au NR@Pt within the assemblies. Following recognition, UCNPs detached from Au NR@Pt, resulting in luminescence restoration to achieve effective in situ imaging and quantifiable detection of target mRNA. The upconversional luminescence intensity of confocal images showed a good linear relationship with intracellular TK1 mRNA ranging from 1.17 to 65.21 fmol/10 μg RNA and a limit of detection (LOD) of 0.67 fmol/10 μg RNA. We believe that our present assay can be broadly applied for detection of endogenous biomolecules at the cellular and tissue levels and restoration of tissue homeostasis in vivo.

Differential cellular responses by oncogenic levels of c-Myc expression in long-term confluent retinal pigment epithelial cells.

PubMed

Wang, Yiping; Cheng, Xiangdong; Samma, Muhammad Kaleem; Kung, Sam K P; Lee, Clement M; Chiu, Sung Kay

2018-06-01

c-Myc is a highly pleiotropic transcription factor known to control cell cycle progression, apoptosis, and cellular transformation. Normally, ectopic expression of c-Myc is associated with promoting cell proliferation or triggering cell death via activating p53. However, it is not clear how the levels of c-Myc lead to different cellular responses. Here, we generated a series of stable RPE cell clones expressing c-Myc at different levels, and found that consistent low level of c-Myc induced cellular senescence by activating AP4 in post-confluent RPE cells, while the cells underwent cell death at high level of c-Myc. In addition, high level of c-Myc could override the effect of AP4 on cellular senescence. Further knockdown of AP4 abrogated senescence-like phenotype in cells expressing low level of c-Myc, and accelerated cell death in cells with medium level of c-Myc, indicating that AP4 was required for cellular senescence induced by low level of c-Myc.

Lack of association between increased mitochondrial DNA4977 deletion and ATP levels of sputum cells from chronic obstructive pulmonary disease patients versus healthy smokers.

PubMed

Karimova, A; Oltulu, Y M; Azaklı, H; Kara, M; Ustek, D; Tutluoglu, B; Onaran, I

2017-05-01

In this study we looked at smokers with and without chronic obstructive pulmonary disease (COPD) patients in order to evaluate the incidence of 4977 base pair (bp) mtDNA (mtDNA 4977 ) deletion and mtDNA copy number in sputum cells and in peripheral blood leukocytes (PBLs) in relation to mitochondrial function and oxidative stress status. Twenty-five COPD patients who were current smokers, 22 smokers and 23 healthy nonsmokers (for only PBLs studies) participated in this study. The 4977-bp deletion was detected in all examined samples within 40 cyles of PCR amplification, using a quantitative real time PCR. The frequency of the mtDNA 4977 was significantly higher in the sputum cells of patients with COPD compared to smokers without COPD (p < 0.0001). This difference was not observed in PBLs. Levels of cellular oxidative stress were significantly higher in the sputum cells of subjects with COPD than in the smoker group. However, mtDNA copy number, mitochondrial membrane potential (ΔΨm) and cellular ATP levels in PBLs and sputum cells were not significantly different between the studied groups. The Pearson analysis revealed no correlations between the accumulation of mtDNA 4977 , and intracellular ATP content and ΔΨm values of the sputum cells, although there was a positive correlation between the increase in the percentage of deleted mtDNA 4977 and the levels of cellular oxidative stress in COPD patients (r = 0.80, p < 0.0001). Our studies may suggest that the accumulation of mtDNA 4977 in the sputum cells of smokers with COPD does not seem to have an important impact on mitochondrial dysfunction in relation to ATP production and ΔΨm when compared to those of healthy smokers.

Quantitative characterization of 3D deformations of cell interactions with soft biomaterials

NASA Astrophysics Data System (ADS)

Franck, Christian

In recent years, the importance of mechanical forces in directing cellular function has been recognized as a significant factor in biological and physiological processes. In fact, these physical forces are now viewed equally as important as biochemical stimuli in controlling cellular response. Not only do these cellular forces, or cell tractions, play an important role in cell migration, they are also significant to many other physiological and pathological processes, both at the tissue and organ level, including wound healing, inflammation, angiogenesis, and embryogenesis. A complete quantification of cell tractions during cell-material interactions can lead to a deeper understanding of the fundamental role these forces play in cell biology. Thus, understanding the function and role of a cell from a mechanical framework can have important implications towards the development of new implant materials and drug treatments. Previous research has contributed significant descriptions of cell-tissue interactions by quantifying cell tractions in two-dimensional environments; however, most physiological processes are three-dimensional in nature. Recent studies have shown morphological differences in cells cultured on two-dimensional substrates versus three-dimensional matrices, and that the intrinsic extracellular matrix interactions and migration behavior are different in three dimensions versus two dimensions. Hence, measurement techniques are needed to investigate cellular behavior in all three dimensions. This thesis presents a full-field imaging technique capable of quantitatively measuring cell traction forces in all three spatial dimensions, and hence addresses the need of a three-dimensional quantitative imaging technique to gain insight into the fundamental role of physical forces in biological processes. The technique combines laser scanning confocal microscopy (LSCM) with digital volume correlation (DVC) to track the motion of fluorescent particles during cell-induced or externally applied deformations. This method is validated by comparing experimentally measured non-uniform deformation fields near hard and soft spherical inclusions under uniaxial compression with the corresponding analytical solution. Utilization of a newly developed computationally efficient stretch-correlation and deconvolution algorithm is shown to improve the overall measurement accuracy, in particular under large deformations. Using this technique, the full three-dimensional substrate displacement fields are experimentally determined during the migration of individual fibroblast cells on polyacrylamide gels. This is the first study to show the highly three-dimensional structure of cell-induced displacement and traction fields. These new findings suggest a three-dimensional push-pull cell motility, which differs from the traditional theories based on two-dimensional data. These results provide new insight into the dynamic cell-matrix force exchange or mechanotransduction of migrating cells, and will aid in the development of new three-dimensional cell motility and adhesion models. As this study reveals, the mechanical interactions of cells and their extracellular matrix appear to be highly three-dimensional. It also shows that the LSCM-DVC technique is well suited for investigating the mechanics of cell-matrix interactions while providing a platform to access detailed information of the intricate biomechanical coupling for many cellular responses. Thus, this method has the capability to provide direct quantitative experimental data showing how cells interact with their surroundings in three dimensions and might stimulate new avenues of scientific thought in understanding the fundamental role physical forces play in regulating cell behavior.

Comparison of reverse transcription-quantitative polymerase chain reaction methods and platforms for single cell gene expression analysis.

PubMed

Fox, Bridget C; Devonshire, Alison S; Baradez, Marc-Olivier; Marshall, Damian; Foy, Carole A

2012-08-15

Single cell gene expression analysis can provide insights into development and disease progression by profiling individual cellular responses as opposed to reporting the global average of a population. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the "gold standard" for the quantification of gene expression levels; however, the technical performance of kits and platforms aimed at single cell analysis has not been fully defined in terms of sensitivity and assay comparability. We compared three kits using purification columns (PicoPure) or direct lysis (CellsDirect and Cells-to-CT) combined with a one- or two-step RT-qPCR approach using dilutions of cells and RNA standards to the single cell level. Single cell-level messenger RNA (mRNA) analysis was possible using all three methods, although the precision, linearity, and effect of lysis buffer and cell background differed depending on the approach used. The impact of using a microfluidic qPCR platform versus a standard instrument was investigated for potential variability introduced by preamplification of template or scaling down of the qPCR to nanoliter volumes using laser-dissected single cell samples. The two approaches were found to be comparable. These studies show that accurate gene expression analysis is achievable at the single cell level and highlight the importance of well-validated experimental procedures for low-level mRNA analysis. Copyright © 2012 Elsevier Inc. All rights reserved.

Specificity of cell–cell adhesion by classical cadherins: Critical role for low-affinity dimerization through β-strand swapping

PubMed Central

Chen, Chien Peter; Posy, Shoshana; Ben-Shaul, Avinoam; Shapiro, Lawrence; Honig, Barry H.

2005-01-01

Cadherins constitute a family of cell-surface proteins that mediate intercellular adhesion through the association of protomers presented from juxtaposed cells. Differential cadherin expression leads to highly specific intercellular interactions in vivo. This cell–cell specificity is difficult to understand at the molecular level because individual cadherins within a given subfamily are highly similar to each other both in sequence and structure, and they dimerize with remarkably low binding affinities. Here, we provide a molecular model that accounts for these apparently contradictory observations. The model is based in part on the fact that cadherins bind to one another by “swapping” the N-terminal β-strands of their adhesive domains. An inherent feature of strand swapping (or, more generally, the domain swapping phenomenon) is that “closed” monomeric conformations act as competitive inhibitors of dimer formation, thus lowering affinities even when the dimer interface has the characteristics of high-affinity complexes. The model describes quantitatively how small affinity differences between low-affinity cadherin dimers are amplified by multiple cadherin interactions to establish large specificity effects at the cellular level. It is shown that cellular specificity would not be observed if cadherins bound with high affinities, thus emphasizing the crucial role of strand swapping in cell–cell adhesion. Numerical estimates demonstrate that the strength of cellular adhesion is extremely sensitive to the concentration of cadherins expressed at the cell surface. We suggest that the domain swapping mechanism is used by a variety of cell-adhesion proteins and that related mechanisms to control affinity and specificity are exploited in other systems. PMID:15937105

Differential KrasV12 protein levels control a switch regulating lung cancer cell morphology and motility

PubMed Central

Schäfer, C.; Mohan, A.; Burford, W.; Driscoll, M. K.; Ludlow, A. T.; Wright, W. E.; Shay, J. W.; Danuser, G.

2016-01-01

Introduction Oncogenic Kras mutations are important drivers of lung cancer development and metastasis. They are known to activate numerous cellular signaling pathways implicated in enhanced proliferation, survival, tumorigenicity and motility during malignant progression. Objectives Most previous studies of Kras in cancer have focused on the comparison of cell states in the absence or presence of oncogenic Kras mutations. Here we show that differential expression of the constitutively active mutation KrasV12 has profound effects on cell morphology and motility that drive metastatic processes. Methods The study relies on lung cancer cell transformation models, patient-derived lung cancer cell lines, and human lung tumor sections combined with molecular biology techniques, live-cell imaging and staining methods. Results Our analysis shows two cell functional states driven by KrasV12 protein levels: a non-motile state associated with high KrasV12 levels and tumorigenicity, and a motile state associated with low KrasV12 levels and cell dissemination. Conversion between the states is conferred by differential activation of a mechano-sensitive double-negative feedback between KrasV12/ERK/Myosin II and matrix-adhesion signaling. KrasV12 expression levels change upon cues such as hypoxia and integrin-mediated cell-matrix adhesion, rendering KrasV12 levels an integrator of micro-environmental signals that translate into cellular function. By live cell imaging of tumor models we observe shedding of mixed high and low KrasV12 expressers forming multi-functional collectives with potentially optimal metastatic properties composed of a highly mobile and a highly tumorigenic unit. Discussion Together these data highlight previously unappreciated roles for the quantitative effects of expression level variation of oncogenic signaling molecules in conferring fundamental alterations in cell function regulation required for cancer progression. PMID:29057096

Reprogramming retinal neurons and standardized quantification of their differentiation in 3-dimensional retinal cultures

PubMed Central

Hiler, Daniel J.; Barabas, Marie E.; Griffiths, Lyra M.; Dyer, Michael A.

2017-01-01

Postmitotic differentiated neurons are among the most difficult cells to reprogram into induced pluripotent stem cells (iPSCs) because they have poor viability when cultured as dissociated cells. Other protocols to reprogram postmitotic neurons have required the inactivation of the p53 tumor suppressor. We describe a method that does not require p53 inactivation and induces reprogramming in cells purified from the retinae of reprogrammable mice in aggregates with wild-type retinal cells. After the first 10 days of reprogramming, the aggregates are then dispersed and plated on irradiated feeder cells to propagate and isolate individual iPSC clones. The reprogramming efficiency of different neuronal populations at any stage of development can be quantitated using this protocol. Reprogramming retinal neurons with this protocol will take 56 days, and these retina-derived iPSCs can undergo retinal differentiation to produce retinae in 34 days. In addition, we describe a quantitative assessment of retinal differentiation from these neuron-derived iPSCs called STEM-RET. The procedure quantitates eye field specification, optic cup formation, and retinal differentiation in 3-dimensional cultures using molecular, cellular and morphological criteria. An advanced level of cell culture experience is required to carry out this protocol. PMID:27658012

Quantitative polymerase chain reaction for transforming growth factor-β applied to a field study of fish health in Chesapeake Bay tributaries

USGS Publications Warehouse

Harms, Craig A.; Ottinger, Christopher A.; Blazer, Vicki S.; Densmore, Christine L.; Pieper, L.H.; Kennedy-Stoskopf, S.

2000-01-01

Fish morbidity and mortality events in Chesapeake Bay tributaries have aroused concern over the health of this important aquatic ecosystem. We applied a recently described method for quantifying mRNA of an immunosuppressive cytokine, transforming growth factor-β (TGF-β), by reverse transcription quantitative-competitive polymerase chain reaction to a field study of fish health in the Chesapeake Basin, and compared the results to those of a traditional cellular immunoassay macrophage bactericidal activity. We selected the white perch (Morone americana) as the sentinel fish species because of its abundance at all of the collection sites. White perch were sampled from Chesapeake Bay tributaries in June, August, and October 1998. Splenic mononuclear cell TGF-β mRNA levels increased and anterior kidney macrophage bactericidal activity decreased, particularly in eastern shore tributaries, from June to August and October. The results of the two assays correlated inversely (Kendall's τ b = -0.600; p = 0.0102). The results indicated both temporal and spatial modulation of white perch immune systems in the Chesapeake Basin, and demonstrated the utility of quantitative PCR for TGF-β as a molecular biomarker for field assessment of teleost fish immune status.

Quantitative analysis of the protein corona on FePt nanoparticles formed by transferrin binding

PubMed Central

Jiang, Xiue; Weise, Stefan; Hafner, Margit; Röcker, Carlheinz; Zhang, Feng; Parak, Wolfgang J.; Nienhaus, G. Ulrich

2010-01-01

Nanoparticles are finding a rapidly expanding range of applications in research and technology, finally entering our daily life in medical, cosmetic or food products. Their ability to invade all regions of an organism including cells and cellular organelles offers new strategies for medical diagnosis and therapy (nanomedicine), but their safe use requires a deep knowledge about their interactions with biological systems at the molecular level. Upon incorporation, nanoparticles are exposed to biological fluids from which they adsorb proteins and other biomolecules to form a ‘protein corona’. These nanoparticle–protein interactions are still poorly understood and quantitative studies to characterize them remain scarce. Here we have quantitatively analysed the adsorption of human transferrin onto small (radius approx. 5 nm) polymer-coated FePt nanoparticles by using fluorescence correlation spectroscopy. Transferrin binds to the negatively charged nanoparticles with an affinity of approximately 26 µM in a cooperative fashion and forms a monolayer with a thickness of 7 nm. By using confocal fluorescence microscopy, we have observed that the uptake of FePt nanoparticles by HeLa cells is suppressed by the protein corona compared with the bare nanoparticles. PMID:19776149

Genetic Complexity and Quantitative Trait Loci Mapping of Yeast Morphological Traits

PubMed Central

Nogami, Satoru; Ohya, Yoshikazu; Yvert, Gaël

2007-01-01

Functional genomics relies on two essential parameters: the sensitivity of phenotypic measures and the power to detect genomic perturbations that cause phenotypic variations. In model organisms, two types of perturbations are widely used. Artificial mutations can be introduced in virtually any gene and allow the systematic analysis of gene function via mutants fitness. Alternatively, natural genetic variations can be associated to particular phenotypes via genetic mapping. However, the access to genome manipulation and breeding provided by model organisms is sometimes counterbalanced by phenotyping limitations. Here we investigated the natural genetic diversity of Saccharomyces cerevisiae cellular morphology using a very sensitive high-throughput imaging platform. We quantified 501 morphological parameters in over 50,000 yeast cells from a cross between two wild-type divergent backgrounds. Extensive morphological differences were found between these backgrounds. The genetic architecture of the traits was complex, with evidence of both epistasis and transgressive segregation. We mapped quantitative trait loci (QTL) for 67 traits and discovered 364 correlations between traits segregation and inheritance of gene expression levels. We validated one QTL by the replacement of a single base in the genome. This study illustrates the natural diversity and complexity of cellular traits among natural yeast strains and provides an ideal framework for a genetical genomics dissection of multiple traits. Our results did not overlap with results previously obtained from systematic deletion strains, showing that both approaches are necessary for the functional exploration of genomes. PMID:17319748

Large-scale protein-protein interaction analysis in Arabidopsis mesophyll protoplasts by split firefly luciferase complementation.

PubMed

Li, Jian-Feng; Bush, Jenifer; Xiong, Yan; Li, Lei; McCormack, Matthew

2011-01-01

Protein-protein interactions (PPIs) constitute the regulatory network that coordinates diverse cellular functions. There are growing needs in plant research for creating protein interaction maps behind complex cellular processes and at a systems biology level. However, only a few approaches have been successfully used for large-scale surveys of PPIs in plants, each having advantages and disadvantages. Here we present split firefly luciferase complementation (SFLC) as a highly sensitive and noninvasive technique for in planta PPI investigation. In this assay, the separate halves of a firefly luciferase can come into close proximity and transiently restore its catalytic activity only when their fusion partners, namely the two proteins of interest, interact with each other. This assay was conferred with quantitativeness and high throughput potential when the Arabidopsis mesophyll protoplast system and a microplate luminometer were employed for protein expression and luciferase measurement, respectively. Using the SFLC assay, we could monitor the dynamics of rapamycin-induced and ascomycin-disrupted interaction between Arabidopsis FRB and human FKBP proteins in a near real-time manner. As a proof of concept for large-scale PPI survey, we further applied the SFLC assay to testing 132 binary PPIs among 8 auxin response factors (ARFs) and 12 Aux/IAA proteins from Arabidopsis. Our results demonstrated that the SFLC assay is ideal for in vivo quantitative PPI analysis in plant cells and is particularly powerful for large-scale binary PPI screens.

Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

DOE PAGES

Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe; ...

2017-04-07

In this study, the increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBAmore » predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.« less

Elucidating dynamic metabolic physiology through network integration of quantitative time-course metabolomics

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

Bordbar, Aarash; Yurkovich, James T.; Paglia, Giuseppe

In this study, the increasing availability of metabolomics data necessitates novel methods for deeper data analysis and interpretation. We present a flux balance analysis method that allows for the computation of dynamic intracellular metabolic changes at the cellular scale through integration of time-course absolute quantitative metabolomics. This approach, termed “unsteady-state flux balance analysis” (uFBA), is applied to four cellular systems: three dynamic and one steady-state as a negative control. uFBA and FBA predictions are contrasted, and uFBA is found to be more accurate in predicting dynamic metabolic flux states for red blood cells, platelets, and Saccharomyces cerevisiae. Notably, only uFBAmore » predicts that stored red blood cells metabolize TCA intermediates to regenerate important cofactors, such as ATP, NADH, and NADPH. These pathway usage predictions were subsequently validated through 13C isotopic labeling and metabolic flux analysis in stored red blood cells. Utilizing time-course metabolomics data, uFBA provides an accurate method to predict metabolic physiology at the cellular scale for dynamic systems.« less

Giardiasis in mice: analysis of humoral and cellular immune responses to Giardia muris.

PubMed

Anders, R F; Roberts-Thomson, I C; Mitchell, G F

1982-01-01

Humoral and cellular immune responses have been evaluated in two inbred strains of mice which differ markedly in their susceptibility to infection with Giardia muris. Serum IgG and IgA antibody levels and IgA levels in intestinal washes were determined by a solid-phase radioimmunoassay using G. muris antigen prepared by sonication of trophozoites, while cell-mediated immunity was assessed by a radiometric ear-assay for delayed-type hypersensitivity. Following infection of BALB/c mice (resistant) and C3H/He mice (susceptible), the IgG and IgA antibody levels in serum progressively increased over the period of study with C3H/He mice having significantly higher titres of IgA antibodies than BALB/c late in the infection. Systemic immunization with G. muris trophozoites resulted in high titres of IgG antibodies in the serum. IgA antibodies were detected in intestinal washes 2 weeks after infection with a subsequent fall in levels in BALB/c mice but a progressive increase levels in C3H/He mice. Prior immunization resulted in IgA antibodies being detected earlier in the intestinal washings after a challenge infection. Delayed-type hypersensitivity to G. muris antigens could not be detected during an infection but a positive response was elicited following antigen priming in mice pretreated with cyclophosphamide. The immune responses evaluated in this study were assessed using a whole G. muris trophozoite sonicate and variations in the quantitative aspects of the responses did not account for observed differences in the course of infection in the two strains of mice.

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. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

MRI-Derived Cellularity Index as a Potential Noninvasive Imaging Biomarker of Prostate Cancer

DTIC Science & Technology

2016-12-01

whole mount pathology sections. Analysis of these UCLA data resulted in four published manuscripts (White et al, Cancer Research 2014; Rakow-Penner...Yamin et al., Clinical Cancer Research , 2016) RSI-MRI mean z-score grouped by pathologic Gleason grade. Mean RSI-MRI cellularity index represented as...Figure 2). A quantitative validation of this approach is now provided in this study. Specifically, in a group of patients with known PCa, ROC

Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

DOE PAGES

Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.; ...

2015-07-06

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less

Identification of Salmonella Typhimurium deubiquitinase SseL substrates by immunoaffinity enrichment and quantitative proteomic analysis

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

Nakayasu, Ernesto S.; Sydor, Michael A.; Brown, Roslyn N.

Ubiquitination is a key protein post-translational modification that regulates many important cellular pathways and whose levels are regulated by equilibrium between the activities of ubiquitin ligases and deubiquitinases. Here we present a method to identify specific deubiquitinase substrates based on treatment of cell lysates with recombinant enzymes, immunoaffinity purification and global quantitative proteomic analysis. As model system to identify substrates, we used a virulence-related deubiquitinase secreted by Salmonella enterica serovar Typhimurium into the host cells, SseL. By using this approach two SseL substrates were identified in RAW 264.7 murine macrophage-like cell line, S100A6 and het-erogeneous nuclear ribonuclear protein K, inmore » addition to the previously reported K63-linked ubiquitin chains. These substrates were further validated by a combination of enzymatic and binding assays. Finally, this method can be used for the systematic identification of substrates of deubiquitinases from other organisms and applied to study their functions in physiology and disease.« less

Single cell model for simultaneous drug delivery and efflux.

PubMed

Yi, C; Saidel, G M; Gratzl, M

1999-01-01

Multidrug resistance (MDR) of some cancer cells is a major challenge for chemotherapy of systemic cancers to overcome. To experimentally uncover the cellular mechanisms leading to MDR, it is necessary to quantitatively assess both drug influx into, and efflux from, the cells exposed to drug treatment. By using a novel molecular microdelivery system to enforce continuous and adjustable drug influx into single cells by controlled diffusion through a gel plug in a micropipet tip, drug resistance studies can now be performed on the single cell level. Our dynamic model of this scheme incorporates drug delivery, diffusive mixing, and accumulation inside the cytoplasm, and efflux by both passive and active membrane transport. Model simulations using available experimental information on these processes can assist in the design of MDR related experiments on single cancer cells which are expected to lead to a quantitative evaluation of mechanisms. Simulations indicate that drug resistance of a cancer cell can be quantified better by its dynamic response than by steady-state analysis.

Overexpression of CaAPX Induces Orchestrated Reactive Oxygen Scavenging and Enhances Cold and Heat Tolerances in Tobacco

PubMed Central

Wang, Jiangying; Wu, Bin; Fan, Zhengqi; Li, Xinlei; Ni, Sui

2017-01-01

Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX (CaAPX) gene from Camellia azalea. Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD, CAT, DHAR, and MDHAR, and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances. PMID:28386551

Immunohistochemistry of catecholamines in the hypothalamic-pituitary-adrenal system with special regard to fatal hypothermia and hyperthermia.

PubMed

Ishikawa, Takaki; Yoshida, Chiemi; Michiue, Tomomi; Perdekamp, Markus Grosse; Pollak, Stefan; Maeda, Hitoshi

2010-05-01

Catecholamines are involved in various stress responses. Previous studies have suggested applicability of the postmortem blood levels to investigations of physical stress responses or toxic/hyperthermic neuronal dysfunction during death process. The present study investigated cellular immunopositivity for adrenaline (Adr), noradrenaline (Nad) and dopamine (DA) in the hypothalamus, adenohypophysis and adrenal medulla with special regard to fatal hypothermia (cold exposure) and hyperthermia (heat stroke) to examine forensic pathological significance. Medicolegal autopsy cases (n=290, within 3 days postmortem) were examined. The proportions of catecholamine (Adr, Nad and DA)-positive cells (% positivity) in each tissue were quantitatively estimated using immunostaining. Hyperthermia cases (n=12) showed a lower neuronal DA-immunopositivity in the hypothalamus than hypothermia cases (n=20), while Nad- and DA-immunopositivities in the adrenal medulla were higher for hyperthermia than for hypothermia. Rates of Nad-immunopositivity in the adrenal medulla were very low for hypothermia. No such difference between hypothermia and hyperthermia was seen in the adenohypophysis. In hypothermia cases, cellular Nad-immunopositivity in the adrenal medulla correlated with the Nad level in cerebrospinal fluid (r=0.591, p<0.01). These observations suggest a characteristic immunohistochemical pattern of systemic stress response to fatal hypothermia and hyperthermia, involving the hypothalamus and adrenal medulla. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

Cidofovir is active against human papillomavirus positive and negative head and neck and cervical tumor cells by causing DNA damage as one of its working mechanisms.

PubMed

Mertens, Barbara; Nogueira, Tatiane; Stranska, Ruzena; Naesens, Lieve; Andrei, Graciela; Snoeck, Robert

2016-07-26

Human papillomavirus (HPV) causes cervical cancer and a large fraction of head and neck squamous cell carcinomas (HNSCC). Cidofovir (CDV) proved efficacious in the treatment of several HPV-induced benign and malignant hyper proliferations. To provide a better insight into how CDV selectively eradicates transformed cells, HPV+ and HPV- cervical carcinoma and HNSCC cell lines were compared to normal cells for antiproliferative effects, CDV metabolism, drug incorporation into cellular DNA, and DNA damage. Incorporation of CDV into cellular DNA was higher in tumor cells than in normal cells and correlated with CDV antiproliferative effects, which were independent of HPV status. Increase in phospho-ATM levels was detected following CDV exposure and higher levels of γ-H2AX (a quantitative marker of double-strand breaks) were measured in tumor cells compared to normal cells. A correlation between DNA damage and CDV incorporation into DNA was found but not between DNA damage and CDV antiproliferative effects. These data indicate that CDV antiproliferative effects result from incorporation of the drug into DNA causing DNA damage. However, the anti-tumor effects of CDV cannot be exclusively ascribed to DNA damage. Furthermore, CDV can be considered a promising broad spectrum anti-cancer agent, not restricted to HPV+ lesions.

Overexpression of CaAPX Induces Orchestrated Reactive Oxygen Scavenging and Enhances Cold and Heat Tolerances in Tobacco.

PubMed

Wang, Jiangying; Wu, Bin; Yin, Hengfu; Fan, Zhengqi; Li, Xinlei; Ni, Sui; He, Libo; Li, Jiyuan

2017-01-01

Ascorbate peroxidase (APX) acts indispensably in synthesizing L-ascorbate (AsA) which is pivotal to plant stress tolerance by detoxifying reactive oxygen species (ROS). Enhanced activity of APX has been shown to be a key step for genetic engineering of improving plant tolerance. However it needs a deeper understanding on the maintenance of cellular ROS homeostasis in response to stress. In this study, we identified and characterized an APX ( CaAPX ) gene from Camellia azalea . Quantitative real-time PCR (qRT-PCR) analysis showed that CaAPX was expressed in all tissues and peaked in immature green fruits; the expression levels were significantly upregulated upon cold and hot stresses. Transgenic plants displayed marked enhancements of tolerance under both cold and heat treatments, and plant growth was correlated with CaAPX expression levels. Furthermore, we monitored the activities of several ROS-scavenging enzymes including Cu/Zn-SOD , CAT , DHAR , and MDHAR , and we showed that stress tolerance was synchronized with elevated activities of ROS-scavenging. Moreover, gene expression analysis of ROS-scavenging enzymes revealed a role of CaAPX to orchestrate ROS signaling in response to temperature stresses. Overall, this study presents a comprehensive characterization of cellular response related to CaAPX expression and provides insights to breed crops with high temperature tolerances.

Multilevel ecotoxicity assessment of environmentally relevant bisphenol A concentrations using the soil invertebrate Eisenia fetida.

PubMed

Babić, Sanja; Barišić, Josip; Bielen, Ana; Bošnjak, Ivana; Sauerborn Klobučar, Roberta; Ujević, Ivana; Strunjak-Perović, Ivančica; Topić Popović, Natalija; Čož-Rakovac, Rozelindra

2016-11-15

Bisphenol A (BPA) presents a serious threat to soil ecosystems, yet its effects on soil-inhabiting organisms are mostly unexplored. Therefore, the impact of environmentally relevant BPA concentrations on a terrestrial model organism, the earthworm Eisenia fetida, was assessed. Animals were cutaneously exposed to 100nM and 10μM BPA up to 10days (10-d). Next, a battery of biomarkers was used for ecotoxicological evaluation on a cellular, tissue and behavioural level. HPLC analysis showed that after a 10-d exposure, BPA accumulation reached a maximum of 2.50μg BPA per g of wet tissue weight. On the cellular level, up to 3-d BPA exposure caused increased lipid oxidation indicating oxidative stress. Histopathological assessment of cell wall and ovaries after 7- and 10-d BPA exposure showed multiple abnormalities, i.e. hyperplasia of epidermis, increased body wall thickness and ovarian atrophy. Detection of these changes was facilitated by a newly proposed semi-quantitative scoring system. Finally, behavioural changes were detected after only 3days of exposure to 100nM BPA. Altogether, the presented multilevel toxicity evaluation indicates high sensitivity of earthworms to low BPA doses. Copyright © 2016 Elsevier B.V. All rights reserved.

Effect of psychological tension on pedestrian counter flow via an extended cost potential field cellular automaton model

NASA Astrophysics Data System (ADS)

Li, Xingli; Guo, Fang; Kuang, Hua; Zhou, Huaguo

2017-12-01

Psychology tells us that the different level of tension may lead to different behavior variation for individuals. In this paper, an extended cost potential field cellular automaton is proposed to simulate pedestrian counter flow under an emergency by considering behavior variation of pedestrian induced by psychological tension. A quantitative formula is introduced to describe behavioral changes caused by psychological tension, which also leads to the increasing cost of discomfort. The numerical simulations are performed under the periodic boundary condition and show that the presented model can capture some essential features of pedestrian counter flow, such as lane formation and segregation phenomenon for normal condition. Furthermore, an interesting feature is found that when pedestrians are in an extremely nervous state, a stable lane formation will be broken by a disordered mixture flow. The psychological nervousness under an emergency is not always negative to moving efficiency and a moderate level of tension will delay the occurrence of jamming phase. In addition, a larger asymmetrical ratio of left walkers to right walkers will improve the critical density related to the jamming phase and retard the occurrence of completely jammed phase. These findings will be helpful in pedestrian control and management under an emergency.

Divergent lactate dehydrogenase isoenzyme profile in cellular compartments of primate forebrain structures.

PubMed

Duka, Tetyana; Collins, Zachary; Anderson, Sarah M; Raghanti, Mary Ann; Ely, John J; Hof, Patrick R; Wildman, Derek E; Goodman, Morris; Grossman, Lawrence I; Sherwood, Chet C

2017-07-01

The compartmentalization and association of lactate dehydrogenase (LDH) with specific cellular structures (e.g., synaptosomal, sarcoplasmic or mitochondrial) may play an important role in brain energy metabolism. Our previous research revealed that LDH in the synaptosomal fraction shifts toward the aerobic isoforms (LDH-B) among the large-brained haplorhine primates compared to strepsirrhines. Here, we further analyzed the subcellular localization of LDH in primate forebrain structures using quantitative Western blotting and ELISA. We show that, in cytosolic and mitochondrial subfractions, LDH-B expression level was relatively elevated and LDH-A declined in haplorhines compared to strepsirrhines. LDH-B expression in mitochondrial fractions of the neocortex was preferentially increased, showing a particularly significant rise in the ratio of LDH-B to LDH-A in chimpanzees and humans. We also found a significant correlation between the protein levels of LDH-B in mitochondrial fractions from haplorhine neocortex and the synaptosomal LDH-B that suggests LDH isoforms shift from a predominance of A-subunits toward B-subunits as part of a system that spatially buffers dynamic energy requirements of brain cells. Our results indicate that there is differential subcellular compartmentalization of LDH isoenzymes that evolved among different primate lineages to meet the energy requirements in neocortical and striatal cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Investigating cellular network heterogeneity and modularity in cancer: a network entropy and unbalanced motif approach.

PubMed

Cheng, Feixiong; Liu, Chuang; Shen, Bairong; Zhao, Zhongming

2016-08-26

Cancer is increasingly recognized as a cellular system phenomenon that is attributed to the accumulation of genetic or epigenetic alterations leading to the perturbation of the molecular network architecture. Elucidation of network properties that can characterize tumor initiation and progression, or pinpoint the molecular targets related to the drug sensitivity or resistance, is therefore of critical importance for providing systems-level insights into tumorigenesis and clinical outcome in the molecularly targeted cancer therapy. In this study, we developed a network-based framework to quantitatively examine cellular network heterogeneity and modularity in cancer. Specifically, we constructed gene co-expressed protein interaction networks derived from large-scale RNA-Seq data across 8 cancer types generated in The Cancer Genome Atlas (TCGA) project. We performed gene network entropy and balanced versus unbalanced motif analysis to investigate cellular network heterogeneity and modularity in tumor versus normal tissues, different stages of progression, and drug resistant versus sensitive cancer cell lines. We found that tumorigenesis could be characterized by a significant increase of gene network entropy in all of the 8 cancer types. The ratio of the balanced motifs in normal tissues is higher than that of tumors, while the ratio of unbalanced motifs in tumors is higher than that of normal tissues in all of the 8 cancer types. Furthermore, we showed that network entropy could be used to characterize tumor progression and anticancer drug responses. For example, we found that kinase inhibitor resistant cancer cell lines had higher entropy compared to that of sensitive cell lines using the integrative analysis of microarray gene expression and drug pharmacological data collected from the Genomics of Drug Sensitivity in Cancer database. In addition, we provided potential network-level evidence that smoking might increase cancer cellular network heterogeneity and further contribute to tyrosine kinase inhibitor (e.g., gefitinib) resistance. In summary, we demonstrated that network properties such as network entropy and unbalanced motifs associated with tumor initiation, progression, and anticancer drug responses, suggesting new potential network-based prognostic and predictive measure in cancer.

Matrix remodeling between cells and cellular interactions with collagen bundle

NASA Astrophysics Data System (ADS)

Kim, Jihan; Sun, Bo

When cells are surrounded by complex environment, they continuously probe and interact with it by applying cellular traction forces. As cells apply traction forces, they can sense rigidity of their local environment and remodel the matrix microstructure simultaneously. Previous study shows that single human carcinoma cell (MDA-MB-231) remodeled its surrounding extracellular matrix (ECM) and the matrix remodeling was reversible. In this study we examined the matrix microstructure between cells and cellular interaction between them using quantitative confocal microscopy. The result shows that the matrix microstructure is the most significantly remodeled between cells consisting of aligned, and densified collagen fibers (collagen bundle)., the result shows that collagen bundle is irreversible and significantly change micromechanics of ECM around the bundle. We further examined cellular interaction with collagen bundle by analyzing dynamics of actin and talin formation along with the direction of bundle. Lastly, we analyzed dynamics of cellular protrusion and migrating direction of cells along the bundle.

Mammalian synthetic biology for studying the cell.

PubMed

Mathur, Melina; Xiang, Joy S; Smolke, Christina D

2017-01-02

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. © 2017 Mathur et al.

Vitamin D endocrine system after short-term space flight

NASA Technical Reports Server (NTRS)

Rhoten, William B. (Principal Investigator); Sergeev, Igor N. (Principal Investigator)

1996-01-01

The exposure of the body to microgravity during space flight causes a series of well-documented changes in Ca(2+) metabolism, yet the cellular/molecular mechanisms leading to these changes are poorly understood. There is some evidence for microgravity-induced alterations in the vitamin D endocrine system, which is known to be primarily involved in the regulation of Ca(2+) metabolism. Vitamin D-dependent Ca(2+) binding proteins, or calbindins, are believed to have a significant role in maintaining cellular Ca(2+) homeostasis. We used immunocytochemical, biochemical and molecular approaches to analyze the expression of calbindin-D(sub 28k) and calbindin-D(sub 9k) in kidneys and intestines of rats flown for 9 days aboard the Spacelab 3 mission. The effects of microgravity on calbindins in rats in space vs. 'grounded' animals (synchronous Animal Enclosure Module controls and tail suspension controls) were compared. Exposure to microgravity resulted in a significant decrease in calbindin-D(sub 28k) content in kidneys and calbindin-D(sub 9k) in the intestine of flight and suspended animals, as measured by enzyme-linked immunosorbent assay (ELISA). Immunocytochemistry (ICC) in combination with quantitative computer image analysis was used to measure in situ the expression of calbindins in kidneys and intestine, and insulin in pancreas. There was a large decrease in the distal tubular cell-associated calbindin-D(sub 28k) and absorptive cell-associated calbindin-D(sub 9k) immunoreactivity in the space and suspension kidneys and intestine, as compared with matched ground controls. No consistent differences in pancreatic insulin immunoreactivity between space, suspension and ground controls was observed. There were significant correlations between results by quantitative ICC and ELISA. Western blot analysis showed no consistent changes in the low levels of intestinal and renal vitamin D receptors. These findings suggest that a decreased expression of calbindins after a short-term exposure to microgravity and modelled weightlessness, may affect cellular Ca(2+) homeostasis and contribute to Ca(2+) and bone metabolism disorders induced by space flight.

Identification of miRNA-103 in the Cellular Fraction of Human Peripheral Blood as a Potential Biomarker for Malignant Mesothelioma – A Pilot Study

PubMed Central

Weber, Daniel G.; Johnen, Georg; Bryk, Oleksandr; Jöckel, Karl-Heinz; Brüning, Thomas

2012-01-01

Background To date, no biomarkers with reasonable sensitivity and specificity for the early detection of malignant mesothelioma have been described. The use of microRNAs (miRNAs) as minimally-invasive biomarkers has opened new opportunities for the diagnosis of cancer, primarily because they exhibit tumor-specific expression profiles and have been commonly observed in blood of both cancer patients and healthy controls. The aim of this pilot study was to identify miRNAs in the cellular fraction of human peripheral blood as potential novel biomarkers for the detection of malignant mesothelioma. Methodology/Principal Findings Using oligonucleotide microarrays for biomarker identification the miRNA levels in the cellular fraction of human peripheral blood of mesothelioma patients and asbestos-exposed controls were analyzed. Using a threefold expression change in combination with a significance level of p<0.05, miR-103 was identified as a potential biomarker for malignant mesothelioma. Quantitative real-time PCR (qRT-PCR) was used for validation of miR-103 in 23 malignant mesothelioma patients, 17 asbestos-exposed controls, and 25 controls from the general population. For discrimination of mesothelioma patients from asbestos-exposed controls a sensitivity of 83% and a specificity of 71% were calculated, and for discrimination of mesothelioma patients from the general population a sensitivity of 78% and a specificity of 76%. Conclusions/Significance The results of this pilot study show that miR-103 is characterized by a promising sensitivity and specificity and might be a potential minimally-invasive biomarker for the diagnosis of mesothelioma. In addition, our results support the concept of using the cellular fraction of human blood for biomarker discovery. However, for early detection of malignant mesothelioma the feasibility of miR-103 alone or in combination with other biomarkers needs to be analyzed in a prospective study. PMID:22253921

[Involvement of cellular immunity and humoral immunity in mixed allergy induced by trichloroethylene].

PubMed

Xu, Xinyun; Li, Xueyu; Liu, Yuefeng

2014-12-01

To investigate whether cellular immunity and humoral immunity are involved in trichlorethylene (TCE)-induced mixed allergy, then provide the scientific basis for the mechanism of this disease. Guinea pigs and rats were tested for this study by application of guinea pig maximization test (GPMT), the animals were randomly divided into negative control, positive control and TCE treatment groups. Animals of these groups were administrated with olive oil, 2, 4-dinitrochlorobenzene (DNCB), and TCE, respectively, by intradermal injection. After TCE administration, rat peripheral blood samples were collected by flow cytometry to detect lymphocytes CD3⁺, CD4⁺, CD8⁺. Guinea pig peripheral blood samples were collected to detect the levels of IgG, IgA, IgM, C3, C4, and the spleens were taken out from guinea pigs after various treatment, mRNA expression of GATA3, T-bet, CTLA4 and Foxp3 in lymphocytes of guinea pig spleen was detected by real-time fluorescent PCR assay. Additionally, TCE allergic dermatitis patients were selected for the study, the peripheral blood samples were collected from the TCE patients group and control group, quantitative PCR was applied to detect mRNA expression of immune-related genes Foxp3, GATA3, CTLA4, T-bet. TCE induced obvious skin allergic reaction in guinea pigs, the sensitization rate was 83.3%, IgG levels in TCE group and positive control increased significantly. Additionally, mRNA expression levels of GATA3, T-bet, CTLA4 significantly elevated in TCE group and positive control, but Foxp3 mRNA levels decreased. The lymphocytes CD3⁺ ratio in TCE group and positive control of rats was higher than that in negative control, we found that there was no statistical difference of CD4⁺, CD8⁺, CD4⁺/CD8⁺ between TCE group and negative control of rats. The mRNA expression levels of Foxp3, GATA3, CTLA4 in TCE patients increased by 115%, 97%, 241%, respectively as compared with the control, T-bet levels decreased by 47%when compared with the control. TCE could induce obvious changes of cellular immunity and humoral immunity in guinea pigs, rats, and TCE patients, these findings indicated that TCE-induced immunological disorder belongs to the mixed allergy with involvment of cellular immunity and humoral immunity, the mixed allergy might be type IV and type II allergy.

Signatures of Evolutionary Adaptation in Quantitative Trait Loci Influencing Trace Element Homeostasis in Liver

PubMed Central

Sabidó, Eduard; Bosch, Elena

2016-01-01

Essential trace elements possess vital functions at molecular, cellular, and physiological levels in health and disease, and they are tightly regulated in the human body. In order to assess variability and potential adaptive evolution of trace element homeostasis, we quantified 18 trace elements in 150 liver samples, together with the expression levels of 90 genes and abundances of 40 proteins involved in their homeostasis. Additionally, we genotyped 169 single nucleotide polymorphism (SNPs) in the same sample set. We detected significant associations for 8 protein quantitative trait loci (pQTL), 10 expression quantitative trait loci (eQTLs), and 15 micronutrient quantitative trait loci (nutriQTL). Six of these exceeded the false discovery rate cutoff and were related to essential trace elements: 1) one pQTL for GPX2 (rs10133290); 2) two previously described eQTLs for HFE (rs12346) and SELO (rs4838862) expression; and 3) three nutriQTLs: The pathogenic C282Y mutation at HFE affecting iron (rs1800562), and two SNPs within several clustered metallothionein genes determining selenium concentration (rs1811322 and rs904773). Within the complete set of significant QTLs (which involved 30 SNPs and 20 gene regions), we identified 12 SNPs with extreme patterns of population differentiation (FST values in the top 5% percentile in at least one HapMap population pair) and significant evidence for selective sweeps involving QTLs at GPX1, SELENBP1, GPX3, SLC30A9, and SLC39A8. Overall, this detailed study of various molecular phenotypes illustrates the role of regulatory variants in explaining differences in trace element homeostasis among populations and in the human adaptive response to environmental pressures related to micronutrients. PMID:26582562

[Study of Cervical Exfoliated Cell's DNA Quantitative Analysis Based on Multi-Spectral Imaging Technology].

PubMed

Wu, Zheng; Zeng, Li-bo; Wu, Qiong-shui

2016-02-01

The conventional cervical cancer screening methods mainly include TBS (the bethesda system) classification method and cellular DNA quantitative analysis, however, by using multiple staining method in one cell slide, which is staining the cytoplasm with Papanicolaou reagent and the nucleus with Feulgen reagent, the study of achieving both two methods in the cervical cancer screening at the same time is still blank. Because the difficulty of this multiple staining method is that the absorbance of the non-DNA material may interfere with the absorbance of DNA, so that this paper has set up a multi-spectral imaging system, and established an absorbance unmixing model by using multiple linear regression method based on absorbance's linear superposition character, and successfully stripped out the absorbance of DNA to run the DNA quantitative analysis, and achieved the perfect combination of those two kinds of conventional screening method. Through a series of experiment we have proved that between the absorbance of DNA which is calculated by the absorbance unmixxing model and the absorbance of DNA which is measured there is no significant difference in statistics when the test level is 1%, also the result of actual application has shown that there is no intersection between the confidence interval of the DNA index of the tetraploid cells which are screened by using this paper's analysis method when the confidence level is 99% and the DNA index's judging interval of cancer cells, so that the accuracy and feasibility of the quantitative DNA analysis with multiple staining method expounded by this paper have been verified, therefore this analytical method has a broad application prospect and considerable market potential in early diagnosis of cervical cancer and other cancers.

Comparison of EBV DNA viral load in whole blood, plasma, B-cells and B-cell culture supernatant.

PubMed

Ouedraogo, David Eric; Bollore, Karine; Viljoen, Johannes; Foulongne, Vincent; Reynes, Jacques; Cartron, Guillaume; Vendrell, Jean-Pierre; Van de Perre, Philippe; Tuaillon, Edouard

2014-05-01

Epstein-Barr virus (EBV) genome quantitation in whole blood is used widely for therapeutic monitoring of EBV-associated disorders in immunosuppressed individuals and in patients with EBV-associated lymphoma. However, the most appropriate biological material to be used for EBV DNA quantitation remains a subject of debate. This study compare the detection rate and levels of EBV DNA from whole blood, plasma, enriched B-cells, and B-cell short-term culture supernatant using quantitative real-time PCR. Samples were collected from 33 subjects with either HIV infection or B-cell lymphoma. Overall, EBV DNA was detected in 100% of enriched B-cell samples, in 82% of B-cell culture supernatants, in 57% of plasma, and 42% of whole blood samples. A significant correlation for EBV viral load was found between enriched B-cell and B-cell culture supernatant material (ρ = 0.92; P < 0.0001), but no significant correlation existed between EBV DNA levels in whole blood and enriched B-cells (ρ = -0.02; P = 0.89), whole blood and plasma (ρ = 0.24; P = 0.24), or enriched B-cells and plasma (ρ = 0.08; P = 0.77). Testing of enriched B-cells appeared to be the most sensitive method for detection of EBV DNA as well as for exploration of the cellular reservoir. Quantitation of EBV DNA in plasma and B-cell culture supernatant may be of interest to assess EBV reactivation dynamics and response to treatment as well as to decipher EBV host-pathogen interactions in various clinical scenarios. © 2013 Wiley Periodicals, Inc.

Movies of cellular and sub-cellular motion by digital holographic microscopy.

PubMed

Mann, Christopher J; Yu, Lingfeng; Kim, Myung K

2006-03-23

Many biological specimens, such as living cells and their intracellular components, often exhibit very little amplitude contrast, making it difficult for conventional bright field microscopes to distinguish them from their surroundings. To overcome this problem phase contrast techniques such as Zernike, Normarsky and dark-field microscopies have been developed to improve specimen visibility without chemically or physically altering them by the process of staining. These techniques have proven to be invaluable tools for studying living cells and furthering scientific understanding of fundamental cellular processes such as mitosis. However a drawback of these techniques is that direct quantitative phase imaging is not possible. Quantitative phase imaging is important because it enables determination of either the refractive index or optical thickness variations from the measured optical path length with sub-wavelength accuracy. Digital holography is an emergent phase contrast technique that offers an excellent approach in obtaining both qualitative and quantitative phase information from the hologram. A CCD camera is used to record a hologram onto a computer and numerical methods are subsequently applied to reconstruct the hologram to enable direct access to both phase and amplitude information. Another attractive feature of digital holography is the ability to focus on multiple focal planes from a single hologram, emulating the focusing control of a conventional microscope. A modified Mach-Zender off-axis setup in transmission is used to record and reconstruct a number of holographic amplitude and phase images of cellular and sub-cellular features. Both cellular and sub-cellular features are imaged with sub-micron, diffraction-limited resolution. Movies of holographic amplitude and phase images of living microbes and cells are created from a series of holograms and reconstructed with numerically adjustable focus, so that the moving object can be accurately tracked with a reconstruction rate of 300ms for each hologram. The holographic movies show paramecium swimming among other microbes as well as displaying some of their intracellular processes. A time lapse movie is also shown for fibroblast cells in the process of migration. Digital holography and movies of digital holography are seen to be useful new tools for visualization of dynamic processes in biological microscopy. Phase imaging digital holography is a promising technique in terms of the lack of coherent noise and the precision with which the optical thickness of a sample can be profiled, which can lead to images with an axial resolution of a few nanometres.

Nandrolone decanoate interferes with testosterone biosynthesis altering blood-testis barrier components.

PubMed

Barone, Rosario; Pitruzzella, Alessandro; Marino Gammazza, Antonella; Rappa, Francesca; Salerno, Monica; Barone, Fulvio; Sangiorgi, Claudia; D'Amico, Daniela; Locorotondo, Nicola; Di Gaudio, Francesca; Cipolloni, Luigi; Di Felice, Valentina; Schiavone, Stefania; Rapisarda, Venerando; Sani, Gabriele; Tambo, Amos; Cappello, Francesco; Turillazzi, Emanuela; Pomara, Cristoforo

2017-08-01

The aim of this study was to investigate whether nandrolone decanoate (ND) use affects testosterone production and testicular morphology in a model of trained and sedentary mice. A group of mice underwent endurance training while another set led a sedentary lifestyle and were freely mobile within cages. All experimental groups were treated with either ND or peanut oil at different doses for 6 weeks. Testosterone serum levels were measured via liquid chromatography-mass spectrometry. Western blot analysis and quantitative real-time PCR were utilized to determine gene and protein expression levels of the primary enzymes implicated in testosterone biosynthesis and gene expression levels of the blood-testis barrier (BTB) components. Immunohistochemistry and immunofluorescence were conducted for testicular morphological evaluation. The study demonstrated that moderate to high doses of ND induced a diminished serum testosterone level and altered the expression level of the key steroidogenic enzymes involved in testosterone biosynthesis. At the morphological level, ND induced degradation of the BTB by targeting the tight junction protein-1 (TJP1). ND stimulation deregulated metalloproteinase-9, metalloproteinase-2 (MMP-2) and the tissue inhibitor of MMP-2. Moreover, ND administration resulted in a mislocalization of mucin-1. In conclusion, ND abuse induces a decline in testosterone production that is unable to regulate the internalization and redistribution of TJP1 and may induce the deregulation of other BTB constituents via the inhibition of MMP-2. ND may well be considered as both a potential inducer of male infertility and a potential risk factor to a low endogenous bioavailable testosterone. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers.

PubMed

Zanivan, Sara; Maione, Federica; Hein, Marco Y; Hernández-Fernaud, Juan Ramon; Ostasiewicz, Pawel; Giraudo, Enrico; Mann, Matthias

2013-12-01

Proteomics has been successfully used for cell culture on dishes, but more complex cellular systems have proven to be challenging and so far poorly approached with proteomics. Because of the complexity of the angiogenic program, we still do not have a complete understanding of the molecular mechanisms involved in this process, and there have been no in depth quantitative proteomic studies. Plating endothelial cells on matrigel recapitulates aspects of vessel growth, and here we investigate this mechanism by using a spike-in SILAC quantitative proteomic approach. By comparing proteomic changes in primary human endothelial cells morphogenesis on matrigel to general adhesion mechanisms in cells spreading on culture dish, we pinpoint pathways and proteins modulated by endothelial cells. The cell-extracellular matrix adhesion proteome depends on the adhesion substrate, and a detailed proteomic profile of the extracellular matrix secreted by endothelial cells identified CLEC14A as a matrix component, which binds to MMRN2. We verify deregulated levels of these proteins during tumor angiogenesis in models of multistage carcinogenesis. This is the most in depth quantitative proteomic study of endothelial cell morphogenesis, which shows the potential of applying high accuracy quantitative proteomics to in vitro models of vessel growth to shed new light on mechanisms that accompany pathological angiogenesis. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD000359.

Navigating the network: signaling cross-talk in hematopoietic cells

PubMed Central

Fraser, Iain D C; Germain, Ronald N

2009-01-01

Recent studies in hematopoietic cells have led to a growing appreciation of the diverse modes of molecular and functional cross-talk between canonical signaling pathways. However, these intersections represent only the tip of the iceberg. Emerging global analytical methods are providing an even richer and more complete picture of the many components that measurably interact in a network manner to produce cellular responses. Here we highlight the pieces in this Focus, emphasize the limitations of the present canonical pathway paradigm, and discuss the value of a systems biology approach using more global, quantitative experimental design and data analysis strategies. Lastly, we urge caution about overly facile interpretation of genome- and proteome-level studies. PMID:19295628

The rapid quantitation of the filamentous blue-green alga plectonema boryanum by the luciferase assay for ATP

NASA Technical Reports Server (NTRS)

Bush, V. N.

1974-01-01

Plectonema boryanum is a filamentous blue green alga. Blue green algae have a procaryotic cellular organization similar to bacteria, but are usually obligate photoautotrophs, obtaining their carbon and energy from photosynthetic mechanism similar to higher plants. This research deals with a comparison of three methods of quantitating filamentous populations: microscopic cell counts, the luciferase assay for ATP and optical density measurements.

Inhibition of glycogen phosphorylation induces changes in cellular proteome and signaling pathways in MIA pancreatic cancer cells

PubMed Central

Ma, Danjun; Wang, Jiarui; Zhao, Yingchun; Lee, Wai-Nang Paul; Xiao, Jing; Go, Vay Liang W.; Wang, Qi; Recker, Robert; Xiao, Gary Guishan

2011-01-01

Objectives Novel quantitative proteomic approaches were used to study the effects of inhibition of glycogen phosphorylase on proteome and signaling pathways in MIA PaCa-2 pancreatic cancer cells. Methods We performed quantitative proteomic analysis in MIA PaCa-2 cancer cells treated with a stratified dose of CP-320626 (25 μM, 50 μM and 100 μM). The effect of metabolic inhibition on cellular protein turnover dynamics was also studied using the modified SILAC method (mSILAC). Results A total of twenty-two protein spots and four phosphoprotein spots were quantitatively analyzed. We found that dynamic expression of total proteins and phosphoproteins was significantly changed in MIA PaCa-2 cells treated with an incremental dose of CP-320626. Functional analyses suggested that most of the proteins differentially expressed were in the pathways of MAPK/ERK and TNF-α/NF-κB. Conclusions Signaling pathways and metabolic pathways share many common cofactors and substrates forming an extended metabolic network. The restriction of substrate through one pathway such as inhibition of glycogen phosphorylation induces pervasive metabolomic and proteomic changes manifested in protein synthesis, breakdown and post-translational modification of signaling molecules. Our results suggest that quantitative proteomic is an important approach to understand the interaction between metabolism and signaling pathways. PMID:22158071

Total internal reflection holographic microscopy (TIRHM) for quantitative phase characterization of cell-substrate adhesion

NASA Astrophysics Data System (ADS)

Ash, William Mason, III

Total Internal Reflection Holographic Microscopy (TIRHM) combines near-field microscopy with digital holography to produce a new form of near-field phase microscopy. Using a prism in TIR as a near-field imager, the presence of microscopic organisms, cell-substrate interfaces, and adhesions, causes relative refractive index (RRI) and frustrated TIR (f-TIR) to modulate the object beam's evanescent wave phase front. Quantitative phase images of test specimens such as Amoeba proteus, Dictyostelium Discoideum and cells such as SKOV-3 ovarian cancer and 3T3 fibroblasts are produced without the need to introduce stains or fluorophores. The angular spectrum method of digital holography to compensate for tilt anamorphism due to the inclined TIR plane is also discussed. The results of this work conclusively demonstrate, for the first time, the integration of near-field microscopy with digital holography. The cellular images presented show a correlation between the physical extent of the Amoeba proteus plasma membrane and the adhesions that are quantitatively profiled by phase cross-sectioning of the holographic images obtained by digital holography. With its ability to quantitatively characterise cellular adhesion and motility, it is anticipated that TIRHM can be a tool for characterizing and combating cancer metastasis, as well as improving our understanding of morphogenesis and embryogenesis itself.

Systems cell biology

PubMed Central

Mast, Fred D.; Ratushny, Alexander V.

2014-01-01

Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology. PMID:25225336

Quantitative mapping of intracellular cations in the human amniotic membrane

NASA Astrophysics Data System (ADS)

Moretto, Ph.; Llabador, Y.; Simonoff, M.; Razafindrabe, L.; Bara, M.; Guiet-Bara, A.

1993-05-01

The effect of magnesium and taurine on the permeability of cell membranes to monovalent cations has been investigated using the Bordeaux nuclear microprobe. PIXE and RBS techniques have been used to provide quantitative measurements and ion distributions in the isolated amniotic membrane. This physiological model for cellular exchanges allowed us to reveal the distribution of most elements involved in cellular pathways and the modifications under different experimental conditions of incubation in physiological fluids. The PIXE microanalysis provided an original viewpoint on these mechanisms. Following this first study, the amnion compact lamina was found to play a role which was not, up to now, taken into account in the interpretation of electrophysiological experimentations. The release of some ionic species, such as K +, from the epithelial cells, during immersion in isotonic fluids, could have been hitherto underestimated.

Multiplexed quantitation of protein expression and phosphorylation based on functionalized soluble nanopolymers

PubMed Central

Pan, Li; Iliuk, Anton; Yu, Shuai; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

We report here for the first time the multiplexed quantitation of phosphorylation and protein expression based on a functionalized soluble nanopolymer. The soluble nanopolymer, pIMAGO, is functionalized with Ti (IV) ions for chelating phosphoproteins in high specificity, and with infrared fluorescent tags for direct, multiplexed assays. The nanopolymer allows for direct competition for epitopes on proteins of interest, thus facilitating simultaneous detection of phosphorylation by pIMAGO and total protein amount by protein antibody in the same well of microplates. The new strategy has a great potential to measure cell signaling events by clearly distinguishing actual phosphorylation signals from protein expression changes, thus providing a powerful tool to accurately profile cellular signal transduction in healthy and disease cells. We anticipate broad applications of this new strategy in monitoring cellular signaling pathways and discovering new signaling events. PMID:23088311

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

NASA Astrophysics Data System (ADS)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Coupled Analysis of In Vitro and Histology Tissue Samples to Quantify Structure-Function Relationship

PubMed Central

Acar, Evrim; Plopper, George E.; Yener, Bülent

2012-01-01

The structure/function relationship is fundamental to our understanding of biological systems at all levels, and drives most, if not all, techniques for detecting, diagnosing, and treating disease. However, at the tissue level of biological complexity we encounter a gap in the structure/function relationship: having accumulated an extraordinary amount of detailed information about biological tissues at the cellular and subcellular level, we cannot assemble it in a way that explains the correspondingly complex biological functions these structures perform. To help close this information gap we define here several quantitative temperospatial features that link tissue structure to its corresponding biological function. Both histological images of human tissue samples and fluorescence images of three-dimensional cultures of human cells are used to compare the accuracy of in vitro culture models with their corresponding human tissues. To the best of our knowledge, there is no prior work on a quantitative comparison of histology and in vitro samples. Features are calculated from graph theoretical representations of tissue structures and the data are analyzed in the form of matrices and higher-order tensors using matrix and tensor factorization methods, with a goal of differentiating between cancerous and healthy states of brain, breast, and bone tissues. We also show that our techniques can differentiate between the structural organization of native tissues and their corresponding in vitro engineered cell culture models. PMID:22479315

Studies of Intercellular Communication and Intracellular Metabolic Responses by Bone Cells to Simulated Weightlessness

NASA Technical Reports Server (NTRS)

Doty, Stephen B.

1997-01-01

Spaceflight affects the weight bearing skeletal tissues by reducing the rate of new bone formation. This effect on the long bones of flown rats has been quantitated but the effect at the cellular level and the mechanism(s) involved are not understood. We are applying electron microscopy, coupled with histochemistry and immunocytochemistry to determine the cellular functions most affected by spaceflight. The emphasis for study of these samples from SLS-1, a 9-day mission, is on the histochemical and structural changes of the endosteal and perivascular osteoblasts found in diaphyseal bone of femur and tibia. Work is still in progress but some findings are described: (1) An expected decrease in alkaline phosphatase activity in osteoblasts from flight animals, but an increase in enzyme activity in the stromal stem cells adjacent to the osteoblast. (2) An increase in osteoclastic TRAP activity in the trabecular bone region in response to spaceflight. (3) A large increase in procollagen containing secretory granules in osteoblasts in the recovery group, and a significant decrease in granule numbers in the flight group.

Quantitative real-time analysis of collective cancer invasion and dissemination

NASA Astrophysics Data System (ADS)

Ewald, Andrew J.

2015-05-01

A grand challenge in biology is to understand the cellular and molecular basis of tissue and organ level function in mammals. The ultimate goals of such efforts are to explain how organs arise in development from the coordinated actions of their constituent cells and to determine how molecularly regulated changes in cell behavior alter the structure and function of organs during disease processes. Two major barriers stand in the way of achieving these goals: the relative inaccessibility of cellular processes in mammals and the daunting complexity of the signaling environment inside an intact organ in vivo. To overcome these barriers, we have developed a suite of tissue isolation, three dimensional (3D) culture, genetic manipulation, nanobiomaterials, imaging, and molecular analysis techniques to enable the real-time study of cell biology within intact tissues in physiologically relevant 3D environments. This manuscript introduces the rationale for 3D culture, reviews challenges to optical imaging in these cultures, and identifies current limitations in the analysis of complex experimental designs that could be overcome with improved imaging, imaging analysis, and automated classification of the results of experimental interventions.

M2-like macrophages are responsible for collagen degradation through a mannose receptor–mediated pathway

PubMed Central

Madsen, Daniel H.; Leonard, Daniel; Masedunskas, Andrius; Moyer, Amanda; Jürgensen, Henrik Jessen; Peters, Diane E.; Amornphimoltham, Panomwat; Selvaraj, Arul; Yamada, Susan S.; Brenner, David A.; Burgdorf, Sven; Engelholm, Lars H.; Behrendt, Niels; Holmbeck, Kenn; Weigert, Roberto

2013-01-01

Tissue remodeling processes critically depend on the timely removal and remodeling of preexisting collagen scaffolds. Nevertheless, many aspects related to the turnover of this abundant extracellular matrix component in vivo are still incompletely understood. We therefore took advantage of recent advances in optical imaging to develop an assay to visualize collagen turnover in situ and identify cell types and molecules involved in this process. Collagen introduced into the dermis of mice underwent cellular endocytosis in a partially matrix metalloproteinase–dependent manner and was subsequently routed to lysosomes for complete degradation. Collagen uptake was predominantly executed by a quantitatively minor population of M2-like macrophages, whereas more abundant Col1a1-expressing fibroblasts and Cx3cr1-expressing macrophages internalized collagen at lower levels. Genetic ablation of the collagen receptors mannose receptor (Mrc1) and urokinase plasminogen activator receptor–associated protein (Endo180 and Mrc2) impaired this intracellular collagen degradation pathway. This study demonstrates the importance of receptor-mediated cellular uptake to collagen turnover in vivo and identifies a key role of M2-like macrophages in this process. PMID:24019537

A Comparative Analysis of Drug-Induced Hepatotoxicity in Clinically Relevant Situations

PubMed Central

Thiel, Christoph; Cordes, Henrik; Fabbri, Lorenzo; Aschmann, Hélène Eloise; Baier, Vanessa; Atkinson, Francis; Blank, Lars Mathias; Kuepfer, Lars

2017-01-01

Drug-induced toxicity is a significant problem in clinical care. A key problem here is a general understanding of the molecular mechanisms accompanying the transition from desired drug effects to adverse events following administration of either therapeutic or toxic doses, in particular within a patient context. Here, a comparative toxicity analysis was performed for fifteen hepatotoxic drugs by evaluating toxic changes reflecting the transition from therapeutic drug responses to toxic reactions at the cellular level. By use of physiologically-based pharmacokinetic modeling, in vitro toxicity data were first contextualized to quantitatively describe time-resolved drug responses within a patient context. Comparatively studying toxic changes across the considered hepatotoxicants allowed the identification of subsets of drugs sharing similar perturbations on key cellular processes, functional classes of genes, and individual genes. The identified subsets of drugs were next analyzed with regard to drug-related characteristics and their physicochemical properties. Toxic changes were finally evaluated to predict both molecular biomarkers and potential drug-drug interactions. The results may facilitate the early diagnosis of adverse drug events in clinical application. PMID:28151932

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Proteomic snapshot of the EGF-induced ubiquitin network

PubMed Central

Argenzio, Elisabetta; Bange, Tanja; Oldrini, Barbara; Bianchi, Fabrizio; Peesari, Raghunath; Mari, Sara; Di Fiore, Pier Paolo; Mann, Matthias; Polo, Simona

2011-01-01

The activity, localization and fate of many cellular proteins are regulated through ubiquitination, a process whereby one or more ubiquitin (Ub) monomers or chains are covalently attached to target proteins. While Ub-conjugated and Ub-associated proteomes have been described, we lack a high-resolution picture of the dynamics of ubiquitination in response to signaling. In this study, we describe the epidermal growth factor (EGF)-regulated Ubiproteome, as obtained by two complementary purification strategies coupled to quantitative proteomics. Our results unveil the complex impact of growth factor signaling on Ub-based intracellular networks to levels that extend well beyond what might have been expected. In addition to endocytic proteins, the EGF-regulated Ubiproteome includes a large number of signaling proteins, ubiquitinating and deubiquitinating enzymes, transporters and proteins involved in translation and transcription. The Ub-based signaling network appears to intersect both housekeeping and regulatory circuitries of cellular physiology. Finally, as proof of principle of the biological relevance of the EGF-Ubiproteome, we demonstrated that EphA2 is a novel, downstream ubiquitinated target of epidermal growth factor receptor (EGFR), critically involved in EGFR biological responses. PMID:21245847

The mechanics behind plant development.

PubMed

Hamant, Olivier; Traas, Jan

2010-01-01

Morphogenesis in living organisms relies on the integration of both biochemical and mechanical signals. During the last decade, attention has been mainly focused on the role of biochemical signals in patterning and morphogenesis, leaving the contribution of mechanics largely unexplored. Fortunately, the development of new tools and approaches has made it possible to re-examine these processes. In plants, shape is defined by two local variables: growth rate and growth direction. At the level of the cell, these variables depend on both the cell wall and turgor pressure. Multidisciplinary approaches have been used to understand how these cellular processes are integrated in the growing tissues. These include quantitative live imaging to measure growth rate and direction in tissues with cellular resolution. In parallel, stress patterns have been artificially modified and their impact on strain and cell behavior been analysed. Importantly, computational models based on analogies with continuum mechanics systems have been useful in interpreting the results. In this review, we will discuss these issues focusing on the shoot apical meristem, a population of stem cells that is responsible for the initiation of the aerial organs of the plant.

Adaptation in Living Systems

NASA Astrophysics Data System (ADS)

Tu, Yuhai; Rappel, Wouter-Jan

2018-03-01

Adaptation refers to the biological phenomenon where living systems change their internal states in response to changes in their environments in order to maintain certain key functions critical for their survival and fitness. Adaptation is one of the most ubiquitous and arguably one of the most fundamental properties of living systems. It occurs throughout all biological scales, from adaptation of populations of species over evolutionary time to adaptation of a single cell to different environmental stresses during its life span. In this article, we review some of the recent progress made in understanding molecular mechanisms of cellular-level adaptation. We take the minimalist (or the physicist) approach and study the simplest systems that exhibit generic adaptive behaviors, namely chemotaxis in bacterium cells (Escherichia coli) and eukaryotic cells (Dictyostelium). We focus on understanding the basic biochemical interaction networks that are responsible for adaptation dynamics. By combining theoretical modeling with quantitative experimentation, we demonstrate universal features in adaptation as well as important differences in different cellular systems. Future work in extending the modeling framework to study adaptation in more complex systems such as sensory neurons is also discussed.

Eicosanoids mediate nodulation reactions to bacterial infections in adults of two 17-year periodical cicadas, Magicicada septendecim and M. cassini.

PubMed

Tunaz, H; Bedick, J C.; Miller, J S.; Hoback, W W.; Rana, R L.; Stanley, D W.

1999-10-01

Nodulation is the first and quantitatively most important cellular defense reaction to bacterial infections in insects. Treating adults of the 17-year periodical cicadas, Magicicada septendecim and M. cassini, with eicosanoid biosynthesis inhibitors immediately prior to intrahemocoelic injections of the bacterium, Serratia marcescens, sharply reduced the nodulation response to bacterial challenges. Separate treatments with specific inhibitors of phospholipase A(2), cyclooxygenase, and lipoxygenase reduced nodulation, supporting our view that nodule formation is a multi-step process in which individual steps are separately mediated by lipoxygenase and cyclooxygenase products. The inhibitory influence of dexamethasone was apparent by 2 h after injection, and nodulation was significantly reduced, relative to control insects, over the following 14 h. The dexamethasone effects were reversed by treating bacteria-challenged insects with the eicosanoid-precursor polyunsaturated fatty acid, arachidonic acid. Low levels of arachidonic acid were detected in fat body phospholipids. These findings in adults of an exopterygote insect species with an unusual life history pattern broaden our hypothesis that eicosanoids mediate cellular immune reactions to bacterial infections in most, if not all, insects.

Combination therapeutics of Nilotinib and radiation in acute lymphoblastic leukemia as an effective method against drug-resistance.

PubMed

Kaveh, Kamran; Takahashi, Yutaka; Farrar, Michael A; Storme, Guy; Guido, Marcucci; Piepenburg, Jamie; Penning, Jackson; Foo, Jasmine; Leder, Kevin Z; Hui, Susanta K

2017-07-01

Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) is characterized by a very poor prognosis and a high likelihood of acquired chemo-resistance. Although tyrosine kinase inhibitor (TKI) therapy has improved clinical outcome, most ALL patients relapse following treatment with TKI due to the development of resistance. We developed an in vitro model of Nilotinib-resistant Ph+ leukemia cells to investigate whether low dose radiation (LDR) in combination with TKI therapy overcome chemo-resistance. Additionally, we developed a mathematical model, parameterized by cell viability experiments under Nilotinib treatment and LDR, to explain the cellular response to combination therapy. The addition of LDR significantly reduced drug resistance both in vitro and in computational model. Decreased expression level of phosphorylated AKT suggests that the combination treatment plays an important role in overcoming resistance through the AKT pathway. Model-predicted cellular responses to the combined therapy provide good agreement with experimental results. Augmentation of LDR and Nilotinib therapy seems to be beneficial to control Ph+ leukemia resistance and the quantitative model can determine optimal dosing schedule to enhance the effectiveness of the combination therapy.

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 (IVIVEs) of toxicity (efficacy) at the cellular level from data generated in cell assays. Overall, this present study may help to optimize the human dose prediction in preclinical and clinical studies, while prescribing drugs with narrow therapeutic windows that are highly bound to extracellular proteins and/or highly ionized at the physiological pH. This may facilitate building a more accurate safety (efficacy) profile for such drugs. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Life cycle thinking and assessment tools on environmentally-benign electronics: Convergent optimization of materials use, end-of-life strategy and environmental policies

NASA Astrophysics Data System (ADS)

Zhou, Xiaoying

The purpose of this study is to integrate the quantitative environmental performance assessment tools and the theory of multi-objective optimization within the boundary of electronic product systems to support the selection among design alternatives in terms of environmental impact, technical criteria, and economic feasibility. To meet with the requirements that result from emerging environmental legislation targeting electronics products, the research addresses an important analytical methodological approach to facilitate environmentally conscious design and end-of-life management with a life cycle viewpoint. A synthesis of diverse assessment tools is applied on a set of case studies: lead-free solder materials selection, cellular phone design, and desktop display technology assessment. In the first part of this work, an in-depth industrial survey of the status and concerns of the U.S. electronics industry on the elimination of lead (Pb) in solders is described. The results show that the trade-offs among environmental consequences, technology challenges, business risks, legislative compliance and stakeholders' preferences must be explicitly, simultaneously, and systematically addressed in the decision-making process used to guide multi-faceted planning of environmental solutions. In the second part of this work, the convergent optimization of the technical cycle, economic cycle and environmental cycle is addressed in a coherent and systematic way using the application of environmentally conscious design of cellular phones. The technical understanding of product structure, components analysis, and materials flow facilitates the development of "Design for Disassembly" guidelines. A bottom-up disassembly analysis on a "bill of materials" based structure at a micro-operational level is utilized to select optimal end-of-life strategies on the basis of economic feasibility. A macro-operational level life cycle model is used to investigate the environmental consequences linking environmental impact with the cellular phone production activities focusing on the upstream manufacturing and end-of-life life cycle stages. The last part of this work, the quantitative elicitation of weighting factors facilitates the comparison of trade-offs in the context of a multi-attribute problem. An integrated analytical approach, Integrated Industrial Ecology Function Deployment (I2-EFD), is proposed to assess alternatives at the design phase of a product system and is validated with the assessment of desktop display technologies and lead-free solder alternatives.

Discrimination of Dysplastic Nevi from Common Melanocytic Nevi by Cellular and Molecular Criteria.

PubMed

Mitsui, Hiroshi; Kiecker, Felix; Shemer, Avner; Cannizzaro, Maria Vittoria; Wang, Claire Q F; Gulati, Nicholas; Ohmatsu, Hanako; Shah, Kejal R; Gilleaudeau, Patricia; Sullivan-Whalen, Mary; Cueto, Inna; McNutt, Neil Scott; Suárez-Fariñas, Mayte; Krueger, James G

2016-10-01

Dysplastic nevi (DNs), also known as Clark's nevi or atypical moles, are distinguished from common melanocytic nevi by variegation in pigmentation and clinical appearance, as well as differences in tissue patterning. However, cellular and molecular differences between DNs and common melanocytic nevi are not completely understood. Using cDNA microarray, quantitative RT-PCR, and immunohistochemistry, we molecularly characterized DNs and analyzed the difference between DNs and common melanocytic nevi. A total of 111 probesets (91 annotated genes, fold change > 2.0 and false discovery rate < 0.25) were differentially expressed between the two lesions. An unexpected finding in DNs was altered differentiation and activation of epidermal keratinocytes with increased expression of hair follicle-related molecules (keratin 25, trichohyalin, ribonuclease, RNase A family, 7) and inflammation-related molecules (S100A7, S100A8) at both genomic and protein levels. The immune microenvironment of DNs was characterized by an increase of T helper type 1 (IFNγ) and T helper type 2 (IL13) cytokines as well as an upregulation of oncostatin M and CXCL1. DUSP3, which regulates cellular senescence, was identified as one of the disease discriminative genes between DNs and common melanocytic nevi by three independent statistical approaches and its altered expression was confirmed by immunohistochemistry. The molecular and cellular changes in which the epidermal-melanin unit undergoes follicular differentiation as well as upregulation of defined cytokines could drive complex immune, epidermal, and pigmentary alterations. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Image analysis in cytology: DNA-histogramming versus cervical smear prescreening.

PubMed

Bengtsson, E W; Nordin, B

1993-01-01

The visual inspection of cellular specimens and histological sections through a light microscope plays an important role in clinical medicine and biomedical research. The human visual system is very good at the recognition of various patterns but less efficient at quantitative assessment of these patterns. Some samples are prepared in great numbers, most notably the screening for cervical cancer, the so-called PAP-smears, which results in hundreds of millions of samples each year, creating a tedious mass inspection task. Numerous attempts have been made over the last 40 years to create systems that solve these two tasks, the quantitative supplement to the human visual system and the automation of mass screening. The most difficult task, the total automation, has received the greatest attention with many large scale projects over the decades. In spite of all these efforts, still no generally accepted automated prescreening device exists on the market. The main reason for this failure is the great pattern recognition capabilities needed to distinguish between cancer cells and all other kinds of objects found in the specimens: cellular clusters, debris, degenerate cells, etc. Improved algorithms, the ever-increasing processing power of computers and progress in biochemical specimen preparation techniques make it likely that eventually useful automated prescreening systems will become available. Meanwhile, much less effort has been put into the development of interactive cell image analysis systems. Still, some such systems have been developed and put into use at thousands of laboratories worldwide. In these the human pattern recognition capability is used to select the fields and objects that are to be analysed while the computational power of the computer is used for the quantitative analysis of cellular DNA content or other relevant markers. Numerous studies have shown that the quantitative information about the distribution of cellular DNA content is of prognostic significance in many types of cancer. Several laboratories are therefore putting these techniques into routine clinical use. The more advanced systems can also study many other markers and cellular features, some known to be of clinical interest, others useful in research. The advances in computer technology are making these systems more generally available through decreasing cost, increasing computational power and improved user interfaces. We have been involved in research and development of both automated and interactive cell analysis systems during the last 20 years. Here some experiences and conclusions from this work will be presented as well as some predictions about what can be expected in the near future.

Restoration of microRNA‑218 increases cellular chemosensitivity to cervical cancer by inhibiting cell‑cycle progression.

PubMed

Dong, Ruofan; Qiu, Haifeng; Du, Guiqiang; Wang, Yuan; Yu, Jinjin; Mao, Caiping

2014-12-01

We previously reported frequent loss of microRNA‑218 (miR‑218) in human cervical cancer, which was associated with tumor progression and poor prognosis. In this study, we investigated whether restoration of the miR‑218 level is a valid strategy for the treatment of cervical cancer. The expression of miR‑218 in cervical cancer samples and cell lines was quantified by reverse transcription TaqMan quantitative (RT‑q)PCR. Overexpression of miR‑218 was achieved by both transient and stable transfection, using a miR‑218 mimic and a miR‑218‑expressing plasmid, respectively. Alterations in cellular proliferation and cell‑cycle progression were measured by the MTT assay and flow cytometry analysis. Nude mice bearing SiHa xenografts were used to investigate the functions of miR‑218 and carboplatin on tumor growth and weight. The expression of cycle‑related proteins was detected by western blotting and immunohistochemical staining. In vitro, miR‑218 significantly inhibited cellular growth in all four cell lines tested (P=0.021 for CaSki, P=0.009 for HeLa, P=0.016 for SiHa, and P=0.029 for C33A). Overexpression of miR‑218 induced G1 phase arrest and reduced expression of cyclin D1 and CDK4. In vivo, restoration of miR‑218 notably inhibited tumor growth and decreased tumor weight. In primary cultured samples, tumors with high levels of miR‑218 were more sensitive to carboplatin (R2=0.3319, P=0.0026); consistently, miR‑218 overexpression suppressed tumor growth, induced cell‑cycle arrest, and reduced the cyclin D1 level. Based on these and previous results, we conclude that restoration of the miR‑218 level inhibits the growth of cervical cancer cells both in vitro and in vivo; furthermore, overexpression of miR‑218 sensitizes cervical cancer cells to carboplatin. Our findings suggest a novel therapy for cervical cancer based on miR‑218, especially in patients with reduced levels of miR‑218.

PdO Doping Tunes Band-Gap Energy Levels as Well as Oxidative Stress Responses to a Co3O4p-Type Semiconductor in Cells and the Lung

PubMed Central

2014-01-01

We demonstrate through PdO doping that creation of heterojunctions on Co3O4 nanoparticles can quantitatively adjust band-gap and Fermi energy levels to study the impact of metal oxide nanoparticle semiconductor properties on cellular redox homeostasis and hazard potential. Flame spray pyrolysis (FSP) was used to synthesize a nanoparticle library in which the gradual increase in the PdO content (0–8.9%) allowed electron transfer from Co3O4 to PdO to align Fermi energy levels across the heterojunctions. This alignment was accompanied by free hole accumulation at the Co3O4 interface and production of hydroxyl radicals. Interestingly, there was no concomitant superoxide generation, which could reflect the hole dominance of a p-type semiconductor. Although the electron flux across the heterojunctions induced upward band bending, the Ec levels of the doped particles showed energy overlap with the biological redox potential (BRP). This allows electron capture from the redox couples that maintain the BRP from −4.12 to −4.84 eV, causing disruption of cellular redox homeostasis and induction of oxidative stress. PdO/Co3O4 nanoparticles showed significant increases in cytotoxicity at 25, 50, 100, and 200 μg/mL, which was enhanced incrementally by PdO doping in BEAS-2B and RAW 264.7 cells. Oxidative stress presented as a tiered cellular response involving superoxide generation, glutathione depletion, cytokine production, and cytotoxicity in epithelial and macrophage cell lines. A progressive series of acute pro-inflammatory effects could also be seen in the lungs of animals exposed to incremental PdO-doped particles. All considered, generation of a combinatorial PdO/Co3O4 nanoparticle library with incremental heterojunction density allowed us to demonstrate the integrated role of Ev, Ec, and Ef levels in the generation of oxidant injury and inflammation by the p-type semiconductor, Co3O4. PMID:24673286

PdO doping tunes band-gap energy levels as well as oxidative stress responses to a Co₃O₄ p-type semiconductor in cells and the lung.

PubMed

Zhang, Haiyuan; Pokhrel, Suman; Ji, Zhaoxia; Meng, Huan; Wang, Xiang; Lin, Sijie; Chang, Chong Hyun; Li, Linjiang; Li, Ruibin; Sun, Bingbing; Wang, Meiying; Liao, Yu-Pei; Liu, Rong; Xia, Tian; Mädler, Lutz; Nel, André E

2014-04-30

We demonstrate through PdO doping that creation of heterojunctions on Co3O4 nanoparticles can quantitatively adjust band-gap and Fermi energy levels to study the impact of metal oxide nanoparticle semiconductor properties on cellular redox homeostasis and hazard potential. Flame spray pyrolysis (FSP) was used to synthesize a nanoparticle library in which the gradual increase in the PdO content (0-8.9%) allowed electron transfer from Co3O4 to PdO to align Fermi energy levels across the heterojunctions. This alignment was accompanied by free hole accumulation at the Co3O4 interface and production of hydroxyl radicals. Interestingly, there was no concomitant superoxide generation, which could reflect the hole dominance of a p-type semiconductor. Although the electron flux across the heterojunctions induced upward band bending, the E(c) levels of the doped particles showed energy overlap with the biological redox potential (BRP). This allows electron capture from the redox couples that maintain the BRP from -4.12 to -4.84 eV, causing disruption of cellular redox homeostasis and induction of oxidative stress. PdO/Co3O4 nanoparticles showed significant increases in cytotoxicity at 25, 50, 100, and 200 μg/mL, which was enhanced incrementally by PdO doping in BEAS-2B and RAW 264.7 cells. Oxidative stress presented as a tiered cellular response involving superoxide generation, glutathione depletion, cytokine production, and cytotoxicity in epithelial and macrophage cell lines. A progressive series of acute pro-inflammatory effects could also be seen in the lungs of animals exposed to incremental PdO-doped particles. All considered, generation of a combinatorial PdO/Co3O4 nanoparticle library with incremental heterojunction density allowed us to demonstrate the integrated role of E(v), E(c), and E(f) levels in the generation of oxidant injury and inflammation by the p-type semiconductor, Co3O4.

Real time quantitative phase microscopy based on single-shot transport of intensity equation (ssTIE) method

NASA Astrophysics Data System (ADS)

Yu, Wei; Tian, Xiaolin; He, Xiaoliang; Song, Xiaojun; Xue, Liang; Liu, Cheng; Wang, Shouyu

2016-08-01

Microscopy based on transport of intensity equation provides quantitative phase distributions which opens another perspective for cellular observations. However, it requires multi-focal image capturing while mechanical and electrical scanning limits its real time capacity in sample detections. Here, in order to break through this restriction, real time quantitative phase microscopy based on single-shot transport of the intensity equation method is proposed. A programmed phase mask is designed to realize simultaneous multi-focal image recording without any scanning; thus, phase distributions can be quantitatively retrieved in real time. It is believed the proposed method can be potentially applied in various biological and medical applications, especially for live cell imaging.

Generation of dense statistical connectomes from sparse morphological data

PubMed Central

Egger, Robert; Dercksen, Vincent J.; Udvary, Daniel; Hege, Hans-Christian; Oberlaender, Marcel

2014-01-01

Sensory-evoked signal flow, at cellular and network levels, is primarily determined by the synaptic wiring of the underlying neuronal circuitry. Measurements of synaptic innervation, connection probabilities and subcellular organization of synaptic inputs are thus among the most active fields of research in contemporary neuroscience. Methods to measure these quantities range from electrophysiological recordings over reconstructions of dendrite-axon overlap at light-microscopic levels to dense circuit reconstructions of small volumes at electron-microscopic resolution. However, quantitative and complete measurements at subcellular resolution and mesoscopic scales to obtain all local and long-range synaptic in/outputs for any neuron within an entire brain region are beyond present methodological limits. Here, we present a novel concept, implemented within an interactive software environment called NeuroNet, which allows (i) integration of sparsely sampled (sub)cellular morphological data into an accurate anatomical reference frame of the brain region(s) of interest, (ii) up-scaling to generate an average dense model of the neuronal circuitry within the respective brain region(s) and (iii) statistical measurements of synaptic innervation between all neurons within the model. We illustrate our approach by generating a dense average model of the entire rat vibrissal cortex, providing the required anatomical data, and illustrate how to measure synaptic innervation statistically. Comparing our results with data from paired recordings in vitro and in vivo, as well as with reconstructions of synaptic contact sites at light- and electron-microscopic levels, we find that our in silico measurements are in line with previous results. PMID:25426033

DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings.

PubMed Central

Quaite, F. E.; Takayanagi, S.; Ruffini, J.; Sutherland, J. C.; Sutherland, B. M.

1994-01-01

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage. PMID:12244228

Proteomic alterations in root tips of Arabidopsis thaliana seedlings under altered gravity conditions

NASA Astrophysics Data System (ADS)

Zheng, H. Q.; Wang, H.

Gravity has a profound influence on plant growth and development Removed the influence of gravitational acceleration by spaceflight caused a wide range of cellular changes in plant Whole seedling that germinated and grown on clinostats showed the absent of gravitropism At the cellular level clinostat treatment has specific effects on plant cells such as induce alterations in cell wall composition increase production of heat-soluble proteins impact on the cellular energy metabolism facilitate a uniform distribution of plastids amyloplasts and increase number and volume of nucleoli A number of recent studies have shown that the exposure of Arabidopsis seedlings and callus cells to gravity stimulation hyper g-forces or clinostat rotation induces alterations in gene expression In our previous study the proteome of the Arabidopsis thaliana callus cells were separated by high resolution two-dimensional electrophoresis 2-DE Image analysis revealed that 80 protein spots showed quantitative and qualitative variations after exposure to clinostat rotation treatment We report here a systematic proteomic approach to investigate the altered gravity responsive proteins in root tip of Arabidopsis thaliana cv Landsberg erecta Three-day-old seedlings were exposed for 12h to a horizontal clinostat rotation H simulated weightlessness altered g-forces by centrifugation 7g hypergravity a vertical clinostat rotation V clinostat control or a stationary control grown conditions Total proteins of roots were extracted

Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells.

PubMed

Norcross, Stevie; Trull, Keelan J; Snaider, Jordan; Doan, Sara; Tat, Kiet; Huang, Libai; Tantama, Mathew

2017-11-22

Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically encoded sensor. Here, we report a new family of genetically encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses.

A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load.

PubMed

Malnati, Mauro S; Scarlatti, Gabriella; Gatto, Francesca; Salvatori, Francesca; Cassina, Giulia; Rutigliano, Teresa; Volpi, Rosy; Lusso, Paolo

2008-01-01

Quantification of human immunodeficiency virus type-1 (HIV-1) proviral DNA is increasingly used to measure the HIV-1 cellular reservoirs, a helpful marker to evaluate the efficacy of antiretroviral therapeutic regimens in HIV-1-infected individuals. Furthermore, the proviral DNA load represents a specific marker for the early diagnosis of perinatal HIV-1 infection and might be predictive of HIV-1 disease progression independently of plasma HIV-1 RNA levels and CD4(+) T-cell counts. The high degree of genetic variability of HIV-1 poses a serious challenge for the design of a universal quantitative assay capable of detecting all the genetic subtypes within the main (M) HIV-1 group with similar efficiency. Here, we describe a highly sensitive real-time PCR protocol that allows for the correct quantification of virtually all group-M HIV-1 strains with a higher degree of accuracy compared with other methods. The protocol involves three stages, namely DNA extraction/lysis, cellular DNA quantification and HIV-1 proviral load assessment. Owing to the robustness of the PCR design, this assay can be performed on crude cellular extracts, and therefore it may be suitable for the routine analysis of clinical samples even in developing countries. An accurate quantification of the HIV-1 proviral load can be achieved within 1 d from blood withdrawal.

Matrix Metalloproteinase 3 Promotes Cellular Anti-Dengue Virus Response via Interaction with Transcription Factor NFκB in Cell Nucleus

PubMed Central

Zuo, Xiangyang; Pan, Wen; Feng, Tingting; Shi, Xiaohong; Dai, Jianfeng

2014-01-01

Dengue virus (DENV), the causative agent of human Dengue hemorrhagic fever, is a mosquito-borne virus of immense global health importance. Characterization of cellular factors promoting or inhibiting DENV infection is important for understanding the mechanism of DENV infection. In this report, MMP3 (stromelysin-1), a secretory endopeptidase that degrades extracellular matrices, has been shown promoting cellular antiviral response against DENV infection. Quantitative RT-PCR and Western Blot showed that the expression of MMP3 was upregulated in DENV-infected RAW264.7 cells. The intracellular viral loads were significantly higher in MMP3 silenced cells compared with controls. The expression level of selective anti-viral cytokines were decreased in MMP3 siRNA treated cells, and the transcription factor activity of NFκB was significantly impaired upon MMP3 silencing during DENV infection. Further, we found that MMP3 moved to cell nucleus upon DENV infection and colocalized with NFκB P65 in nucleus. Co-immunoprecipitation analysis suggested that MMP3 directly interacted with NFκB in nucleus during DENV infection and the C-terminal hemopexin-like domain of MMP3 was required for the interaction. This study suggested a novel role of MMP3 in nucleus during viral infection and provided new evidence for MMPs in immunomodulation. PMID:24416274

Exposure of differentiated airway epithelial cells to volatile smoke in vitro.

PubMed

Beisswenger, Christoph; Platz, Juliane; Seifart, Carola; Vogelmeier, Claus; Bals, Robert

2004-01-01

Cigarette smoke (CS) is the predominant pathogenetic factor in the development of chronic bronchitis and chronic obstructive pulmonary disease. The knowledge about the cellular and molecular mechanisms underlying the smoke-induced inflammation in epithelial cells is limited. The aim of this study was to develop an in vitro model to monitor the effects of volatile CS on differentiated airway epithelial cells. The airway epithelial cell line MM-39 and primary human bronchial epithelial cells were cultivated as air-liquid interface cultures and exposed directly to volatile CS. We used two types of exposure models, one using ambient air, the other using humidified and warm air. Cytokine levels were measured by quantitative PCR and ELISA. Phosphorylation of p38 MAP kinase was assessed by Western blot analysis. To reduce the smoke-induced inflammation, antisense oligonucleotides directed against the p65 subunit of NF-kappaB were applied. Exposure of epithelia to cold and dry air resulted in a significant inflammatory response. In contrast, exposure to humidified warm air did not elicit a cellular response. Stimulation with CS resulted in upregulation of mRNA for IL-6 and IL-8 and protein release. Exposure to CS combined with heat-inactivated bacteria synergistically increased levels of the cytokines. Reactions of differentiated epithelial cells to smoke are mediated by the MAP kinase p38 and the transcription factor NF-kappaB. We developed an exposure model to examine the consequences of direct exposure of differentiated airway epithelial cells to volatile CS. The model enables to measure the cellular reactions to smoke exposure and to determine the outcome of therapeutic interventions. Copyright 2004 S. Karger AG, Basel

Partial Resistance of Carrot to Alternaria dauci Correlates with In Vitro Cultured Carrot Cell Resistance to Fungal Exudates

PubMed Central

Voisine, Linda; Gatto, Julia; Hélesbeux, Jean-Jacques; Séraphin, Denis; Peña-Rodriguez, Luis M.; Richomme, Pascal; Boedo, Cora; Yovanopoulos, Claire; Gyomlai, Melvina; Briard, Mathilde; Simoneau, Philippe; Poupard, Pascal; Berruyer, Romain

2014-01-01

Although different mechanisms have been proposed in the recent years, plant pathogen partial resistance is still poorly understood. Components of the chemical warfare, including the production of plant defense compounds and plant resistance to pathogen-produced toxins, are likely to play a role. Toxins are indeed recognized as important determinants of pathogenicity in necrotrophic fungi. Partial resistance based on quantitative resistance loci and linked to a pathogen-produced toxin has never been fully described. We tested this hypothesis using the Alternaria dauci – carrot pathosystem. Alternaria dauci, causing carrot leaf blight, is a necrotrophic fungus known to produce zinniol, a compound described as a non-host selective toxin. Embryogenic cellular cultures from carrot genotypes varying in resistance against A. dauci were confronted with zinniol at different concentrations or to fungal exudates (raw, organic or aqueous extracts). The plant response was analyzed through the measurement of cytoplasmic esterase activity, as a marker of cell viability, and the differentiation of somatic embryos in cellular cultures. A differential response to toxicity was demonstrated between susceptible and partially resistant genotypes, with a good correlation noted between the resistance to the fungus at the whole plant level and resistance at the cellular level to fungal exudates from raw and organic extracts. No toxic reaction of embryogenic cultures was observed after treatment with the aqueous extract or zinniol used at physiological concentration. Moreover, we did not detect zinniol in toxic fungal extracts by UHPLC analysis. These results suggest that strong phytotoxic compounds are present in the organic extract and remain to be characterized. Our results clearly show that carrot tolerance to A. dauci toxins is one component of its partial resistance. PMID:24983469

Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy.

PubMed

Rappaz, Benjamin; Cano, Elena; Colomb, Tristan; Kühn, Jonas; Depeursinge, Christian; Simanis, Viesturs; Magistretti, Pierre J; Marquet, Pierre

2009-01-01

Digital holography microscopy (DHM) is an optical technique which provides phase images yielding quantitative information about cell structure and cellular dynamics. Furthermore, the quantitative phase images allow the derivation of other parameters, including dry mass production, density, and spatial distribution. We have applied DHM to study the dry mass production rate and the dry mass surface density in wild-type and mutant fission yeast cells. Our study demonstrates the applicability of DHM as a tool for label-free quantitative analysis of the cell cycle and opens the possibility for its use in high-throughput screening.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

PubMed

Calderón-Celis, Francisco; Encinar, Jorge Ruiz; Sanz-Medel, Alfredo

2017-07-31

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review. © 2017 Wiley Periodicals, Inc.

Systems cell biology.

PubMed

Mast, Fred D; Ratushny, Alexander V; Aitchison, John D

2014-09-15

Systems cell biology melds high-throughput experimentation with quantitative analysis and modeling to understand many critical processes that contribute to cellular organization and dynamics. Recently, there have been several advances in technology and in the application of modeling approaches that enable the exploration of the dynamic properties of cells. Merging technology and computation offers an opportunity to objectively address unsolved cellular mechanisms, and has revealed emergent properties and helped to gain a more comprehensive and fundamental understanding of cell biology. © 2014 Mast et al.

Molecular counting of membrane receptor subunits with single-molecule localization microscopy

NASA Astrophysics Data System (ADS)

Krüger, Carmen; Fricke, Franziska; Karathanasis, Christos; Dietz, Marina S.; Malkusch, Sebastian; Hummer, Gerhard; Heilemann, Mike

2017-02-01

We report on quantitative single-molecule localization microscopy, a method that next to super-resolved images of cellular structures provides information on protein copy numbers in protein clusters. This approach is based on the analysis of blinking cycles of single fluorophores, and on a model-free description of the distribution of the number of blinking events. We describe the experimental and analytical procedures, present cellular data of plasma membrane proteins and discuss the applicability of this method.

Sitagliptin attenuates sympathetic innervation via modulating reactive oxygen species and interstitial adenosine in infarcted rat hearts.

PubMed

Lee, Tsung-Ming; Chen, Wei-Ting; Yang, Chen-Chia; Lin, Shinn-Zong; Chang, Nen-Chung

2015-02-01

We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats. © 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Analysis of cellular signal transduction from an information theoretic approach.

PubMed

Uda, Shinsuke; Kuroda, Shinya

2016-03-01

Signal transduction processes the information of various cellular functions, including cell proliferation, differentiation, and death. The information for controlling cell fate is transmitted by concentrations of cellular signaling molecules. However, how much information is transmitted in signaling pathways has thus far not been investigated. Shannon's information theory paves the way to quantitatively analyze information transmission in signaling pathways. The theory has recently been applied to signal transduction, and mutual information of signal transduction has been determined to be a measure of information transmission. We review this work and provide an overview of how signal transduction transmits informational input and exerts biological output. Copyright © 2015 Elsevier Ltd. All rights reserved.

Three-color FRET expands the ability to quantify the interactions of several proteins involved in actin filament nucleation

NASA Astrophysics Data System (ADS)

Wallrabe, Horst; Sun, Yuansheng; Fang, Xiaolan; Periasamy, Ammasi; Bloom, George

2012-03-01

With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFPNWASP/ Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1- F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

PubMed

Rice, G C; Bump, E A; Shrieve, D C; Lee, W; Kovacs, M

1986-12-01

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking.

PubMed

Budayeva, Hanna G; Cristea, Ileana M

2016-10-01

Human sirtuin 2 (SIRT2) is an NAD + -dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Human Sirtuin 2 Localization, Transient Interactions, and Impact on the Proteome Point to Its Role in Intracellular Trafficking*

PubMed Central

Budayeva, Hanna G.; Cristea, Ileana M.

2016-01-01

Human sirtuin 2 (SIRT2) is an NAD+-dependent deacetylase that primarily functions in the cytoplasm, where it can regulate α-tubulin acetylation levels. SIRT2 is linked to cancer progression, neurodegeneration, and infection with bacteria or viruses. However, the current knowledge about its interactions and the means through which it exerts its functions has remained limited. Here, we aimed to gain a better understanding of its cellular functions by characterizing SIRT2 subcellular localization, the identity and relative stability of its protein interactions, and its impact on the proteome of primary human fibroblasts. To assess the relative stability of SIRT2 interactions, we used immunoaffinity purification in conjunction with both label-free and metabolic labeling quantitative mass spectrometry. In addition to the expected associations with cytoskeleton proteins, including its known substrate TUBA1A, our results reveal that SIRT2 specifically interacts with proteins functioning in membrane trafficking, secretory processes, and transcriptional regulation. By quantifying their relative stability, we found most interactions to be transient, indicating a dynamic SIRT2 environment. We discover that SIRT2 localizes to the ER-Golgi intermediate compartment (ERGIC), and that this recruitment requires an intact ER-Golgi trafficking pathway. Further expanding these findings, we used microscopy and interaction assays to establish the interaction and coregulation of SIRT2 with liprin-β1 scaffolding protein (PPFiBP1), a protein with roles in focal adhesions disassembly. As SIRT2 functions may be accomplished via interactions, enzymatic activity, and transcriptional regulation, we next assessed the impact of SIRT2 levels on the cellular proteome. SIRT2 knockdown led to changes in the levels of proteins functioning in membrane trafficking, including some of its interaction partners. Altogether, our study expands the knowledge of SIRT2 cytoplasmic functions to define a previously unrecognized involvement in intracellular trafficking pathways, which may contribute to its roles in cellular homeostasis and human diseases. PMID:27503897

Calcium Sensor, NCS-1, Promotes Tumor Aggressiveness and Predicts Patient Survival.

PubMed

Moore, Lauren M; England, Allison; Ehrlich, Barbara E; Rimm, David L

2017-07-01

Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional Ca 2+ -binding protein that affects a range of cellular processes beyond those related to neurons. Functional characterization of NCS-1 in neuronal model systems suggests that NCS-1 may influence oncogenic processes. To this end, the biological role of NCS-1 was investigated by altering its endogenous expression in MCF-7 and MB-231 breast cancer cells. Overexpression of NCS-1 resulted in a more aggressive tumor phenotype demonstrated by a marked increase in invasion and motility, and a decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitaxel-induced cell death in a manner that was independent of cellular proliferation. To determine the association between NCS-1 and clinical outcome, NCS-1 expression was measured in two independent breast cancer cohorts by the Automated Quantitative Analysis method of quantitative immunofluorescence. Elevated levels of NCS-1 were significantly correlated with shorter survival rates. Furthermore, multivariate analysis demonstrated that NCS-1 status was prognostic, independent of estrogen receptor, progesterone receptor, HER2, and lymph node status. These findings indicate that NCS-1 plays a role in the aggressive behavior of a subset of breast cancers and has therapeutic or biomarker potential. Implications: NCS-1, a calcium-binding protein, is associated with clinicopathologic features of aggressiveness in breast cancer cells and worse outcome in two breast cancer patient cohorts. Mol Cancer Res; 15(7); 942-52. ©2017 AACR . ©2017 American Association for Cancer Research.

Comprehensive Analysis of Proteomic Differences between Escherichia coli K-12 and B Strains Using Multiplexed Isobaric Tandem Mass Tag (TMT) Labeling.

PubMed

Han, Mee-Jung

2017-11-28

The Escherichia coli K-12 and B strains are among the most frequently used bacterial hosts for scientific research and biotechnological applications. However, omics analyses have revealed that E. coli K-12 and B exhibit notably different genotypic and phenotypic attributes, even though they were derived from the same ancestor. In a previous study, we identified a limited number of proteins from the two strains using two-dimensional gel electrophoresis and tandem mass spectrometry (MS/MS). In this study, an in-depth analysis of the physiological behavior of the E. coli K-12 and B strains at the proteomic level was performed using six-plex isobaric tandem mass tag-based quantitative MS. Additionally, the best lysis buffer for increasing the efficiency of protein extraction was selected from three tested buffers prior to the quantitative proteomic analysis. This study identifies the largest number of proteins in the two E. coli strains reported to date and is the first to show the dynamics of these proteins. Notable differences in proteins associated with key cellular properties, including some metabolic pathways, the biosynthesis and degradation of amino acids, membrane integrity, cellular tolerance, and motility, were found between the two representative strains. Compared with previous studies, these proteomic results provide a more holistic view of the overall state of E. coli cells based on a single proteomic study and reveal significant insights into why the two strains show distinct phenotypes. Additionally, the resulting data provide in-depth information that will help fine-tune processes in the future.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

PubMed Central

Almassalha, Luay M.; Bauer, Greta M.; Chandler, John E.; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K.; Subramanian, Hariharan; Chandel, Navdeep S.; Szleifer, Igal; Backman, Vadim

2016-01-01

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells. PMID:27702891

Novel image analysis methods for quantification of in situ 3-D tendon cell and matrix strain.

PubMed

Fung, Ashley K; Paredes, J J; Andarawis-Puri, Nelly

2018-01-23

Macroscopic tendon loads modulate the cellular microenvironment leading to biological outcomes such as degeneration or repair. Previous studies have shown that damage accumulation and the phases of tendon healing are marked by significant changes in the extracellular matrix, but it remains unknown how mechanical forces of the extracellular matrix are translated to mechanotransduction pathways that ultimately drive the biological response. Our overarching hypothesis is that the unique relationship between extracellular matrix strain and cell deformation will dictate biological outcomes, prompting the need for quantitative methods to characterize the local strain environment. While 2-D methods have successfully calculated matrix strain and cell deformation, 3-D methods are necessary to capture the increased complexity that can arise due to high levels of anisotropy and out-of-plane motion, particularly in the disorganized, highly cellular, injured state. In this study, we validated the use of digital volume correlation methods to quantify 3-D matrix strain using images of naïve tendon cells, the collagen fiber matrix, and injured tendon cells. Additionally, naïve tendon cell images were used to develop novel methods for 3-D cell deformation and 3-D cell-matrix strain, which is defined as a quantitative measure of the relationship between matrix strain and cell deformation. The results support that these methods can be used to detect strains with high accuracy and can be further extended to an in vivo setting for observing temporal changes in cell and matrix mechanics during degeneration and healing. Copyright © 2017. Published by Elsevier Ltd.

Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast

PubMed Central

Dephoure, Noah; Hwang, Sunyoung; O'Sullivan, Ciara; Dodgson, Stacie E; Gygi, Steven P; Amon, Angelika; Torres, Eduardo M

2014-01-01

Aneuploidy causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. Here, we examined the consequences of aneuploidy on the proteome of aneuploid budding yeast strains. We show that although protein levels largely scale with gene copy number, subunits of multi-protein complexes are notable exceptions. Posttranslational mechanisms attenuate their expression when their encoding genes are in excess. Our proteomic analyses further revealed a novel aneuploidy-associated protein expression signature characteristic of altered metabolism and redox homeostasis. Indeed aneuploid cells harbor increased levels of reactive oxygen species (ROS). Interestingly, increased protein turnover attenuates ROS levels and this novel aneuploidy-associated signature and improves the fitness of most aneuploid strains. Our results show that aneuploidy causes alterations in metabolism and redox homeostasis. Cells respond to these alterations through both transcriptional and posttranscriptional mechanisms. DOI: http://dx.doi.org/10.7554/eLife.03023.001 PMID:25073701

mRNA levels of enzymes and receptors implicated in arachidonic acid metabolism in gliomas.

PubMed

De Armas, Rafael; Durand, Karine; Guillaudeau, Angélique; Weinbreck, Nicolas; Robert, Sandrine; Moreau, Jean-Jacques; Caire, François; Acosta, Gisela; Pebet, Matias; Chaunavel, Alain; Marin, Benoît; Labrousse, François; Denizot, Yves

2010-07-01

Gliomas are tumors of the central nervous system derived from glial cells. They show cellular heterogeneity and lack specific diagnostic markers. Although a possible role for the eicosanoid cascade has been suggested in glioma tumorigenesis, the relationship between enzymes and receptors implicated in arachidonic acid metabolism, with histological tumor type has not yet been determined. Quantitative real-time reverse transcription-polymerase chain reaction was performed to measure and compare transcript levels of enzymes and receptors implicated in both lipoxygenase and cyclooxygenase pathways between oligodendrogliomas, astrocytomas, glioblastomas and mixed oligoastrocytomas. Arachidonic acid metabolism-related enzymes and receptor transcripts (i) were underexpressed in classical oligodendrogliomas compared to astrocytomas and/or glioblastomas, (ii) differed between astrocytomas and glioblastomas and (iii) had an intermediate expression in mixed oligoastrocytomas. mRNA levels of enzymes and receptors implicated both in lipoxygenase and cyclooxygenase pathways differed significantly in gliomas according to the histological type. Copyright 2010 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Peptide-based, two-fluorophore, ratiometric probe for quantifying mobile zinc in biological solutions.

PubMed

Zhang, Daniel Y; Azrad, Maria; Demark-Wahnefried, Wendy; Frederickson, Christopher J; Lippard, Stephen J; Radford, Robert J

2015-02-20

Small-molecule fluorescent sensors are versatile agents for detecting mobile zinc in biology. Capitalizing on the abundance of validated mobile zinc probes, we devised a strategy for repurposing existing intensity-based sensors for quantitative applications. Using solid-phase peptide synthesis, we conjugated a zinc-sensitive Zinpyr-1 derivative and a zinc-insensitive 7-hydroxycoumarin derivative onto opposite ends of a rigid P9K peptide scaffold to create HcZ9, a ratiometric fluorescent probe for mobile zinc. A plate reader-based assay using HcZ9 was developed, the accuracy of which is comparable to that of atomic absorption spectroscopy. We investigated zinc accumulation in prostatic cells and zinc levels in human seminal fluid. When normal and tumorigenic cells are bathed in zinc-enriched media, cellular mobile zinc is buffered and changes slightly, but total zinc levels increase significantly. Quantification of mobile and total zinc levels in human seminal plasma revealed that the two are positively correlated with a Pearson's coefficient of 0.73.

Temporal proteomic analysis of HIV infection reveals remodelling of the host phosphoproteome by lentiviral Vif variants

PubMed Central

Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J

2016-01-01

Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223

Dynamic expression of viral and cellular microRNAs in infectious mononucleosis caused by primary Epstein-Barr virus infection in children.

PubMed

Gao, Liwei; Ai, Junhong; Xie, Zhengde; Zhou, Chen; Liu, Chunyan; Zhang, Hui; Shen, Kunling

2015-12-03

Epstein-Barr virus (EBV) was the first virus identified to encode microRNAs (miRNAs). Both of viral and human cellular miRNAs are important in EBV infection. However, the dynamic expression profile of miRNAs during primary EBV infection was unknown. This study aimed to investigate the dynamic expression profile of viral and cellular miRNAs in infectious mononucleosis (IM) caused by primary EBV infection. The levels of viral and cellular miRNAs were measured in fifteen pediatric IM patients at three different time-points. Fifteen healthy children who were seropositive for EBV were enrolled in the control group. Relative expression levels of miRNAs were detected by quantitative real-time PCR (qPCR) assay. EBV-miR-BHRF1-1, 1-2-3P, miR-BART13-1, 19-3p, 11-3P, 12-1, and 16-1 in IM patients of early phase were significantly higher than in healthy children. Most cellular miRNAs of B cells, such as hsa-miR-155-5p, -34a-5p, -18b-5p, -181a-5p, and -142-5p were up-regulated; while most of cellular miRNAs of CD8 + T cells, such as hsa-miR-223, -29c-3p, -181a, -200a-3p, miR-155-5p, -146a, and -142-5p were down-regulated in IM patients. With disease progression, nearly all of EBV-miRNAs decreased, especially miR-BHRF1, but at a slower rate than EBV DNA loads. Most of the cellular miRNAs of B cells, including hsa-miR-134-5p, -18b-5p, -34a-5p, and -196a-5p increased with time. However, most of the cellular miRNAs of CD8 + T cells, including hsa-let-7a-5p, -142-3p, -142-5p, and -155-5p decreased with time. Additionally, hsa-miR-155-5p of B cells and hsa-miR-18b-5p of CD8+ T cells exhibited a positive correlation with miR-BHRF1-2-5P and miR-BART2-5P (0.96 ≤ r ≤ 0.99, P < 0.05). Finally, hsa-miR-181a-5p of B cells had positive correlation with miR-BART4-3p, 4-5P, 16-1, and 22 (0.97 ≤ r ≤ 0.99, P < 0.05). Our study is the first to describe the expression profile of viral and cellular miRNAs in IM caused by primary EBV infection. These results might be the basis of investigating the pathogenic mechanism of EBV-related diseases and bring new insights into their diagnosis and treatment.

Liquid chromatography-tandem mass spectrometric assay for the T790M mutant EGFR inhibitor osimertinib (AZD9291) in human plasma.

PubMed

Rood, Johannes J M; van Bussel, Mark T J; Schellens, Jan H M; Beijnen, Jos H; Sparidans, Rolf W

2016-09-15

A method for the quantitative analysis by ultra-performance liquid chromatography-tandem mass spectrometry of the highly selective irreversible covalent inhibitor of EGFR-TK, osimertinib in human plasma was developed and validated, using pazopanib as an internal standard. The validation was performed in a range from 1 to 1000ng/ml, with the lowest level corresponding to the lower limit of quantitation. Gradient elution was performed on a 1.8μm particle trifunctional bonded C18 column by 1% (v/v) formic acid in water, and acetonitrile as mobile phase. The analyte was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer after positive ionization with the heated electrospray interface. Within-day precisions ranged from 3.4 to 10.3%, and between-day precisions from 3.8 to 10.4%, accuracies were 95.5-102.8%. Plasma (either lithium heparin or sodium EDTA) pretreatment was performed by salting-out assisted liquid-liquid extraction using acetonitrile and magnesium sulfate. This method was used to analyze the osimertinib blood plasma levels of five adult patients with metastatic T790M mutated non-small cellular lung carcinoma for therapeutic drug monitoring purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

Modeling cognitive deficits following neurodegenerative diseases and traumatic brain injuries with deep convolutional neural networks.

PubMed

Lusch, Bethany; Weholt, Jake; Maia, Pedro D; Kutz, J Nathan

2018-06-01

The accurate diagnosis and assessment of neurodegenerative disease and traumatic brain injuries (TBI) remain open challenges. Both cause cognitive and functional deficits due to focal axonal swellings (FAS), but it is difficult to deliver a prognosis due to our limited ability to assess damaged neurons at a cellular level in vivo. We simulate the effects of neurodegenerative disease and TBI using convolutional neural networks (CNNs) as our model of cognition. We utilize biophysically relevant statistical data on FAS to damage the connections in CNNs in a functionally relevant way. We incorporate energy constraints on the brain by pruning the CNNs to be less over-engineered. Qualitatively, we demonstrate that damage leads to human-like mistakes. Our experiments also provide quantitative assessments of how accuracy is affected by various types and levels of damage. The deficit resulting from a fixed amount of damage greatly depends on which connections are randomly injured, providing intuition for why it is difficult to predict impairments. There is a large degree of subjectivity when it comes to interpreting cognitive deficits from complex systems such as the human brain. However, we provide important insight and a quantitative framework for disorders in which FAS are implicated. Copyright © 2018 Elsevier Inc. All rights reserved.

Quantitative proteomics links metabolic pathways to specific developmental stages of the plant-pathogenic oomycete Phytophthora capsici.

PubMed

Pang, Zhili; Srivastava, Vaibhav; Liu, Xili; Bulone, Vincent

2017-04-01

The oomycete Phytophthora capsici is a plant pathogen responsible for important losses to vegetable production worldwide. Its asexual reproduction plays an important role in the rapid propagation and spread of the disease in the field. A global proteomics study was conducted to compare two key asexual life stages of P. capsici, i.e. the mycelium and cysts, to identify stage-specific biochemical processes. A total of 1200 proteins was identified using qualitative and quantitative proteomics. The transcript abundance of some of the enriched proteins was also analysed by quantitative real-time polymerase chain reaction. Seventy-three proteins exhibited different levels of abundance between the mycelium and cysts. The proteins enriched in the mycelium are mainly associated with glycolysis, the tricarboxylic acid (or citric acid) cycle and the pentose phosphate pathway, providing the energy required for the biosynthesis of cellular building blocks and hyphal growth. In contrast, the proteins that are predominant in cysts are essentially involved in fatty acid degradation, suggesting that the early infection stage of the pathogen relies primarily on fatty acid degradation for energy production. The data provide a better understanding of P. capsici biology and suggest potential metabolic targets at the two different developmental stages for disease control. © 2016 BSPP AND JOHN WILEY & SONS LTD.

Comparative analysis of the surface exposed proteome of two canine osteosarcoma cell lines and normal canine osteoblasts

PubMed Central

2013-01-01

Background Osteosarcoma (OSA) is the most common primary bone tumor of dogs and carries a poor prognosis despite aggressive treatment. An improved understanding of the biology of OSA is critically needed to allow for development of novel diagnostic, prognostic, and therapeutic tools. The surface-exposed proteome (SEP) of a cancerous cell includes a multifarious array of proteins critical to cellular processes such as proliferation, migration, adhesion, and inter-cellular communication. The specific aim of this study was to define a SEP profile of two validated canine OSA cell lines and a normal canine osteoblast cell line utilizing a biotinylation/streptavidin system to selectively label, purify, and identify surface-exposed proteins by mass spectrometry (MS) analysis. Additionally, we sought to validate a subset of our MS-based observations via quantitative real-time PCR, Western blot and semi-quantitative immunocytochemistry. Our hypothesis was that MS would detect differences in the SEP composition between the OSA and the normal osteoblast cells. Results Shotgun MS identified 133 putative surface proteins when output from all samples were combined, with good consistency between biological replicates. Eleven of the MS-detected proteins underwent analysis of gene expression by PCR, all of which were actively transcribed, but varied in expression level. Western blot of whole cell lysates from all three cell lines was effective for Thrombospondin-1, CYR61 and CD44, and indicated that all three proteins were present in each cell line. Semi-quantitative immunofluorescence indicated that CD44 was expressed at much higher levels on the surface of the OSA than the normal osteoblast cell lines. Conclusions The results of the present study identified numerous differences, and similarities, in the SEP of canine OSA cell lines and normal canine osteoblasts. The PCR, Western blot, and immunocytochemistry results, for the subset of proteins evaluated, were generally supportive of the mass spectrometry data. These methods may be applied to other cell lines, or other biological materials, to highlight unique and previously unrecognized differences between samples. While this study yielded data that may prove useful for OSA researchers and clinicians, further refinements of the described techniques are expected to yield greater accuracy and produce a more thorough SEP analysis. PMID:23758893

The genetics and transcriptional profiles of the cellulose synthase-like HvCslF gene family in barley.

PubMed

Burton, Rachel A; Jobling, Stephen A; Harvey, Andrew J; Shirley, Neil J; Mather, Diane E; Bacic, Antony; Fincher, Geoffrey B

2008-04-01

Cellulose synthase-like CslF genes have been implicated in the biosynthesis of (1,3;1,4)-beta-d-glucans, which are major cell wall constituents in grasses and cereals. Seven CslF genes from barley (Hordeum vulgare) can be divided into two classes on the basis of intron-exon arrangements. Four of the HvCslF genes have been mapped to a single locus on barley chromosome 2H, in a region corresponding to a major quantitative trait locus for grain (1,3;1,4)-beta-d-glucan content. The other HvCslF genes map to chromosomes 1H, 5H, and 7H, and in two cases the genes are close to other quantitative trait loci for grain (1,3;1,4)-beta-d-glucan content. Spatial and temporal patterns of transcription of the seven genes have been defined through quantitative polymerase chain reaction. In developing barley coleoptiles HvCslF6 mRNA is most abundant. Transcript levels are maximal in 4- to 5-d coleoptiles, at a time when (1,3;1,4)-beta-d-glucan content of coleoptile cell walls also reaches maximal levels. In the starchy endosperm of developing grain, HvCslF6 and HvCslF9 transcripts predominate. Two peaks of transcription are apparent. One occurs just after endosperm cellularization, 4 to 8 d after pollination, while the second occurs much later in grain development, more than 20 d after pollination. Marked varietal differences in transcription of the HvCslF genes are observed during endosperm development. Given the commercial importance of cereal (1,3;1,4)-beta-d-glucans in human nutrition, in stock feed, and in malting and brewing, the observation that only two genes, HvCslF6 and HvCslF9, are transcribed at high levels in developing grain is of potential relevance for the future manipulation of grain (1,3;1,4)-beta-d-glucan levels.

Effects of cell phone radiation on lipid peroxidation, glutathione and nitric oxide levels in mouse brain during epileptic seizure.

PubMed

Esmekaya, Meric Arda; Tuysuz, Mehmet Zahid; Tomruk, Arın; Canseven, Ayse G; Yücel, Engin; Aktuna, Zuhal; Keskil, Semih; Seyhan, Nesrin

2016-09-01

The objective of the this study was to evaluate the effects of cellular phone radiation on oxidative stress parameters and oxide levels in mouse brain during pentylenetetrazole (PTZ) induced epileptic seizure. Eight weeks old mice were used in the study. Animals were distributed in the following groups: Group I: Control group treated with PTZ, Group II: 15min cellular phone radiation+PTZ treatment+30min cellular phone radiation, Group III: 30min cellular phone radiation+PTZ treatment+30min cellular phone radiation. The RF radiation was produced by a 900MHz cellular phone. Lipid peroxidation, which is the indicator of oxidative stress was quantified by measuring the formation of thiobarbituric acid reactive substances (TBARS). The glutathione (GSH) levels were determined by the Ellman method. Tissue total nitric oxide (NOx) levels were obtained using the Griess assay. Lipid peroxidation and NOx levels of brain tissue increased significantly in group II and III compared to group I. On the contrary, GSH levels were significantly lower in group II and III than group I. However, no statistically significant alterations in any of the endpoints were noted between group II and Group III. Overall, the experimental findings demonstrated that cellular phone radiation may increase the oxidative damage and NOx level during epileptic activity in mouse brain. Copyright © 2016 Elsevier B.V. All rights reserved.

A quantitative study of exocytosis of titanium dioxide nanoparticles from neural stem cells

NASA Astrophysics Data System (ADS)

Wang, Yanli; Wu, Qiuxia; Sui, Keke; Chen, Xin-Xin; Fang, Jie; Hu, Xuefeng; Wu, Minghong; Liu, Yuanfang

2013-05-01

Nanoparticles (NPs) have been widely studied and applied in biomedicine and other fields. It is important to know the basic process of interaction between NPs and cells in terms of cellular endocytosis and exocytosis. However, little attention has been paid to the cellular exocytosis of NPs. Herein, using a multi-step cellular subculture method, we ascertain quantitatively the endocytosis and exocytosis of widely used TiO2 NPs using the neural stem cells (NSC) as a cellular model and ICP-AES as an analytic measure. Irrespective of the type and dose of TiO2 NPs, approximately 30% of the total TiO2 NPs entered NSCs after 48 h incubation. In the first 24 h after removing TiO2NPs, from the culture medium, about 35.0%, 34.6% and 41.7% of NP1 (50 nm), NP2 (30 nm) and NTs (nanotubes, 100 nm × 4-6 nm) were released (exocytosed) from cells, respectively. The release decreased over time, and became negligible at 72 h. Exocytosis did not happen during cell division. In addition, our results suggested that both endocytosis and exocytosis of TiO2NPs were energy-dependent processes, and NPs uptake by cells was influenced by serum proteins. Furthermore, we achieved primary dynamic confocal imaging of the exocytosis, allowing tracking of TiO2 NPs from NSCs. These findings may benefit studies on nanotoxicology and nanomedicine of TiO2 NPs.Nanoparticles (NPs) have been widely studied and applied in biomedicine and other fields. It is important to know the basic process of interaction between NPs and cells in terms of cellular endocytosis and exocytosis. However, little attention has been paid to the cellular exocytosis of NPs. Herein, using a multi-step cellular subculture method, we ascertain quantitatively the endocytosis and exocytosis of widely used TiO2 NPs using the neural stem cells (NSC) as a cellular model and ICP-AES as an analytic measure. Irrespective of the type and dose of TiO2 NPs, approximately 30% of the total TiO2 NPs entered NSCs after 48 h incubation. In the first 24 h after removing TiO2NPs, from the culture medium, about 35.0%, 34.6% and 41.7% of NP1 (50 nm), NP2 (30 nm) and NTs (nanotubes, 100 nm × 4-6 nm) were released (exocytosed) from cells, respectively. The release decreased over time, and became negligible at 72 h. Exocytosis did not happen during cell division. In addition, our results suggested that both endocytosis and exocytosis of TiO2NPs were energy-dependent processes, and NPs uptake by cells was influenced by serum proteins. Furthermore, we achieved primary dynamic confocal imaging of the exocytosis, allowing tracking of TiO2 NPs from NSCs. These findings may benefit studies on nanotoxicology and nanomedicine of TiO2 NPs. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr00796k

An uncooked vegan diet shifts the profile of human fecal microflora: computerized analysis of direct stool sample gas-liquid chromatography profiles of bacterial cellular fatty acids.

PubMed Central

Peltonen, R; Ling, W H; Hänninen, O; Eerola, E

1992-01-01

The effect of an uncooked extreme vegan diet on fecal microflora was studied by direct stool sample gas-liquid chromatography (GLC) of bacterial cellular fatty acids and by quantitative bacterial culture by using classical microbiological techniques of isolation, identification, and enumeration of different bacterial species. Eighteen volunteers were divided randomly into two groups. The test group received an uncooked vegan diet for 1 month and a conventional diet of mixed Western type for the other month of the study. The control group consumed a conventional diet throughout the study period. Stool samples were collected. Bacterial cellular fatty acids were extracted directly from the stool samples and measured by GLC. Computerized analysis of the resulting fatty acid profiles was performed. Such a profile represents all bacterial cellular fatty acids in a sample and thus reflects its microflora and can be used to detect changes, differences, or similarities of bacterial flora between individual samples or sample groups. GLC profiles changed significantly in the test group after the induction and discontinuation of the vegan diet but not in the control group at any time, whereas quantitative bacterial culture did not detect any significant change in fecal bacteriology in either of the groups. The results suggest that an uncooked extreme vegan diet alters the fecal bacterial flora significantly when it is measured by direct stool sample GLC of bacterial fatty acids. PMID:1482187

Glucose supplement reverses the fasting-induced suppression of cellular immunity in Mongolian gerbils (Meriones unguiculatus).

PubMed

Xu, De-Li; Wang, De-Hua

2011-10-01

Glucose plays an important role in immunity. Three day fasting will decrease cellular immunity and blood glucose levels in Mongolian gerbils (Meriones unguiculatus). In the present study, we tested the hypothesis that glucose supplement can reverse the fasting-induced suppression in cellular immunity in gerbils. Twenty-eight male gerbils were selected and randomly divided into fed and fasting groups. Half of the gerbils in each group were then provided with either 10% glucose water or pure water. After 66 h, each gerbil was injected with phytohaemagglutinin (PHA) solution to challenge cellular immunity. Results showed that glucose supplement restored blood glucose levels in fasted gerbils to those of the fed controls. It also recovered cellular immunity, body fat mass and serum leptin levels in fasted gerbils to the values of the fed controls. Blood glucose levels were positively correlated with body fat mass, leptin levels and cellular immune responses. Thymus and spleen masses, and white blood cells in fasted gerbils were not affected by glucose supplement. In general, our data demonstrate that glucose supplement could reverse fasting-induced suppression of cellular immunity in Mongolian gerbils. Copyright © 2011 Elsevier GmbH. All rights reserved.

The Regulation of a Post-Translational Peptide Acetyltransferase: Strategies for Selectively Modifying the Biological Activity of Neural and Endocrine Peptides

DTIC Science & Technology

1988-02-01

quantitatively miror pathway. Only two of the enzymes which process 8-endorphin have been firmly identified, peptide acetyltransferase and... quantitatively minor. This implied that perhaps peptide acetyltransferase is not a critical determinant of the bioactivity of B-endorphin in brain. If so...provided us with a more difinitive understanding of the role of processing enzyme regulation in the overall biochemical and cellular response of the

A tunable ratiometric pH sensor based on carbon nanodots for the quantitative measurement of the intracellular pH of whole cells.

PubMed

Shi, Wen; Li, Xiaohua; Ma, Huimin

2012-06-25

The whole picture: Carbon nanodots labeled with two fluorescent dyes have been developed as a tunable ratiometric pH sensor to measure intracellular pH. The nanosensor shows good biocompatibility and cellular dispersibility. Quantitative determinations on intact HeLa cells and pH fluctuations associated with oxidative stress were performed. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of fluorescent dye substrates for functional characterization of ABC multidrug transporters at a single cell level.

PubMed

Nerada, Zsuzsanna; Hegyi, Zoltán; Szepesi, Áron; Tóth, Szilárd; Hegedüs, Csilla; Várady, György; Matula, Zsolt; Homolya, László; Sarkadi, Balázs; Telbisz, Ágnes

2016-09-01

ABC multidrug transporters are key players in cancer multidrug resistance and in determining the ADME-Tox properties of drugs and xenobiotics. The most sensitive and specific detection of these transporters is based on functional assays. Assessment of the transporter-dependent reduction of cellular uptake of the fluorescent dyes, such as Hoechst 33342 (Ho) and more recently DyeCycle Violet (DCV), have been widely advocated for the characterization of both ABCB1 and ABCG2 multidrug transporters. Detailed comparison of these supravital DNA-binding dyes revealed that DCV is less toxic to ABCG2- and ABCB1-expressing cells than Ho. ATPase measurements imply that DCV and Ho are similarly handled by ABCB1, whereas ABCG2 seems to transport DVC more effectively. In addition, we have developed an image-based high content microscopy screening method for simultaneous in situ measurement of the cellular activity and expression of the ABCG2 multidrug transporter. We demonstrated the applicability of this method for identifying ABCG2-positive cells in heterogeneous cell population by a single dye uptake measurement. These results may promote multidrug transporter studies at a single cell level and allow the quantitative detection of clinically important drug-resistant sub-populations. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

PubMed Central

Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

2016-01-01

Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells.

PubMed

Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

2009-08-01

Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements

PubMed Central

Uhlirova, Hana; Kılıç, Kıvılcım; Tian, Peifang; Sakadžić, Sava; Thunemann, Martin; Desjardins, Michèle; Saisan, Payam A.; Nizar, Krystal; Yaseen, Mohammad A.; Hagler, Donald J.; Vandenberghe, Matthieu; Djurovic, Srdjan; Andreassen, Ole A.; Silva, Gabriel A.; Masliah, Eliezer; Vinogradov, Sergei; Buxton, Richard B.; Einevoll, Gaute T.; Boas, David A.; Dale, Anders M.; Devor, Anna

2016-01-01

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed ‘bottom-up’ forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the ‘ground truth’ for the development of new tools for tackling the inverse problem—estimation of neuronal activity from multimodal non-invasive imaging data. This article is part of the themed issue ‘Interpreting BOLD: a dialogue between cognitive and cellular neuroscience’. PMID:27574309

Synergistic effects of micropatterned biodegradable conduits and Schwann cells on sciatic nerve regeneration

NASA Astrophysics Data System (ADS)

Rutkowski, Gregory E.; Miller, Cheryl A.; Jeftinija, Srdija; Mallapragada, Surya K.

2004-09-01

This paper describes a novel biodegradable conduit that provides a combination of physical, chemical and biological cues at the cellular level to facilitate peripheral nerve regeneration. The conduit consists of a porous poly(D,L-lactic acid) (PDLLA) tubular support structure with a micropatterned inner lumen. Schwann cells were pre-seeded into the lumen to provide additional trophic support. Conduits with micropatterned inner lumens pre-seeded with Schwann cells (MS) were fabricated and compared with three types of conduits used as controls: M (conduits with micropatterned inner lumens without pre-seeded Schwann cells), NS (conduits without micropatterned inner lumens pre-seeded with Schwann cells) and N (conduits without micropatterned inner lumens, without pre-seeded Schwann cells). The conduits were implanted in rats with 1 cm sciatic nerve transections and the regeneration and functional recovery were compared in the four different cases. The number or size of regenerated axons did not vary significantly among the different conduits. The time of recovery, and the sciatic function index, however, were significantly enhanced using the MS conduits, based on qualitative observations as well as quantitative measurements using walking track analysis. This demonstrates that biodegradable micropatterned conduits pre-seeded with Schwann cells that provide a combination of physical, chemical and biological guidance cues for regenerating axons at the cellular level offer a better alternative for repairing sciatic nerve transactions than conventional biodegradable conduits.

Quantitative expression and localization of cysteine and aspartic proteases in human abdominal aortic aneurysms

PubMed Central

Lohoefer, Fabian; Reeps, Christian; Lipp, Christina; Rudelius, Martina; Haertl, Felix; Matevossian, Edouard; Zernecke, Alma; Eckstein, Hans-Henning; Pelisek, Jaroslav

2014-01-01

Cysteine and aspartic proteases possess high elastolytic activity and might contribute to the degradation of the abdominal aortic aneurysm (AAA) wall. The aim of this study was to analyze, in detail, the proteases (cathepsins B, D, K, L and S, and inhibitor cystatin C) found in human AAA and healthy aortic tissue samples. The vessel walls from AAA patients (n=36) and nonaneurysmal aortae (n=10) were retrieved using conventional surgical repair and autopsy methods. Serum samples from the same AAA patients and 10 healthy volunteers were also collected. Quantitative expression analyses were performed at the mRNA level using real-time reverse transcriptase-PCR (RT–PCR). Furthermore, analyses at the protein level included western blot and immunoprecipitation analyses. Cellular sources of cysteine/aspartic proteases and cystatin C were identified by immunohistochemistry (IHC). All cysteine/aspartic proteases and cystatin C were detected in the AAA and control samples. Using quantitative RT–PCR, a significant increase in expression was observed for cathepsins B (P=0.021) and L (P=0.018), compared with the controls. Cathepsin B and cystatin C were also detected in the serum of AAA patients. Using IHC, smooth muscle cells (SMCs) and macrophages were positive for all of the tested cathepsins, as well as cystatin C; in addition, the lymphocytes were mainly positive for cathepsin B, followed by cathepsins D and S. All cysteine/aspartic proteases analyzed in our study were detected in the AAA and healthy aorta. The highest expression was found in macrophages and SMCs. Consequently, cysteine/aspartic proteases might play a substantial role in AAA. PMID:24833013

Quantitative Analysis of the KSHV Transcriptome Following Primary Infection of Blood and Lymphatic Endothelial Cells

PubMed Central

Bruce, A. Gregory; Barcy, Serge; DiMaio, Terri; Gan, Emilia; Garrigues, H. Jacques; Lagunoff, Michael; Rose, Timothy M.

2017-01-01

The transcriptome of the Kaposi’s sarcoma-associated herpesvirus (KSHV/HHV8) after primary latent infection of human blood (BEC), lymphatic (LEC) and immortalized (TIME) endothelial cells was analyzed using RNAseq, and compared to long-term latency in BCBL-1 lymphoma cells. Naturally expressed transcripts were obtained without artificial induction, and a comprehensive annotation of the KSHV genome was determined. A set of unique coding sequence (UCDS) features and a process to resolve overlapping transcripts were developed to accurately quantitate transcript levels from specific promoters. Similar patterns of KSHV expression were detected in BCBL-1 cells undergoing long-term latent infections and in primary latent infections of both BEC and LEC cultures. High expression levels of poly-adenylated nuclear (PAN) RNA and spliced and unspliced transcripts encoding the K12 Kaposin B/C complex and associated microRNA region were detected, with an elevated expression of a large set of lytic genes in all latently infected cultures. Quantitation of non-overlapping regions of transcripts across the complete KSHV genome enabled for the first time accurate evaluation of the KSHV transcriptome associated with viral latency in different cell types. Hierarchical clustering applied to a gene correlation matrix identified modules of co-regulated genes with similar correlation profiles, which corresponded with biological and functional similarities of the encoded gene products. Gene modules were differentially upregulated during latency in specific cell types indicating a role for cellular factors associated with differentiated and/or proliferative states of the host cell to influence viral gene expression. PMID:28335496

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

PubMed

Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

2015-04-01

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

PubMed

Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

2015-10-30

Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

Cellular water distribution, transport, and its investigation methods for plant-based food material.

PubMed

Khan, Md Imran H; Karim, M A

2017-09-01

Heterogeneous and hygroscopic characteristics of plant-based food material make it complex in structure, and therefore water distribution in its different cellular environments is very complex. There are three different cellular environments, namely the intercellular environment, the intracellular environment, and the cell wall environment inside the food structure. According to the bonding strength, intracellular water is defined as loosely bound water, cell wall water is categorized as strongly bound water, and intercellular water is known as free water (FW). During food drying, optimization of the heat and mass transfer process is crucial for the energy efficiency of the process and the quality of the product. For optimizing heat and mass transfer during food processing, understanding these three types of waters (strongly bound, loosely bound, and free water) in plant-based food material is essential. However, there are few studies that investigate cellular level water distribution and transport. As there is no direct method for determining the cellular level water distributions, various indirect methods have been applied to investigate the cellular level water distribution, and there is, as yet, no consensus on the appropriate method for measuring cellular level water in plant-based food material. Therefore, the main aim of this paper is to present a comprehensive review on the available methods to investigate the cellular level water, the characteristics of water at different cellular levels and its transport mechanism during drying. The effect of bound water transport on quality of food product is also discussed. This review article presents a comparative study of different methods that can be applied to investigate cellular water such as nuclear magnetic resonance (NMR), bioelectric impedance analysis (BIA), differential scanning calorimetry (DSC), and dilatometry. The article closes with a discussion of current challenges to investigating cellular water. Copyright © 2017 Elsevier Ltd. All rights reserved.

NMR-based Metabolomics for Cancer Research

EPA Science Inventory

Metabolomics is considered as a complementary tool to other omics platforms to provide a snapshot of the cellular biochemistry and physiology taking place at any instant. Metabolmics approaches have been widely used to provide comprehensive and quantitative analyses of the metabo...

INTERNALIZATION AND FATE OF INDIVIDUAL MANUFACTURED NANOMATERIAL WITHIN LIVING CELLS

EPA Science Inventory

Using quantitative fluorescence imaging with single molecule sensitivity, combined with molecular biology techniques, we have been investigating the cellular interactions and fate of one nanoparticle or nanoscale aggregate at a time, identifying molecular interactions and cellula...

Comparison between the early cellular response to electron radiation and the production of tumors

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

Sinclair, Ian P.

1972-11-01

The aim of the study is to quantitate radiation-induced cell population changes in various epithelial components of rat skin and to correlate them, if possible with the subsequent production of tumors.

Letting the cat out of the bag: a personal journey in Biophysics.

PubMed

Bustamante, Carlos J

2014-10-08

When the author arrived in Berkeley, in the mid 1970s, to study Biophysics he soon felt as if he was engaging himself in a somewhat marginal activity. Biology was then entering another of its cyclical periods of annotation that was to culminate with the human genome project. Two decades later, however, at the end of this process, it had become clear that two main tasks were acquiring a central importance in biological research: a renewed push for a quantitative, precise description of biological systems at the molecular level, and efforts towards an integrated understanding of the operation, control, and coordination of cellular processes. Today, these have become two of the most fertile research areas in Biophysics.

Application of biospeckles for assessment of structural and cellular changes in muscle tissue

NASA Astrophysics Data System (ADS)

Maksymenko, Oleksandr P.; Muravsky, Leonid I.; Berezyuk, Mykola I.

2015-09-01

A modified spatial-temporal speckle correlation technique for operational assessment of structural changes in muscle tissues after slaughtering is considered. Coefficient of biological activity as a quantitative indicator of structural changes of biochemical processes in biological tissues is proposed. The experimental results have shown that this coefficient properly evaluates the biological activity of pig and chicken muscle tissue samples. Studying the degradation processes in muscle tissue during long-time storage in a refrigerator by measuring the spatial-temporal dynamics of biospeckle patterns is carried out. The reduction of the bioactivity level of refrigerated muscle tissue samples connected with the initiation of muscle fiber cracks and ruptures, reduction of sarcomeres, nuclei deformation, nuclear chromatin diminishing, and destruction of mitochondria is analyzed.

PCR array analysis of gene expression profiles in chronic active Epstein-Barr virus infection.

PubMed

Murakami, Masanao; Hashida, Yumiko; Imajoh, Masayuki; Maeda, Akihiko; Kamioka, Mikio; Senda, Yasutaka; Sato, Tetsuya; Fujieda, Mikiya; Wakiguchi, Hiroshi; Daibata, Masanori

2014-07-01

To determine the host cellular gene expression profiles in chronic active Epstein-Barr virus infection (CAEBV), peripheral blood samples were obtained from three patients with CAEBV and investigated using a PCR array analysis that focused on T-cell/B-cell activation. We identified six genes with expression levels that were tenfold higher in CAEBV patients compared with those in healthy controls. These results were verified by quantitative reverse transcription-PCR. We identified four highly upregulated genes, i.e., IL-10, IL-2, IFNGR1, and INHBA. These genes may be involved in inflammatory responses and cell proliferation, and they may contribute to the development and progression of CAEBV. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Calibration of the optical torque wrench.

PubMed

Pedaci, Francesco; Huang, Zhuangxiong; van Oene, Maarten; Dekker, Nynke H

2012-02-13

The optical torque wrench is a laser trapping technique that expands the capability of standard optical tweezers to torque manipulation and measurement, using the laser linear polarization to orient tailored microscopic birefringent particles. The ability to measure torque of the order of kBT (∼4 pN nm) is especially important in the study of biophysical systems at the molecular and cellular level. Quantitative torque measurements rely on an accurate calibration of the instrument. Here we describe and implement a set of calibration approaches for the optical torque wrench, including methods that have direct analogs in linear optical tweezers as well as introducing others that are specifically developed for the angular variables. We compare the different methods, analyze their differences, and make recommendations regarding their implementations.

Selection of stable reference genes for quantitative rt-PCR comparisons of mouse embryonic and extra-embryonic stem cells.

PubMed

Veazey, Kylee J; Golding, Michael C

2011-01-01

Isolation and culture of both embryonic and tissue specific stem cells provide an enormous opportunity to study the molecular processes driving development. To gain insight into the initial events underpinning mammalian embryogenesis, pluripotent stem cells from each of the three distinct lineages present within the preimplantation blastocyst have been derived. Embryonic (ES), trophectoderm (TS) and extraembryonic endoderm (XEN) stem cells possess the developmental potential of their founding lineages and seemingly utilize distinct epigenetic modalities to program gene expression. However, the basis for these differing cellular identities and epigenetic properties remain poorly defined.Quantitative reverse transcription-polymerase chain reaction (qPCR) is a powerful and efficient means of rapidly comparing patterns of gene expression between different developmental stages and experimental conditions. However, careful, empirical selection of appropriate reference genes is essential to accurately measuring transcriptional differences. Here we report the quantitation and evaluation of fourteen commonly used references genes between ES, TS and XEN stem cells. These included: Actb, B2m, Hsp70, Gapdh, Gusb, H2afz, Hk2, Hprt, Pgk1, Ppia, Rn7sk, Sdha, Tbp and Ywhaz. Utilizing three independent statistical analysis, we identify Pgk1, Sdha and Tbp as the most stable reference genes between each of these stem cell types. Furthermore, we identify Sdha, Tbp and Ywhaz as well as Ywhaz, Pgk1 and Hk2 as the three most stable reference genes through the in vitro differentiation of embryonic and trophectoderm stem cells respectively.Understanding the transcriptional and epigenetic regulatory mechanisms controlling cellular identity within these distinct stem cell types provides essential insight into cellular processes controlling both embryogenesis and stem cell biology. Normalizing quantitative RT-PCR measurements using the geometric mean CT values obtained for the identified mRNAs, offers a reliable method to assess differing patterns of gene expression between the three founding stem cell lineages present within the mammalian preimplantation embryo.

Analyzing the texture changes in the quantitative phase maps of adipocytes

NASA Astrophysics Data System (ADS)

Roitshtain, Darina; Sharabani-Yosef, Orna; Gefen, Amit; Shaked, Natan T.

2016-03-01

We present a new analysis tool for studying texture changes in the quantitative phase maps of live cells acquired by wide-field interferometry. The sensitivity of wide-field interferometry systems to small changes in refractive index enables visualizing cells and inner cell organelles without the using fluorescent dyes or other cell-invasive approaches, which may affect the measurement and require external labeling. Our label-free texture-analysis tool is based directly on the optical path delay profile of the sample and does not necessitate decoupling refractive index and thickness in the cell quantitative phase profile; thus, relevant parameters can be calculated using a single-frame acquisition. Our experimental system includes low-coherence wide-field interferometer, combined with simultaneous florescence microscopy system for validation. We used this system and analysis tool for studying lipid droplets formation in adipocytes. The latter demonstration is relevant for various cellular functions such as lipid metabolism, protein storage and degradation to viral replication. These processes are functionally linked to several physiological and pathological conditions, including obesity and metabolic diseases. Quantification of these biological phenomena based on the texture changes in the cell phase map has a potential as a new cellular diagnosis tool.

In vivo confocal microscopy of the cornea: New developments in image acquisition, reconstruction and analysis using the HRT-Rostock Corneal Module

PubMed Central

Petroll, W. Matthew; Robertson, Danielle M.

2015-01-01

The optical sectioning ability of confocal microscopy allows high magnification images to be obtained from different depths within a thick tissue specimen, and is thus ideally suited to the study of intact tissue in living subjects. In vivo confocal microscopy has been used in a variety of corneal research and clinical applications since its development over 25 years ago. In this article we review the latest developments in quantitative corneal imaging with the Heidelberg Retinal Tomograph with Rostock Corneal Module (HRT-RCM). We provide an overview of the unique strengths and weaknesses of the HRT-RCM. We discuss techniques for performing 3-D imaging with the HRT-RCM, including hardware and software modifications that allow full thickness confocal microscopy through focusing (CMTF) of the cornea, which can provide quantitative measurements of corneal sublayer thicknesses, stromal cell and extracellular matrix backscatter, and depth dependent changes in corneal keratocyte density. We also review current approaches for quantitative imaging of the subbasal nerve plexus, which require a combination of advanced image acquisition and analysis procedures, including wide field mapping and 3-D reconstruction of nerve structures. The development of new hardware, software, and acquisition techniques continues to expand the number of applications of the HRT-RCM for quantitative in vivo corneal imaging at the cellular level. Knowledge of these rapidly evolving strategies should benefit corneal clinicians and basic scientists alike. PMID:25998608

An Ibm PC/AT-Based Image Acquisition And Processing System For Quantitative Image Analysis

NASA Astrophysics Data System (ADS)

Kim, Yongmin; Alexander, Thomas

1986-06-01

In recent years, a large number of applications have been developed for image processing systems in the area of biological imaging. We have already finished the development of a dedicated microcomputer-based image processing and analysis system for quantitative microscopy. The system's primary function has been to facilitate and ultimately automate quantitative image analysis tasks such as the measurement of cellular DNA contents. We have recognized from this development experience, and interaction with system users, biologists and technicians, that the increasingly widespread use of image processing systems, and the development and application of new techniques for utilizing the capabilities of such systems, would generate a need for some kind of inexpensive general purpose image acquisition and processing system specially tailored for the needs of the medical community. We are currently engaged in the development and testing of hardware and software for a fairly high-performance image processing computer system based on a popular personal computer. In this paper, we describe the design and development of this system. Biological image processing computer systems have now reached a level of hardware and software refinement where they could become convenient image analysis tools for biologists. The development of a general purpose image processing system for quantitative image analysis that is inexpensive, flexible, and easy-to-use represents a significant step towards making the microscopic digital image processing techniques more widely applicable not only in a research environment as a biologist's workstation, but also in clinical environments as a diagnostic tool.

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

PubMed

Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

2015-05-01

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

Classification of Normal and Apoptotic Cells from Fluorescence Microscopy Images Using Generalized Polynomial Chaos and Level Set Function.

PubMed

Du, Yuncheng; Budman, Hector M; Duever, Thomas A

2016-06-01

Accurate automated quantitative analysis of living cells based on fluorescence microscopy images can be very useful for fast evaluation of experimental outcomes and cell culture protocols. In this work, an algorithm is developed for fast differentiation of normal and apoptotic viable Chinese hamster ovary (CHO) cells. For effective segmentation of cell images, a stochastic segmentation algorithm is developed by combining a generalized polynomial chaos expansion with a level set function-based segmentation algorithm. This approach provides a probabilistic description of the segmented cellular regions along the boundary, from which it is possible to calculate morphological changes related to apoptosis, i.e., the curvature and length of a cell's boundary. These features are then used as inputs to a support vector machine (SVM) classifier that is trained to distinguish between normal and apoptotic viable states of CHO cell images. The use of morphological features obtained from the stochastic level set segmentation of cell images in combination with the trained SVM classifier is more efficient in terms of differentiation accuracy as compared with the original deterministic level set method.

Quantitative readout of optically encoded gold nanorods using an ordinary dark-field microscope.

PubMed

Mercatelli, Raffaella; Ratto, Fulvio; Centi, Sonia; Soria, Silvia; Romano, Giovanni; Matteini, Paolo; Quercioli, Franco; Pini, Roberto; Fusi, Franco

2013-10-21

In this paper we report on a new use for dark-field microscopy in order to retrieve two-dimensional maps of optical parameters of a thin sample such as a cryptograph, a histological section, or a cell monolayer. In particular, we discuss the construction of quantitative charts of light absorbance and scattering coefficients of a polyvinyl alcohol film that was embedded with gold nanorods and then etched using a focused mode-locked Ti:Sapphire oscillator. Individual pulses from this laser excite plasmonic oscillations of the gold nanorods, thus triggering plastic deformations of the particles and their environment, which are confined within a few hundred nm of the light focus. In turn, these deformations modify the light absorbance and scattering landscape, which can be measured with optical resolution in a dark-field microscope equipped with an objective of tuneable numerical aperture. This technique may prove to be valuable for various applications, such as the fast readout of optically encoded data or to model functional interactions between light and biological tissue at the level of cellular organelles, including the photothermolysis of cancer.

Quantitative Aspects of Single Molecule Microscopy

PubMed Central

Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

2015-01-01

Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

Morphological imaging and quantification of axial xylem tissue in Fraxinus excelsior L. through X-ray micro-computed tomography.

PubMed

Koddenberg, Tim; Militz, Holger

2018-05-05

The popularity of X-ray based imaging methods has continued to increase in research domains. In wood research, X-ray micro-computed tomography (XμCT) is useful for structural studies examining the three-dimensional and complex xylem tissue of trees qualitatively and quantitatively. In this study, XμCT made it possible to visualize and quantify the spatial xylem organization of the angiosperm species Fraxinus excelsior L. on the microscopic level. Through image analysis, it was possible to determine morphological characteristics of the cellular axial tissue (vessel elements, fibers, and axial parenchyma cells) three-dimensionally. X-ray imaging at high resolutions provides very distinct visual insight into the xylem structure. Numerical analyses performed through semi-automatic procedures made it possible to quickly quantify cell characteristics (length, diameter, and volume of cells). Use of various spatial resolutions (0.87-5 μm) revealed boundaries users should be aware of. Nevertheless, our findings, both qualitative and quantitative, demonstrate XμCT to be a valuable tool for studying the spatial cell morphology of F. excelsior. Copyright © 2018. Published by Elsevier Ltd.

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.

Wheat germ agglutinin-conjugated PLGA nanoparticles for enhanced intracellular delivery of paclitaxel to colon cancer cells.

PubMed

Wang, Chunxia; Ho, Paul C; Lim, Lee Yong

2010-11-15

The purpose of this study was to investigate the potentiation of the anticancer activity and enhanced cellular retention of paclitaxel-loaded PLGA nanoparticles after surface conjugation with wheat germ agglutinin (WGA) against colon cancer cells. Glycosylation patterns of representative colon cancer cells confirmed the higher expression levels of WGA-binding glycoproteins in the Caco-2 and HT-29 cells, than in the CCD-18Co cells. Cellular uptake and in vitro cytotoxicity of WNP (final formulation) against colon cell lines was evaluated alongside control formulations. Confocal microscopy and quantitative analysis of intracellular paclitaxel were used to monitor the endocytosis and retention of nanoparticles inside the cells. WNP showed enhanced anti-proliferative activity against Caco-2 and HT-29 cells compared to corresponding nanoparticles without WGA conjugation (PNP). The greater efficacy of WNP was associated with higher cellular uptake and sustained intracellular retention of paclitaxel, which in turn was attributed to the over-expression of N-acetyl-D-glucosamine-containing glycoprotein on the colon cell membrane. WNP also demonstrated increased intracellular retention in the Caco-2 (30% of uptake) and HT-29 (40% of uptake) cells, following post-uptake incubation with fresh medium, compared to the unconjugated PNP nanoparticles (18% in Caco-2) and (27% in HT-29), respectively. Cellular trafficking study of WNP showed endocytosed WNP could successful escape from the endo-lysosome compartment and release into the cytosol with increasing incubation time. It may be concluded that WNP has the potential to be applied as a targeted delivery platform for paclitaxel in the treatment of colon cancer. Copyright © 2010 Elsevier B.V. All rights reserved.

Quantitative assessment of surface functionality effects on microglial uptake and retention of PAMAM dendrimers

NASA Astrophysics Data System (ADS)

Liaw, Kevin; Gök, Ozgul; DeRidder, Louis B.; Kannan, Sujatha; Kannan, Rangaramanujam M.

2018-04-01

Dendrimers are a promising class of polymeric nanoparticles for delivery of therapeutics and diagnostics. Polyamidoamine (PAMAM) dendrimers have shown significant efficacy in many animal models, with performance dependent on surface functionalities. Understanding the effects of end groups on biological interactions is critical for rational design of dendrimer-mediated therapies. In this study, we quantify the cellular trafficking kinetics (endocytosis and exocytosis) of generation 4 neutral (D4-OH), cationic (D4-NH2), anionic (D3.5-COOH), and generation 6 neutral (D6-OH) PAMAM dendrimers to investigate the nanoscale effects of surface functionality and size on cellular interactions. Resting and LPS-activated microglia were studied due to their central roles in dendrimer therapies for central nervous system disorders. D4-OH exhibits greater cellular uptake and lower retention than the larger D6-OH. D4-OH and D3.5-COOH exhibit similar trafficking kinetics, while D4-NH2 exhibits significant membrane interactions, resulting in faster cell association but lower internalization. Cationic charge may also enhance vesicular escape for greater cellular retention and preferential partitioning to nuclei. LPS activation further improves uptake of dendrimers, with smaller and cationic dendrimers experiencing the greatest increases in uptake compared to resting microglia. These studies have implications for the dependence of trafficking pathway on dendrimer properties and inform the design of dendrimer constructs tailored to specific therapeutic needs. Cationic dendrimers are ideal for delivering genetic materials to nuclei, but toxicity may be a limiting factor. Smaller, neutral dendrimers are best suited for delivering high levels of therapeutics in acute neuroinflammation, while larger or cationic dendrimers provide robust retention for sustained release of therapeutics in longer-term diseases.

Quantitative RT-PCR and immunoblot analyses reveal acclimated A2 noradrenergic neuron substrate fuel transporter, glucokinase, phospho-AMPK, and dopamine-β-hydroxylase responses to hypoglycemia.

PubMed

Cherian, Ajeesh Koshy; Briski, Karen P

2011-07-01

Cellular metabolic stasis is monitored in discrete brain sites, including the dorsal vagal complex (DVC), where A2 noradrenergic neurons perform this sensory function. Single-cell qPCR and high-sensitivity immunoblotting were used to determine if A2 neurons adapt to chronic hypoglycemia by increasing substrate fuel transporter expression, and whether such adjustments coincide with decreased cellular energy instability during this systemic metabolic stress. Tyrosine hydroxylase-immunolabeled neurons were laser-microdissected from the caudal DVC 2 hr after single or serial neutral protamine Hagedorn insulin (NPH) dosing. Preceding hypoglycemia suppressed basal A2 MCT2, GLUT3, and GLUT4 profiles and diminished MCT2, GLUT4, and glucokinase responses to recurring hypoglycemia. Acute NPH caused a robust increase in A2 phospho-AMPK protein levels; baseline phospho-AMPK expression was elevated after 3 days of insulin treatment but only slight augmented after a fourth NPH injection. Transcripts encoding the catecholamine biosynthetic enzyme dopamine-β-hydroxylase were unaffected by acute NPH but were diminished by serial insulin dosing. This evidence for diminished basal A2 glucose and lactate uptake and attenuated phospho-AMPK-mediated detection of hypoglycemia-associated energy deficits suggests that these cells acclimate to chronic hypoglycemia by adopting a new metabolic steady state characterized by energy paucity and reduced sensitivity to hypoglycemia. Because dopamine-β-hydroxylase mRNA was reduced after serial, but not single NPH dosing, A2 neurotransmitter biosynthesis may be impervious to acute hypoglycemia but inhibited when posthypoglycemic metabolic deficiency is exacerbated by recurring hypoglycemia. This research suggests that chronic hypoglycemia-associated adjustments in A2-sensory neurotransmission may reflect cellular energetic debilitation rather than adaptive attenuation of cellular metabolic imbalance. Copyright © 2011 Wiley-Liss, Inc.

The third dimension bridges the gap between cell culture and live tissue.

PubMed

Pampaloni, Francesco; Reynaud, Emmanuel G; Stelzer, Ernst H K

2007-10-01

Moving from cell monolayers to three-dimensional (3D) cultures is motivated by the need to work with cellular models that mimic the functions of living tissues. Essential cellular functions that are present in tissues are missed by 'petri dish'-based cell cultures. This limits their potential to predict the cellular responses of real organisms. However, establishing 3D cultures as a mainstream approach requires the development of standard protocols, new cell lines and quantitative analysis methods, which include well-suited three-dimensional imaging techniques. We believe that 3D cultures will have a strong impact on drug screening and will also decrease the use of laboratory animals, for example, in the context of toxicity assays.

Multiscale Feature Analysis of Salivary Gland Branching Morphogenesis

PubMed Central

Baydil, Banu; Daley, William P.; Larsen, Melinda; Yener, Bülent

2012-01-01

Pattern formation in developing tissues involves dynamic spatio-temporal changes in cellular organization and subsequent evolution of functional adult structures. Branching morphogenesis is a developmental mechanism by which patterns are generated in many developing organs, which is controlled by underlying molecular pathways. Understanding the relationship between molecular signaling, cellular behavior and resulting morphological change requires quantification and categorization of the cellular behavior. In this study, tissue-level and cellular changes in developing salivary gland in response to disruption of ROCK-mediated signaling by are modeled by building cell-graphs to compute mathematical features capturing structural properties at multiple scales. These features were used to generate multiscale cell-graph signatures of untreated and ROCK signaling disrupted salivary gland organ explants. From confocal images of mouse submandibular salivary gland organ explants in which epithelial and mesenchymal nuclei were marked, a multiscale feature set capturing global structural properties, local structural properties, spectral, and morphological properties of the tissues was derived. Six feature selection algorithms and multiway modeling of the data was performed to identify distinct subsets of cell graph features that can uniquely classify and differentiate between different cell populations. Multiscale cell-graph analysis was most effective in classification of the tissue state. Cellular and tissue organization, as defined by a multiscale subset of cell-graph features, are both quantitatively distinct in epithelial and mesenchymal cell types both in the presence and absence of ROCK inhibitors. Whereas tensor analysis demonstrate that epithelial tissue was affected the most by inhibition of ROCK signaling, significant multiscale changes in mesenchymal tissue organization were identified with this analysis that were not identified in previous biological studies. We here show how to define and calculate a multiscale feature set as an effective computational approach to identify and quantify changes at multiple biological scales and to distinguish between different states in developing tissues. PMID:22403724

Recursive expectation-maximization clustering: A method for identifying buffering mechanisms composed of phenomic modules

NASA Astrophysics Data System (ADS)

Guo, Jingyu; Tian, Dehua; McKinney, Brett A.; Hartman, John L.

2010-06-01

Interactions between genetic and/or environmental factors are ubiquitous, affecting the phenotypes of organisms in complex ways. Knowledge about such interactions is becoming rate-limiting for our understanding of human disease and other biological phenomena. Phenomics refers to the integrative analysis of how all genes contribute to phenotype variation, entailing genome and organism level information. A systems biology view of gene interactions is critical for phenomics. Unfortunately the problem is intractable in humans; however, it can be addressed in simpler genetic model systems. Our research group has focused on the concept of genetic buffering of phenotypic variation, in studies employing the single-cell eukaryotic organism, S. cerevisiae. We have developed a methodology, quantitative high throughput cellular phenotyping (Q-HTCP), for high-resolution measurements of gene-gene and gene-environment interactions on a genome-wide scale. Q-HTCP is being applied to the complete set of S. cerevisiae gene deletion strains, a unique resource for systematically mapping gene interactions. Genetic buffering is the idea that comprehensive and quantitative knowledge about how genes interact with respect to phenotypes will lead to an appreciation of how genes and pathways are functionally connected at a systems level to maintain homeostasis. However, extracting biologically useful information from Q-HTCP data is challenging, due to the multidimensional and nonlinear nature of gene interactions, together with a relative lack of prior biological information. Here we describe a new approach for mining quantitative genetic interaction data called recursive expectation-maximization clustering (REMc). We developed REMc to help discover phenomic modules, defined as sets of genes with similar patterns of interaction across a series of genetic or environmental perturbations. Such modules are reflective of buffering mechanisms, i.e., genes that play a related role in the maintenance of physiological homeostasis. To develop the method, 297 gene deletion strains were selected based on gene-drug interactions with hydroxyurea, an inhibitor of ribonucleotide reductase enzyme activity, which is critical for DNA synthesis. To partition the gene functions, these 297 deletion strains were challenged with growth inhibitory drugs known to target different genes and cellular pathways. Q-HTCP-derived growth curves were used to quantify all gene interactions, and the data were used to test the performance of REMc. Fundamental advantages of REMc include objective assessment of total number of clusters and assignment to each cluster a log-likelihood value, which can be considered an indicator of statistical quality of clusters. To assess the biological quality of clusters, we developed a method called gene ontology information divergence z-score (GOid_z). GOid_z summarizes total enrichment of GO attributes within individual clusters. Using these and other criteria, we compared the performance of REMc to hierarchical and K-means clustering. The main conclusion is that REMc provides distinct efficiencies for mining Q-HTCP data. It facilitates identification of phenomic modules, which contribute to buffering mechanisms that underlie cellular homeostasis and the regulation of phenotypic expression.

Metabolic regulation of histone acetyltransferases by endogenous Acyl-CoA cofactors | Center for Cancer Research

Cancer.gov

Unraveling the metabolic regulation of lysine acetyltransferases (KATs). Montgomery et al. detail the application of a competitive chemoproteomic strategy to quantitatively characterize the interactions of acyl-CoA metabolites with cellular KAT enzymes.

Building robust functionality in synthetic circuits using engineered feedback regulation.

PubMed

Chen, Susan; Harrigan, Patrick; Heineike, Benjamin; Stewart-Ornstein, Jacob; El-Samad, Hana

2013-08-01

The ability to engineer novel functionality within cells, to quantitatively control cellular circuits, and to manipulate the behaviors of populations, has many important applications in biotechnology and biomedicine. These applications are only beginning to be explored. In this review, we advocate the use of feedback control as an essential strategy for the engineering of robust homeostatic control of biological circuits and cellular populations. We also describe recent works where feedback control, implemented in silico or with biological components, was successfully employed for this purpose. Copyright © 2013 Elsevier Ltd. All rights reserved.

Direct real-time measurement of intra-oocyte nitric oxide concentration in vivo.

PubMed

Goud, Pravin T; Goud, Anuradha P; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P; Saed, Ghassan M; Zhang, Xueji; Abu-Soud, Husam M

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70-75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process.

Direct Real-Time Measurement of Intra-Oocyte Nitric Oxide Concentration In Vivo

PubMed Central

Goud, Pravin T.; Goud, Anuradha P.; Najafi, Tohid; Gonik, Bernard; Diamond, Michael P.; Saed, Ghassan M.; Zhang, Xueji; Abu-Soud, Husam M.

2014-01-01

Nitric oxide (NO) is reported to play significant a role in oocyte activation and maturation, implantation, and early embryonic development. Previously we have shown that NO forms an important component of the oocyte microenvironment, and functions effectively to delay oocyte aging. Thus, precise information about intra-oocyte NO concentrations [NO] will result in designing more accurate treatment plans in assisted reproduction. In this work, the direct, real-time and quantitative intra-oocyte [NO] was measured utilizing an L-shaped amperometric integrated NO-selective electrode. This method not only provides an elegant and convenient approach to real-time the measurement of NO in physiological environments, but also mimics the loss of NO caused by rapid NO diffusion combined with its reactivity in the biological milieu. This experiment suggests that the NO levels of oocytes obtained from young animals are significantly higher than those of oocytes obtained from old animals. Additionally the NO levels stay constant during the measurements; however, the intra-oocyte [NO] is reduced significantly (70–75% reduction) in response to L-NAME incubation, suggesting that NO measurements are truly NOS based rather than caused by an unknown interfering substance in our system. We believe this first demonstration of the direct quantitative measurement of [NO] in situ in an intact cellular complex should be useful in tracking real-time and rapid changes at nanomolar levels. Moreover, this finding confirms and extends our previous work showing that supplementation with NO delays the oocyte aging process. PMID:24887331

Common and Specific Protein Accumulation Patterns in Different Albino/Pale-Green Mutants Reveals Regulon Organization at the Proteome Level1[W

PubMed Central

Motohashi, Reiko; Rödiger, Anja; Agne, Birgit; Baerenfaller, Katja; Baginsky, Sacha

2012-01-01

Research interest in proteomics is increasingly shifting toward the reverse genetic characterization of gene function at the proteome level. In plants, several distinct gene defects perturb photosynthetic capacity, resulting in the loss of chlorophyll and an albino or pale-green phenotype. Because photosynthesis is interconnected with the entire plant metabolism and its regulation, all albino plants share common characteristics that are determined by the switch from autotrophic to heterotrophic growth. Reverse genetic characterizations of such plants often cannot distinguish between specific consequences of a gene defect from generic effects in response to perturbations in photosynthetic capacity. Here, we set out to define common and specific features of protein accumulation in three different albino/pale-green plant lines. Using quantitative proteomics, we report a common molecular phenotype that connects the loss of photosynthetic capacity with other chloroplast and cellular functions, such as protein folding and stability, plastid protein import, and the expression of stress-related genes. Surprisingly, we do not find significant differences in the expression of key transcriptional regulators, suggesting that substantial regulation occurs at the posttranscriptional level. We examine the influence of different normalization schemes on the quantitative proteomics data and report all identified proteins along with their fold changes and P values in albino plants in comparison with the wild type. Our analysis provides initial guidance for the distinction between general and specific adaptations of the proteome in photosynthesis-impaired plants. PMID:23027667

Comparison between chondroprotective effects of glucosamine, curcumin, and diacerein in IL-1beta-stimulated C-28/I2 chondrocytes.

PubMed

Toegel, S; Wu, S Q; Piana, C; Unger, F M; Wirth, M; Goldring, M B; Gabor, F; Viernstein, H

2008-10-01

To compare the effects of glucosamine (GlcN), curcumin, and diacerein in immortalized human C-28/I2 chondrocytes at the cellular and the gene expression level. This study aimed to provide insights into the proposed beneficial effects of these agents and to assess the applicability of the C-28/I2 cell line as a model for the evaluation of chondroprotective action. Interleukin-1beta (IL-1beta)-stimulated C-28/I2 cells were cultured in the presence of GlcN, curcumin, and diacerein prior to the evaluation of parameters such as viability, morphology and proliferation. The impact of GlcN, curcumin, and diacerein on gene expression was determined using quantitative real-time RT-PCR (qPCR). At the transcriptional level, 5 mM GlcN and 50 microM diacerein increased the expression of cartilage-specific genes such as aggrecan (AGC) and collagen type II (COL2), while reducing collagen type I (COL1) mRNA levels. Moreover, the IL-1beta-mediated shift in gene expression pattern was antagonized by GlcN and diacerein. These effects were associated with a significant reduction in cellular proliferation and the development of chondrocyte-specific cell morphology. In contrast, curcumin was not effective at lower concentrations but even damaged the cells at higher amounts. Both GlcN and diacerein promoted a differentiated chondrocytic phenotype of immortalized human C-28/I2 chondrocytes by altering proliferation, morphology, and COL2/COL1 mRNA ratios. Moreover, both agents antagonized inhibitory effects of IL-1beta by enhancing AGC and COL2 as well as by reducing COL1 mRNA levels.

Alterations of proteins in MDCK cells during acute potassium deficiency.

PubMed

Peerapen, Paleerath; Ausakunpipat, Nardtaya; Chanchaem, Prangwalai; Thongboonkerd, Visith

2016-06-01

Chronic K(+) deficiency can cause hypokalemic nephropathy associated with metabolic alkalosis, polyuria, tubular dilatation, and tubulointerstitial injury. However, effects of acute K(+) deficiency on the kidney remained unclear. This study aimed to explore such effects by evaluating changes in levels of proteins in renal tubular cells during acute K(+) deficiency. MDCK cells were cultivated in normal K(+) (NK) (K(+)=5.3 mM), low K(+) (LK) (K(+)=2.5 mM), or K(+) depleted (KD) (K(+)=0 mM) medium for 24 h and then harvested. Cellular proteins were resolved by two-dimensional gel electrophoresis (2-DE) and visualized by SYPRO Ruby staining (5 gels per group). Spot matching and quantitative intensity analysis revealed a total 48 protein spots that had significantly differential levels among the three groups. Among these, 46 and 30 protein spots had differential levels in KD group compared to NK and LK groups, respectively. Comparison between LK and NK groups revealed only 10 protein spots that were differentially expressed. All of these differentially expressed proteins were successfully identified by Q-TOF MS and/or MS/MS analyses. The altered levels of heat shock protein 90 (HSP90), ezrin, lamin A/C, tubulin, chaperonin-containing TCP1 (CCT1), and calpain 1 were confirmed by Western blot analysis. Global protein network analysis showed three main functional networks, including 1) cell growth and proliferation, 2) cell morphology, cellular assembly and organization, and 3) protein folding in which the altered proteins were involved. Further investigations on these networks may lead to better understanding of pathogenic mechanisms of low K(+)-induced renal injury. Copyright © 2016 Elsevier B.V. All rights reserved.

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

Almeida, Luciana O.; Goto, Renata N.; Neto, Marinaldo P.C.

We hypothesized that SET, a protein accumulated in some cancer types and Alzheimer disease, is involved in cell death through mitochondrial mechanisms. We addressed the mRNA and protein levels of the mitochondrial uncoupling proteins UCP1, UCP2 and UCP3 (S and L isoforms) by quantitative real-time PCR and immunofluorescence as well as other mitochondrial involvements, in HEK293 cells overexpressing the SET protein (HEK293/SET), either in the presence or absence of oxidative stress induced by the pro-oxidant t-butyl hydroperoxide (t-BHP). SET overexpression in HEK293 cells decreased UCP1 and increased UCP2 and UCP3 (S/L) mRNA and protein levels, whilst also preventing lipid peroxidationmore » and decreasing the content of cellular ATP. SET overexpression also (i) decreased the area of mitochondria and increased the number of organelles and lysosomes, (ii) increased mitochondrial fission, as demonstrated by increased FIS1 mRNA and FIS-1 protein levels, an apparent accumulation of DRP-1 protein, and an increase in the VDAC protein level, and (iii) reduced autophagic flux, as demonstrated by a decrease in LC3B lipidation (LC3B-II) in the presence of chloroquine. Therefore, SET overexpression in HEK293 cells promotes mitochondrial fission and reduces autophagic flux in apparent association with up-regulation of UCP2 and UCP3; this implies a potential involvement in cellular processes that are deregulated such as in Alzheimer's disease and cancer. - Highlights: • SET, UCPs and autophagy prevention are correlated. • SET action has mitochondrial involvement. • UCP2/3 may reduce ROS and prevent autophagy. • SET protects cell from ROS via UCP2/3.« less

Quantitative genetic analysis of cellular adhesion molecules: the Fels Longitudinal Study.

PubMed

Lee, Miryoung; Czerwinski, Stefan A; Choh, Audrey C; Demerath, Ellen W; Sun, Shumei S; Chumlea, Wm C; Towne, Bradford; Siervogel, Roger M

2006-03-01

Circulating concentrations of inflammatory markers predict cardiovascular disease (CVD) risk and are closely associated with obesity. However, little is known concerning genetic influences on serum levels of inflammatory markers. In this study, we estimated the heritability (h2) of soluble cellular adhesion molecule (sCAM) concentrations and examined the correlational architecture between different sCAMs. The study population included 234 men and 270 women aged 18-76 years, belonging to 121 families participating in the Fels Longitudinal Study. Serum levels of soluble intercellular adhesion molecule-1 (sICAM-1), vascular cell adhesion molecule-1 (sVCAM-1), E-selectin (sESEL-1) and P-selectin (sPSEL-1) were assayed using commercially available kits. A variance components-based maximum likelihood method was used to estimate the h2 of the different serum inflammatory markers while simultaneously adjusting for the effects of known CVD risk factors, such as age and smoking. Additionally, we used bivariate extensions of these methods to estimate genetic and random environmental correlations among sCAMs. Levels of sCAMs were significantly heritable: h2=0.24+/-0.10 for sICAM-1, h2=0.22+/-0.10 for sVCAM-1, h2=0.50+/-0.11 for sESEL-1, and h2=0.46+/-0.10 for sPSEL-1. In addition, a significant genetic correlation (rho(G)=0.63) was found between sICAM-1 and sVCAM-1 indicating some degree of shared genetic control. In the Fels Longitudinal Study, the levels of four sCAMs are significantly influenced by genetic effects, and sICAM-1 shares a common genetic background with sVCAM-1.

Postnatal odorant exposure induces peripheral olfactory plasticity at the cellular level.

PubMed

Cadiou, Hervé; Aoudé, Imad; Tazir, Bassim; Molinas, Adrien; Fenech, Claire; Meunier, Nicolas; Grosmaitre, Xavier

2014-04-02

Mammalian olfactory sensory neurons (OSNs) form the primary elements of the olfactory system. Inserted in the olfactory mucosa lining of the nasal cavity, they are exposed to the environment and their lifespan is brief. Several reports say that OSNs are regularly regenerated during the entire life and that odorant environment affects the olfactory epithelium. However, little is known about the impact of the odorant environment on OSNs at the cellular level and more precisely in the context of early postnatal olfactory exposure. Here we exposed MOR23-green fluorescent protein (GFP) and M71-GFP mice to lyral or acetophenone, ligands for MOR23 or M71, respectively. Daily postnatal exposure to lyral induces plasticity in the population of OSNs expressing MOR23. Their density decreases after odorant exposure, whereas the amount of MOR23 mRNA and protein remain stable in the whole epithelium. Meanwhile, quantitative PCR indicates that each MOR23 neuron has higher levels of olfactory receptor transcripts and also expresses more CNGA2 and phosphodiesterase 1C, fundamental olfactory transduction pathway proteins. Transcript levels return to baseline after 4 weeks recovery. Patch-clamp recordings reveal that exposed MOR23 neurons respond to lyral with higher sensitivity and broader dynamic range while the responses' kinetics were faster. These effects are specific to the odorant-receptor pair lyral-MOR23: there was no effect of acetophenone on MOR23 neurons and no effect of acetophenone and lyral on the M71 population. Together, our results clearly demonstrate that OSNs undergo specific anatomical, molecular, and functional adaptation when chronically exposed to odorants in the early stage of life.

Quantitative biology of single neurons

PubMed Central

Eberwine, James; Lovatt, Ditte; Buckley, Peter; Dueck, Hannah; Francis, Chantal; Kim, Tae Kyung; Lee, Jaehee; Lee, Miler; Miyashiro, Kevin; Morris, Jacqueline; Peritz, Tiina; Schochet, Terri; Spaethling, Jennifer; Sul, Jai-Yoon; Kim, Junhyong

2012-01-01

The building blocks of complex biological systems are single cells. Fundamental insights gained from single-cell analysis promise to provide the framework for understanding normal biological systems development as well as the limits on systems/cellular ability to respond to disease. The interplay of cells to create functional systems is not well understood. Until recently, the study of single cells has concentrated primarily on morphological and physiological characterization. With the application of new highly sensitive molecular and genomic technologies, the quantitative biochemistry of single cells is now accessible. PMID:22915636

Highly dynamic cellular-level response of symbiotic coral to a sudden increase in environmental nitrogen.

PubMed

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

2013-05-14

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 (15)N 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. The methodology applied, combining transmission electron microscopy with nanoscale secondary-ion mass spectrometry (NanoSIMS) imaging of coral tissue labeled with stable isotope tracers, allows quantification and submicrometric localization of metabolic fluxes in an intact symbiosis. This study opens the way for investigations of physiological adaptations of symbiotic systems to nutrient availability and for increasing knowledge of global nitrogen and carbon biogeochemical cycling.

Na+ extrusion from the cytosol and tissue-specific Na+ sequestration in roots confer differential salt stress tolerance between durum and bread wheat.

PubMed

Wu, Honghong; Shabala, Lana; Azzarello, Elisa; Huang, Yuqing; Pandolfi, Camilla; Su, Nana; Wu, Qi; Cai, Shengguan; Bazihizina, Nadia; Wang, Lu; Zhou, Meixue; Mancuso, Stefano; Chen, Zhonghua; Shabala, Sergey

2018-06-11

The progress in plant breeding for salinity stress tolerance is handicapped by the lack of understanding of the specificity of salt stress signalling and adaptation at the cellular and tissue levels. In this study, we used electrophysiological, fluorescence imaging, and real-time quantitative PCR tools to elucidate the essentiality of the cytosolic Na+ extrusion in functionally different root zones (elongation, meristem, and mature) in a large number of bread and durum wheat accessions. We show that the difference in the root's ability for vacuolar Na+ sequestration in the mature zone may explain differential salinity stress tolerance between salt-sensitive durum and salt-tolerant bread wheat species. Bread wheat genotypes also had on average 30% higher capacity for net Na+ efflux from the root elongation zone, providing the first direct evidence for the essentiality of the root salt exclusion trait at the cellular level. At the same time, cytosolic Na+ accumulation in the root meristem was significantly higher in bread wheat, leading to the suggestion that this tissue may harbour a putative salt sensor. This hypothesis was then tested by investigating patterns of Na+ distribution and the relative expression level of several key genes related to Na+ transport in leaves in plants with intact roots and in those in which the root meristems were removed. We show that tampering with this sensing mechanism has resulted in a salt-sensitive phenotype, largely due to compromising the plant's ability to sequester Na+ in mesophyll cell vacuoles. The implications of these findings for plant breeding for salinity stress tolerance are discussed.

Cyr61 as mediator of Src signaling in triple negative breast cancer cells

PubMed Central

Molinari, Agnese; Wagner, Kay-Uwe; Losada, Jesús Pérez; Ciordia, Sergio; Albar, Juan Pablo; Martín-Pérez, Jorge

2015-01-01

SFKs are involved in tumorigenesis and metastasis. Here we analyzed c-Src contribution to initial steps of metastasis by tetracycline-dependent expression of a specific shRNA-c-Src, which suppressed c-Src mRNA and protein levels in metastatic MDA-MB-231 cells. c-Src suppression did not alter cell proliferation or survival, but it significantly reduced anchorage-independent growth. Concomitantly with diminished tyrosine-phosphorylation/activation of Fak, caveolin-1, paxillin and p130CAS, c-Src depletion also inhibited cellular migration, invasion and transendothelial migration. Quantitative proteomic analyses of the secretome showed that Cyr61 levels, which were detected in the exosomal fraction, were diminished upon shRNA-c-Src expression. In contrast, Cyr61 expression was unaltered inside cells. Cyr61 partially colocalized with cis-Golgi gp74 marker and with exosomal marker CD63, but c-Src depletion did not alter their cellular distribution. In SUM159PT cells, transient c-Src suppression also reduced secreted exosomal Cyr61 levels. Furthermore, conditional expression of a c-Src dominant negative mutant (SrcDN, c-Src-K295M/Y527F) in MDA-MB-231 and in SUM159PT diminished secreted Cyr61 as well. Cyr61 transient suppression in MDA-MB-231 inhibited invasion and transendothelial migration. Finally, in both MDA-MB-231 and SUM159PT, a neutralizing Cyr61 antibody restrained migration. Collectively, these results suggest that c-Src regulates secreted proteins, including the exosomal Cyr61, which are involved in modulating the metastatic potential of triple negative breast cancer cells. PMID:25980494

Untargeted metabolomics studies employing NMR and LC-MS reveal metabolic coupling between Nanoarcheum equitans and its archaeal host Ignicoccus hospitalis.

PubMed

Hamerly, Timothy; Tripet, Brian P; Tigges, Michelle; Giannone, Richard J; Wurch, Louie; Hettich, Robert L; Podar, Mircea; Copié, Valerie; Bothner, Brian

2015-08-01

Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC-MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with information about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hospitalis that are impacted by the presence of N. equitans , including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equitans 's consumption of a significant fraction of the metabolite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans . Combining LC-MS and NMR metabolomics datasets improved coverage of the metabolome and enhanced the identification and quantitation of cellular metabolites.

Untargeted metabolomics studies employing NMR and LC-MS reveal metabolic coupling between Nanoarcheum equitans and its archaeal host Ignicoccus hospitalis

PubMed Central

Hamerly, Timothy; Tripet, Brian P.; Tigges, Michelle; Giannone, Richard J.; Wurch, Louie; Hettich, Robert L.; Podar, Mircea; Copié, Valerie; Bothner, Brian

2014-01-01

Interspecies interactions are the basis of microbial community formation and infectious diseases. Systems biology enables the construction of complex models describing such interactions, leading to a better understanding of disease states and communities. However, before interactions between complex organisms can be understood, metabolic and energetic implications of simpler real-world host-microbe systems must be worked out. To this effect, untargeted metabolomics experiments were conducted and integrated with proteomics data to characterize key molecular-level interactions between two hyperthermophilic microbial species, both of which have reduced genomes. Metabolic changes and transfer of metabolites between the archaea Ignicoccus hospitalis and Nanoarcheum equitans were investigated using integrated LC-MS and NMR metabolomics. The study of such a system is challenging, as no genetic tools are available, growth in the laboratory is challenging, and mechanisms by which they interact are unknown. Together with information about relative enzyme levels obtained from shotgun proteomics, the metabolomics data provided useful insights into metabolic pathways and cellular networks of I. hospitalis that are impacted by the presence of N. equitans, including arginine, isoleucine, and CTP biosynthesis. On the organismal level, the data indicate that N. equitans exploits metabolites generated by I. hospitalis to satisfy its own metabolic needs. This finding is based on N. equitans’s consumption of a significant fraction of the metabolite pool in I. hospitalis that cannot solely be attributed to increased biomass production for N. equitans. Combining LC-MS and NMR metabolomics datasets improved coverage of the metabolome and enhanced the identification and quantitation of cellular metabolites. PMID:26273237

Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells.

PubMed

Weber, Christoph; Schreiber, Thiemo B; Daub, Henrik

2012-02-02

Small molecule inhibitors of protein kinases have emerged as a major class of therapeutic agents for the treatment of hematological malignancies. Both in vitro studies and patient case reports suggest therapeutic potential of the clinical kinase inhibitors erlotinib and gefitinib in acute myeloid leukemia (AML). The drugs' cellular modes of action in AML warrant further investigation as their primary therapeutic target, the epidermal growth factor receptor, is not expressed. We therefore performed SILAC-based quantitative mass spectrometry analyses to a depth of 10,975 distinct phosphorylation sites to characterize the phosphoproteome of KG1 AML cells and its regulation upon erlotinib and gefitinib treatment. Less than 50 site-specific phosphorylations changed significantly, indicating rather specific interference with AML cell signaling. Many drug-induced changes occurred within a network of tyrosine phosphorylated proteins that included Src family kinases (SFKs) and the tyrosine kinases Btk and Syk. We further performed quantitative chemical proteomics in KG1 cell extracts and identified SFKs and Btk as direct cellular targets of both erlotinib and gefitinib. Taken together, our data suggest that cellular perturbation of SFKs and/or Btk translates into rather specific signal transduction inhibition, which in turn contributes to the antileukemic activity of erlotinib and gefitinib in AML. Copyright © 2011 Elsevier B.V. All rights reserved.

Research highlights: Microtechnologies for engineering the cellular environment.

PubMed

Tseng, Peter; Kunze, Anja; Kittur, Harsha; Di Carlo, Dino

2014-04-07

In this issue we highlight recent microtechnology-enabled approaches to control the physical and biomolecular environment around cells: (1) developing micropatterned surfaces to quantify cell affinity choices between two adhesive patterns, (2) controlling topographical cues to align cells and improve reprogramming to a pluripotent state, and (3) controlling gradients of biomolecules to maintain pluripotency in embryonic stem cells. Quantitative readouts of cell-surface affinity in environments with several cues should open up avenues in tissue engineering where self-assembly of complex multi-cellular structures is possible by precisely engineering relative adhesive cues in three dimensional constructs. Methods of simple and local epigenetic modification of chromatin structure with microtopography and biomolecular gradients should also be of use in regenerative medicine, as well as in high-throughput quantitative analysis of external signals that impact and can be used to control cells. Overall, approaches to engineer the cellular environment will continue to be an area of further growth in the microfluidic and lab on a chip community, as the scale of the technologies seamlessly matches that of biological systems. However, because of regulations and other complexities with tissue engineered therapies, these micro-engineering approaches will likely first impact organ-on-a-chip technologies that are poised to improve drug discovery pipelines.

Micro-patterned agarose gel devices for single-cell high-throughput microscopy of E. coli cells.

PubMed

Priest, David G; Tanaka, Nobuyuki; Tanaka, Yo; Taniguchi, Yuichi

2017-12-21

High-throughput microscopy of bacterial cells elucidated fundamental cellular processes including cellular heterogeneity and cell division homeostasis. Polydimethylsiloxane (PDMS)-based microfluidic devices provide advantages including precise positioning of cells and throughput, however device fabrication is time-consuming and requires specialised skills. Agarose pads are a popular alternative, however cells often clump together, which hinders single cell quantitation. Here, we imprint agarose pads with micro-patterned 'capsules', to trap individual cells and 'lines', to direct cellular growth outwards in a straight line. We implement this micro-patterning into multi-pad devices called CapsuleHotel and LineHotel for high-throughput imaging. CapsuleHotel provides ~65,000 capsule structures per mm 2 that isolate individual Escherichia coli cells. In contrast, LineHotel provides ~300 line structures per mm that direct growth of micro-colonies. With CapsuleHotel, a quantitative single cell dataset of ~10,000 cells across 24 samples can be acquired and analysed in under 1 hour. LineHotel allows tracking growth of > 10 micro-colonies across 24 samples simultaneously for up to 4 generations. These easy-to-use devices can be provided in kit format, and will accelerate discoveries in diverse fields ranging from microbiology to systems and synthetic biology.

Correlative atomic force microscopy quantitative imaging-laser scanning confocal microscopy quantifies the impact of stressors on live cells in real-time.

PubMed

Bhat, Supriya V; Sultana, Taranum; Körnig, André; McGrath, Seamus; Shahina, Zinnat; Dahms, Tanya E S

2018-05-29

There is an urgent need to assess the effect of anthropogenic chemicals on model cells prior to their release, helping to predict their potential impact on the environment and human health. Laser scanning confocal microscopy (LSCM) and atomic force microscopy (AFM) have each provided an abundance of information on cell physiology. In addition to determining surface architecture, AFM in quantitative imaging (QI) mode probes surface biochemistry and cellular mechanics using minimal applied force, while LSCM offers a window into the cell for imaging fluorescently tagged macromolecules. Correlative AFM-LSCM produces complimentary information on different cellular characteristics for a comprehensive picture of cellular behaviour. We present a correlative AFM-QI-LSCM assay for the simultaneous real-time imaging of living cells in situ, producing multiplexed data on cell morphology and mechanics, surface adhesion and ultrastructure, and real-time localization of multiple fluorescently tagged macromolecules. To demonstrate the broad applicability of this method for disparate cell types, we show altered surface properties, internal molecular arrangement and oxidative stress in model bacterial, fungal and human cells exposed to 2,4-dichlorophenoxyacetic acid. AFM-QI-LSCM is broadly applicable to a variety of cell types and can be used to assess the impact of any multitude of contaminants, alone or in combination.

Toward Multiscale Models of Cyanobacterial Growth: A Modular Approach

PubMed Central

Westermark, Stefanie; Steuer, Ralf

2016-01-01

Oxygenic photosynthesis dominates global primary productivity ever since its evolution more than three billion years ago. While many aspects of phototrophic growth are well understood, it remains a considerable challenge to elucidate the manifold dependencies and interconnections between the diverse cellular processes that together facilitate the synthesis of new cells. Phototrophic growth involves the coordinated action of several layers of cellular functioning, ranging from the photosynthetic light reactions and the electron transport chain, to carbon-concentrating mechanisms and the assimilation of inorganic carbon. It requires the synthesis of new building blocks by cellular metabolism, protection against excessive light, as well as diurnal regulation by a circadian clock and the orchestration of gene expression and cell division. Computational modeling allows us to quantitatively describe these cellular functions and processes relevant for phototrophic growth. As yet, however, computational models are mostly confined to the inner workings of individual cellular processes, rather than describing the manifold interactions between them in the context of a living cell. Using cyanobacteria as model organisms, this contribution seeks to summarize existing computational models that are relevant to describe phototrophic growth and seeks to outline their interactions and dependencies. Our ultimate aim is to understand cellular functioning and growth as the outcome of a coordinated operation of diverse yet interconnected cellular processes. PMID:28083530

A Quantitative Study of Oxygen as a Metabolic Regulator

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; LaManna, Joseph C.; Cabrera, Marco E.

1999-01-01

An acute reduction in oxygen (O2) delivery to a tissue is generally associated with a decrease in phosphocreatine, increases in ADP, NADH/NAD, and inorganic phosphate, increased rates of glycolysis and lactate production, and reduced rates of pyruvate and fatty acid oxidation. However, given the complexity of the human bioenergetic system and its components, it is difficult to determine quantitatively how cellular metabolic processes interact to maintain ATP homeostasis during stress (e.g., hypoxia, ischemia, and exercise). Of special interest is the determination of mechanisms relating tissue oxygenation to observed metabolic responses at the tissue, organ, and whole body levels and the quantification of how changes in tissue O2 availability affect the pathways of ATP synthesis and the metabolites that control these pathways. In this study, we extend a previously developed mathematical model of human bioenergetics to provide a physicochemical framework that permits quantitative understanding of O2 as a metabolic regulator. Specifically, the enhancement permits studying the effects of variations in tissue oxygenation and in parameters controlling the rate of cellular respiration on glycolysis, lactate production, and pyruvate oxidation. The whole body is described as a bioenergetic system consisting of metabolically distinct tissue/organ subsystems that exchange materials with the blood. In order to study the dynamic response of each subsystem to stimuli, we solve the ordinary differential equations describing the temporal evolution of metabolite levels, given the initial concentrations. The solver used in the present study is the packaged code LSODE, as implemented in the NASA Lewis kinetics and sensitivity analysis code, LSENS. A major advantage of LSENS is the efficient procedures supporting systematic sensitivity analysis, which provides the basic methods for studying parameter sensitivities (i.e., changes in model behavior due to parameter variation). Sensitivity analysis establishes relationships between model predictions and problem parameters (i.e., initial concentrations, rate coefficients, etc). It helps determine the effects of uncertainties or changes in these input parameters on the predictions, which ultimately are compared with experimental observations in order to validate the model. Sensitivity analysis can identify parameters that must be determined accurately because of their large effect on the model predictions and parameters that need not be known with great precision because they have little or no effect on the solution. This capability may prove to be important in optimizing the design of experiments, thereby reducing the use of animals. This approach can be applied to study the metabolic effects of reduced oxygen delivery to cardiac muscle due to local myocardial ischemia and the effects of acute hypoxia on brain metabolism. Other important applications of sensitivity analysis include identification of quantitatively relevant pathways and biochemical species within an overall mechanism, when examining the effects of a genetic anomaly or pathological state on energetic system components and whole system behavior.

Total cellular glycomics allows characterizing cells and streamlining the discovery process for cellular biomarkers.

PubMed

Fujitani, Naoki; Furukawa, Jun-ichi; Araki, Kayo; Fujioka, Tsuyoshi; Takegawa, Yasuhiro; Piao, Jinhua; Nishioka, Taiki; Tamura, Tomohiro; Nikaido, Toshio; Ito, Makoto; Nakamura, Yukio; Shinohara, Yasuro

2013-02-05

Although many of the frequently used pluripotency biomarkers are glycoconjugates, a glycoconjugate-based exploration of novel cellular biomarkers has proven difficult due to technical difficulties. This study reports a unique approach for the systematic overview of all major classes of oligosaccharides in the cellular glycome. The proposed method enabled mass spectrometry-based structurally intensive analyses, both qualitatively and quantitatively, of cellular N- and O-linked glycans derived from glycoproteins, glycosaminoglycans, and glycosphingolipids, as well as free oligosaccharides of human embryonic stem cells (hESCs), induced pluripotent stem cells (hiPSCs), and various human cells derived from normal and carcinoma cells. Cellular total glycomes were found to be highly cell specific, demonstrating their utility as unique cellular descriptors. Structures of glycans of all classes specifically observed in hESCs and hiPSCs tended to be immature in general, suggesting the presence of stem cell-specific glycosylation spectra. The current analysis revealed the high similarity of the total cellular glycome between hESCs and hiPSCs, although it was suggested that hESCs are more homogeneous than hiPSCs from a glycomic standpoint. Notably, this study enabled a priori identification of known pluripotency biomarkers such as SSEA-3, -4, and -5 and Tra-1-60/81, as well as a panel of glycans specifically expressed by hESCs and hiPSCs.

The arginylation branch of the N-end rule pathway positively regulates cellular autophagic flux and clearance of proteotoxic proteins

PubMed Central

Jiang, Yanxialei; Lee, Jeeyoung; Lee, Jung Hoon; Lee, Joon Won; Kim, Ji Hyeon; Choi, Won Hoon; Yoo, Young Dong; Cha-Molstad, Hyunjoo; Kim, Bo Yeon; Kwon, Yong Tae; Noh, Sue Ah; Kim, Kwang Pyo; Lee, Min Jae

2016-01-01

ABSTRACT The N-terminal amino acid of a protein is an essential determinant of ubiquitination and subsequent proteasomal degradation in the N-end rule pathway. Using para-chloroamphetamine (PCA), a specific inhibitor of the arginylation branch of the pathway (Arg/N-end rule pathway), we identified that blocking the Arg/N-end rule pathway significantly impaired the fusion of autophagosomes with lysosomes. Under ER stress, ATE1-encoded Arg-tRNA-protein transferases carry out the N-terminal arginylation of the ER heat shock protein HSPA5 that initially targets cargo proteins, along with SQSTM1, to the autophagosome. At the late stage of autophagy, however, proteasomal degradation of arginylated HSPA5 might function as a critical checkpoint for the proper progression of autophagic flux in the cells. Consistently, the inhibition of the Arg/N-end rule pathway with PCA significantly elevated levels of MAPT and huntingtin aggregates, accompanied by increased numbers of LC3 and SQSTM1 puncta. Cells treated with the Arg/N-end rule inhibitor became more sensitized to proteotoxic stress-induced cytotoxicity. SILAC-based quantitative proteomics also revealed that PCA significantly alters various biological pathways, including cellular responses to stress, nutrient, and DNA damage, which are also closely involved in modulation of autophagic responses. Thus, our results indicate that the Arg/N-end rule pathway may function to actively protect cells from detrimental effects of cellular stresses, including proteotoxic protein accumulation, by positively regulating autophagic flux. PMID:27560450

Effect of omega-3 polyunsaturated fatty acids on the cytoskeleton: an open-label intervention study.

PubMed

Schmidt, Simone; Willers, Janina; Riecker, Sabine; Möller, Katharina; Schuchardt, Jan Philipp; Hahn, Andreas

2015-02-14

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) show beneficial effects on cardiovascular health and cognitive functions, but the underlying molecular mechanisms are not completely understood. Because of the fact that cytoskeleton dynamics affect almost every cellular process, the regulation of cytoskeletal dynamics could be a new pathway by which n-3 PUFAs exert their effects on cellular level. A 12-week open-label intervention study with 12 healthy men was conducted to determine the effects of 2.7 g/d n-3 PUFA on changes in mRNA expression of cytoskeleton-associated genes by quantitative real-time PCR in whole blood. Furthermore, the actin content in red blood cells was analyzed by immunofluorescence imaging. N-3 PUFA supplementation resulted in a significant down-regulation of cytoskeleton-associated genes, in particular three GTPases (RAC1, RHOA, CDC42), three kinases (ROCK1, PAK2, LIMK), two Wiskott-Aldrich syndrome proteins (WASL, WASF2) as well as actin related protein 2/3 complex (ARPC2, ARPC3) and cofilin (CFL1). Variability in F-actin content between subjects was high; reduced actin content was only reduced within group evaluation. Reduced cytoskeleton-associated gene expression after n-3 PUFA supplementation suggests that regulation of cytoskeleton dynamics might be an additional way by which n-3 PUFAs exert their cellular effects. Concerning F-actin, this analysis did not reveal unmistakable results impeding a generalized conclusion.

Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity.

PubMed

Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

2016-06-01

ZnO nanoparticles (NPs) are reported to show a high degree of cancer cell selectivity with potential use in cancer imaging and therapy. Questions remain about the mode by which the ZnO NPs cause cell death, whether they exert an intra- or extracellular effect, and the resistance among different cancer cell types to ZnO NP exposure. The present study quantifies the variability between the cellular toxicity, dynamics of cellular uptake, and dissolution of bare and RGD (Arg-Gly-Asp)-targeted ZnO NPs by MDA-MB-231 cells. Compared to bare ZnO NPs, RGD-targeting of the ZnO NPs to integrin αvβ3 receptors expressed on MDA-MB-231 cells appears to increase the toxicity of the ZnO NPs to breast cancer cells at lower doses. Confocal microscopy of live MDA-MB-231 cells confirms uptake of both classes of ZnO NPs with a commensurate rise in intracellular Zn(2+) concentration prior to cell death. The response of the cells within the population to intracellular Zn(2+) is highly heterogeneous. In addition, the results emphasize the utility of dynamic and quantitative imaging in understanding cell uptake and processing of targeted therapeutic ZnO NPs at the cellular level by heterogeneous cancer cell populations, which can be crucial for the development of optimized treatment strategies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A sensitive and accurate quantification method for the detection of hepatitis B virus covalently closed circular DNA by the application of a droplet digital polymerase chain reaction amplification system.

PubMed

Mu, Di; Yan, Liang; Tang, Hui; Liao, Yong

2015-10-01

To develop a sensitive and accurate assay system for the quantification of covalently closed circular HBV DNA (cccDNA) for future clinical monitoring of cccDNA fluctuation during antiviral therapy in the liver of infected patients. A droplet digital PCR (ddPCR)-based assay system detected template DNA input at the single copy level (or ~10(-5) pg of plasmid HBV DNA) by using serially diluted plasmid HBV DNA samples. Compared with the conventional quantitative PCR assay in the detection of cccDNA, which required at least 50 ng of template DNA input, a parallel experiment applying a ddPCR system demonstrates that the lowest detection limit of cccDNA from HepG2.215 cellular DNA samples is around 1 ng, which is equivalent to 0.54 ± 0.94 copies of cccDNA. In addition, we demonstrated that the addition of cccDNA-safe exonuclease and utilization of cccDNA-specific primers in the ddPCR assay system significantly improved the detection accuracy of HBV cccDNA from HepG2.215 cellular DNA samples. The ddPCR-based cccDNA detection system is a sensitive and accurate assay for the quantification of cccDNA in HBV-transfected HepG2.215 cellular DNA samples and may represent an important method for future application in monitoring cccDNA fluctuation during antiviral therapy.

Quantitation of the cellular content of saliva and buccal swab samples.

PubMed

Theda, Christiane; Hwang, Seo Hye; Czajko, Anna; Loke, Yuk Jing; Leong, Pamela; Craig, Jeffrey M

2018-05-02

Buccal swabs and saliva are the two most common oral sampling methods used for medical research. Often, these samples are used interchangeably, despite previous evidence that both contain buccal cells and blood leukocytes in different proportions. For some research, such as epigenetic studies, the cell types contributing to the analysis are highly relevant. We collected such samples from twelve children and twenty adults and, using Papanicolaou staining, measured the proportions of epithelial cells and leukocytes through microscopy. To our knowledge, no studies have compared cellular heterogeneity in buccal swab and saliva samples from adults and children. We confirmed that buccal swabs contained a higher proportion of epithelial cells than saliva and that children have a greater proportion of such cells in saliva compared to adults. At this level of resolution, buccal swabs and saliva contained similar epithelial cell subtypes. Gingivitis in children was associated with a higher proportion of leukocytes in saliva samples but not in buccal swabs. Compared to more detailed and costly methods such as flow cytometry or deconvolution methods used in epigenomic analysis, the procedure described here can serve as a simple and low-cost method to characterize buccal and saliva samples. Microscopy provides a low-cost tool to alert researchers to the presence of oral inflammation which may affect a subset of their samples. This knowledge might be highly relevant to their specific research questions, may assist with sample selection and thus might be crucial information despite the ability of data deconvolution methods to correct for cellular heterogeneity.

Size of submicrometric and nanometric particles affect cellular uptake and biological activity of macrophages in vitro.

PubMed

Leclerc, L; Rima, W; Boudard, D; Pourchez, J; Forest, V; Bin, V; Mowat, P; Perriat, P; Tillement, O; Grosseau, P; Bernache-Assollant, D; Cottier, M

2012-08-01

Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

PubMed Central

Baudoin, Jean-Pierre; Jerome, W. Gray; Kübel, Christian; de Jonge, Niels

2013-01-01

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters. PMID:23383042

Exon microarray analysis of human dorsolateral prefrontal cortex in alcoholism.

PubMed

Manzardo, Ann M; Gunewardena, Sumedha; Wang, Kun; Butler, Merlin G

2014-06-01

Alcohol abuse is associated with cellular and biochemical disturbances that impact upon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To further characterize the genetic influences in alcoholism and the effects of alcohol consumption on gene expression, we used a highly sensitive exon microarray to examine mRNA expression in human frontal cortex of alcoholics and control males. Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmann area 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. Affymetrix Human Exon 1.0 ST array was performed according to standard procedures and the results analyzed at the gene level. Microarray findings were validated using quantitative reverse transcription polymerase chain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis (IPA). Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g., CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g., ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurological disease and development including cellular assembly and organization impacting on psychological disorders. Our data in alcoholism support a reduction in expression of dlPFC mRNA for genes involved with neuronal growth, differentiation, and signaling that targets white matter of the brain. Copyright © 2014 by the Research Society on Alcoholism.

Multifidus Muscle Changes After Back Injury Are Characterized by Structural Remodeling of Muscle, Adipose and Connective Tissue, but Not Muscle Atrophy: Molecular and Morphological Evidence.

PubMed

Hodges, Paul W; James, Gregory; Blomster, Linda; Hall, Leanne; Schmid, Annina; Shu, Cindy; Little, Chris; Melrose, James

2015-07-15

Longitudinal case-controlled animal study. To investigate putative cellular mechanisms to explain structural changes in muscle and adipose and connective tissues of the back muscles after intervertebral disc (IVD) injury. Structural back muscle changes are ubiquitous with back pain/injury and considered relevant for outcome, but their exact nature, time course, and cellular mechanisms remain elusive. We used an animal model that produces phenotypic back muscle changes after IVD injury to study these issues at the cellular/molecular level. Multifidus muscle was harvested from both sides of the spine at L1-L2 and L3-L4 IVDs in 27 castrated male sheep at 3 (n = 10) or 6 (n = 17) months after a surgical anterolateral IVD injury at both levels. Ten control sheep underwent no surgery (3 mo, n = 4; 6 mo, n = 6). Tissue was harvested at L4 for histological analysis of cross-sectional area of muscle and adipose and connective tissue (whole muscle), plus immunohistochemistry to identify proportion and cross-sectional area of individual muscle fiber types in the deepest fascicle. Quantitative polymerase chain reaction measured gene expression of typical cytokines/signaling molecules at L2. Contrary to predictions, there was no multifidus muscle atrophy (whole muscle or individual fiber). There was increased adipose and connective tissue (fibrotic proliferation) cross-sectional area and slow-to-fast muscle fiber transition at 6 but not 3 months. Within the multifidus muscle, increases in the expression of several cytokines (tumor necrosis factor α and interleukin-1β) and molecules that signal trophic/atrophic processes for the 3 tissue types (e.g., growth factor pathway [IGF-1, PI3k, Akt1, mTOR], potent tissue modifiers [calcineurin, PCG-1α, and myostatin]) were present. This study provides cellular evidence that refutes the presence of multifidus muscle atrophy accompanying IVD degeneration at this intermediate time point. Instead, adipose/connective tissue increased in parallel with the expression of the genes that provide putative mechanisms for multifidus structural remodeling. This provides novel targets for pharmacological and physical interventions. N/A.

A general method for bead-enhanced quantitation by flow cytometry

PubMed Central

Montes, Martin; Jaensson, Elin A.; Orozco, Aaron F.; Lewis, Dorothy E.; Corry, David B.

2009-01-01

Flow cytometry provides accurate relative cellular quantitation (percent abundance) of cells from diverse samples, but technical limitations of most flow cytometers preclude accurate absolute quantitation. Several quantitation standards are now commercially available which, when added to samples, permit absolute quantitation of CD4+ T cells. However, these reagents are limited by their cost, technical complexity, requirement for additional software and/or limited applicability. Moreover, few studies have validated the use of such reagents in complex biological samples, especially for quantitation of non-T cells. Here we show that addition to samples of known quantities of polystyrene fluorescence standardization beads permits accurate quantitation of CD4+ T cells from complex cell samples. This procedure, here termed single bead-enhanced cytofluorimetry (SBEC), was equally capable of enumerating eosinophils as well as subcellular fragments of apoptotic cells, moieties with very different optical and fluorescent characteristics. Relative to other proprietary products, SBEC is simple, inexpensive and requires no special software, suggesting that the method is suitable for the routine quantitation of most cells and other particles by flow cytometry. PMID:17067632

Tensegrity, cellular biophysics, and the mechanics of living systems

PubMed Central

Ingber, Donald E.; Wang, Ning; Stamenović, Dimitrije

2014-01-01

The recent convergence between physics and biology has led many physicists to enter the fields of cell and developmental biology. One of the most exciting areas of interest has been the emerging field of mechanobiology that centers on how cells control their mechanical properties, and how physical forces regulate cellular biochemical responses, a process that is known as mechanotransduction. In this article, we review the central role that tensegrity (tensional integrity) architecture, which depends on tensile prestress for its mechanical stability, plays in biology. We describe how tensional prestress is a critical governor of cell mechanics and function, and how use of tensegrity by cells contributes to mechanotransduction. Theoretical tensegrity models are also described that predict both quantitative and qualitative behaviors of living cells, and these theoretical descriptions are placed in context of other physical models of the cell. In addition, we describe how tensegrity is used at multiple size scales in the hierarchy of life — from individual molecules to whole living organisms — to both stabilize three-dimensional form and to channel forces from the macroscale to the nanoscale, thereby facilitating mechanochemical conversion at the molecular level. PMID:24695087

A positional code and anisotropic forces control tissue remodeling in Drosophila

NASA Astrophysics Data System (ADS)

Zallen, Jennifer

A major challenge in developmental biology is to understand how tissue-scale changes in organism structure arise from events that occur on a cellular and molecular level. We are using cell biological, biophysical, and quantitative live-embryo imaging approaches to understand how genes encode the forces that shape tissues, and to identify the mechanisms that modulate cell behavior in response to local forces. In many animals, the elongated head-to-tail body axis is achieved by rapid and coordinated movements of hundreds of cells. We found that in the fruit fly, these cell movements are regulated by subcellular asymmetries in the localization of proteins that generate contractile and adhesive forces between cells. Asymmetries in the force-generating machinery are in turn controlled by a positional code of spatial information provided by an ancient family of Toll-related receptors that are widely used for pathogen recognition by the innate immune system. I will describe how this spatial system systematically orients local cell movements and collective rosette-like clusters in the Drosophila embryo. Rosettes have now also been shown to shape the body axis in chicks, frogs, and mice, demonstrating that rosette behaviors are a general mechanism linking cellular asymmetry to tissue reorganization.

Controlled Endolysosomal Release of Agents by pH-responsive Polymer Blend Particles.

PubMed

Zhan, Xi; Tran, Kenny K; Wang, Liguo; Shen, Hong

2015-07-01

A key step of delivering extracellular agents to its intracellular target is to escape from endosomal/lysosomal compartments, while minimizing the release of digestive enzymes that may compromise cellular functions. In this study, we examined the intracellular distribution of both fluorecent cargoes and enzymes by a particle delivery platform made from the controlled blending of poly(lactic-co-glycolic acid) (PLGA) and a random pH-sensitive copolymer. We utilized both microscopic and biochemical methods to semi-quantitatively assess how the composition of blend particles affects the level of endosomal escape of cargos of various sizes and enzymes into the cytosolic space. We demonstrated that these polymeric particles enabled the controlled delivery of cargos into the cytosolic space that was more dependent on the cargo size and less on the composition of blend particles. Blend particles did not induce the rupture of endosomal/lysosomal compartments and released less than 20% of endosomal/lysosomal enzymes. This study provides insight into understanding the efficacy and safety of a delivery system for intracellular delivery of biologics and drugs. Blend particles offer a potential platform to target intracellular compartments while potentially minimizing cellular toxicity.

Controlled endolysosomal release of agents by pH-responsive polymer blend particles

PubMed Central

Zhan, Xi; Tran, Kenny K.; Wang, Liguo; Shen, Hong

2015-01-01

Purpose A key step of delivering extracellular agents to its intracellular target is to escape from endosomal/lysosomal compartments, while minimizing the release of digestive enzymes that may compromise cellular functions. In this study, we examined the intracellular distribution of both fluorecent cargoes and enzymes by a particle delivery platform made from the controlled blending of poly (lactic-co-glycolic acid) (PLGA) and a random pH-sensitive copolymer. Methods We utilized both microscopic and biochemical methods to semi-quantitatively assess how the composition of blend particles affects the level of endosomal escape of cargos of various sizes and enzymes into the cytosolic space. Results We demonstrated that these polymeric particles enabled the controlled delivery of cargos into the cytosolic space that was more dependent on the cargo size and less on the composition of blend particles. Blend particles did not induce the rupture of endosomal/lysosomal compartments and released less than 20% of endosomal/lysosomal enzymes. Conclusions This study provides insight into understanding the efficacy and safety of a delivery system for intracellular delivery of biologics and drugs. Blend particles offer a potential platform to target intracellular compartments while potentially minimizing cellular toxicity. PMID:25592550

Imaging of cellular spread on a three-dimensional scaffold by means of a novel cell-labeling technique for high-resolution computed tomography.

PubMed

Thimm, Benjamin W; Hofmann, Sandra; Schneider, Philipp; Carretta, Roberto; Müller, Ralph

2012-03-01

Computed tomography (CT) represents a truly three-dimensional (3D) imaging technique that can provide high-resolution images on the cellular level. Thus, one approach to detect single cells is X-ray absorption-based CT, where cells are labeled with a dense, opaque material providing the required contrast for CT imaging. Within the present work, a novel cell-labeling method has been developed showing the feasibility of labeling fixed cells with iron oxide (FeO) particles for subsequent CT imaging and quantitative morphometry. A biotin-streptavidin detection system was exploited to bind FeO particles to its target endothelial cells. The binding of the particles was predominantly close to the cell centers on 2D surfaces as shown by light microscopy, scanning electron microscopy, and CT. When cells were cultured on porous, 3D polyurethane surfaces, significantly more FeO particles were detected compared with surfaces without cells and FeO particle labeling using CT. Here, we report on the implementation and evaluation of a novel cell detection method based on high-resolution CT. This system has potential in cell tracking for 3D in vitro imaging in the future.

Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast

PubMed Central

Dong, Biqin; Almassalha, Luay M.; Stypula-Cyrus, Yolanda; Urban, Ben E.; Chandler, John E.; Nguyen, The-Quyen; Sun, Cheng; Zhang, Hao F.; Backman, Vadim

2016-01-01

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging. PMID:27535934

Profiling antibody responses by multiparametric analysis of primary B cells

PubMed Central

Story, Craig M.; Papa, Eliseo; Hu, Chih-Chi Andrew; Ronan, Jehnna L.; Herlihy, Kara; Ploegh, Hidde L.; Love, J. Christopher

2008-01-01

Determining the efficacy of a vaccine generally relies on measuring neutralizing antibodies in sera. This measure cannot elucidate the mechanisms responsible for the development of immunological memory at the cellular level, however. Quantitative profiles that detail the cellular origin, extent, and diversity of the humoral (antibody-based) immune response would improve both the assessment and development of vaccines. Here, we describe a novel approach to collect multiparametric datasets that describe the specificity, isotype, and apparent affinity of the antibodies secreted from large numbers of individual primary B cells (≈103-104). The antibody/antigen binding curves obtained by this approach can be used to classify closely related populations of cells using algorithms for data clustering, and the relationships among populations can be visualized graphically using affinity heatmaps. The technique described was used to evaluate the diversity of antigen-specific antibody-secreting cells generated during an in vivo humoral response to a series of immunizations designed to mimic a multipart vaccination. Profiles correlating primary antibody-producing cells with the molecular characteristics of their secreted antibodies should facilitate both the evaluation of candidate vaccines and, broadly, studies on the repertoires of antibodies generated in response to infectious or autoimmune diseases. PMID:19004776

SYMPOSIUM SESSION PROPOSAL: INCORPORATION OF MODE OF ACTION INTO MECHANISTICALLY-BASED QUANTITATIVE MODELS

EPA Science Inventory

The biological processes by which environmental pollutants induce adverse health effects is most likely regulated by complex interactions dependent upon the route of exposure, dose, kinetics of distribution, and multiple cellular responses. To further complicate deciphering thes...

20180312 - Mechanistic Modeling of Developmental Defects through Computational Embryology (SOT)

EPA Science Inventory

Significant advances in the genome sciences, in automated high-throughput screening (HTS), and in alternative methods for testing enable rapid profiling of chemical libraries for quantitative effects on diverse cellular activities. While a surfeit of HTS data and information is n...

Quantitative and histological assessment of maternal-fetal transmission of Trypanosoma cruzi in guinea pigs: An experimental model of congenital Chagas disease

PubMed Central

Torres-Vargas, Jatziri; Jiménez-Coello, Matilde; Guzmán-Marín, Eugenia; Acosta-Viana, Karla Y.; Yadon, Zaida E.; Gutiérrez-Blanco, Eduardo; Guillermo-Cordero, José Leonardo

2018-01-01

Objective We evaluated the effect of Trypanosoma cruzi infection on fertility, gestation outcome, and maternal-fetal transmission in guinea pigs (Cavia porcellus). Methods Animals were infected with T. cruzi H4 strain (TcI lineage) before gestation (IBG) or during gestation (IDG). Tissue and sera samples of dams and fetuses were obtained near parturition. Results All IBG and IDG dams were seropositive by two tests, and exhibited blood parasite load of 1.62±2.2 and 50.1±62 parasites/μl, respectively, by quantitative PCR. Histological evaluation showed muscle fiber degeneration and cellular necrosis in all infected dams. Parasite nests were not detected in infected dams by histology. However, qPCR analysis detected parasites-eq/g heart tissue of 153±104.7 and 169.3±129.4 in IBG and IDG dams, respectively. All fetuses of infected dams were positive for anti-parasite IgG antibodies and tissue parasites by qPCR, but presented a low level of tissue inflammatory infiltrate. Fetuses of IDG (vs. IBG) dams exhibited higher degree of muscle fiber degeneration and cellular necrosis in the heart and skeletal tissues. The placental tissue exhibited no inflammatory lesions and amastigote nests, yet parasites-eq/g of 381.2±34.3 and 79.2±84.9 were detected in IDG and IBG placentas, respectively. Fetal development was compromised, and evidenced by a decline in weight, crow-rump length, and abdominal width in both groups. Conclusions T. cruzi TcI has a high capacity of congenital transmission even when it was inoculated at a very low dose before or during gestation. Tissue lesions, parasite load, and fetal under development provide evidence for high virulence of the parasite during pregnancy. Despite finding of high parasite burden by qPCR, placentas were protected from cellular damage. Our studies offer an experimental model to study the efficacy of vaccines and drugs against congenital transmission of T. cruzi. These results also call for T. cruzi screening in pregnant women and adequate follow up of the newborns in endemic areas. PMID:29364882

Cellular Automata

NASA Astrophysics Data System (ADS)

Gutowitz, Howard

1991-08-01

Cellular automata, dynamic systems in which space and time are discrete, are yielding interesting applications in both the physical and natural sciences. The thirty four contributions in this book cover many aspects of contemporary studies on cellular automata and include reviews, research reports, and guides to recent literature and available software. Chapters cover mathematical analysis, the structure of the space of cellular automata, learning rules with specified properties: cellular automata in biology, physics, chemistry, and computation theory; and generalizations of cellular automata in neural nets, Boolean nets, and coupled map lattices. Current work on cellular automata may be viewed as revolving around two central and closely related problems: the forward problem and the inverse problem. The forward problem concerns the description of properties of given cellular automata. Properties considered include reversibility, invariants, criticality, fractal dimension, and computational power. The role of cellular automata in computation theory is seen as a particularly exciting venue for exploring parallel computers as theoretical and practical tools in mathematical physics. The inverse problem, an area of study gaining prominence particularly in the natural sciences, involves designing rules that possess specified properties or perform specified task. A long-term goal is to develop a set of techniques that can find a rule or set of rules that can reproduce quantitative observations of a physical system. Studies of the inverse problem take up the organization and structure of the set of automata, in particular the parameterization of the space of cellular automata. Optimization and learning techniques, like the genetic algorithm and adaptive stochastic cellular automata are applied to find cellular automaton rules that model such physical phenomena as crystal growth or perform such adaptive-learning tasks as balancing an inverted pole. Howard Gutowitz is Collaborateur in the Service de Physique du Solide et Résonance Magnetique, Commissariat a I'Energie Atomique, Saclay, France.

A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system

PubMed Central

2014-01-01

Background Copper is essential for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. Results We found that addition of copper (Cu) to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains. Cu’s positive effect on respiratory growth was quantitatively reduced in deletion strains representing 73 different genes, the function of which identify increased iron uptake as a cause of the increase in growth rate. Conversely, these effects were enhanced in strains representing 93 genes. Many of these strains exhibited respiratory defects that were specifically rescued by supplementing the growth medium with Cu. Among the genes identified are known and direct regulators of copper homeostasis, genes required to maintain low vacuolar pH, and genes where evidence supporting a functional link with Cu has been heretofore lacking. Roughly half of the genes are conserved in man, and several of these are associated with Mendelian disorders, including the Cu-imbalance syndromes Menkes and Wilson’s disease. We additionally demonstrate that pharmacological agents, including the approved drug disulfiram, can rescue Cu-deficiencies of both environmental and genetic origin. Conclusions A functional screen in yeast has expanded the list of genes required for Cu-dependent fitness, revealing a complex cellular system with implications for human health. Respiratory fitness defects arising from perturbations in this system can be corrected with pharmacological agents that increase intracellular copper concentrations. PMID:24708151

Quantitative Biology of Exercise-Induced Signal Transduction Pathways.

PubMed

Liu, Timon Cheng-Yi; Liu, Gang; Hu, Shao-Juan; Zhu, Ling; Yang, Xiang-Bo; Zhang, Quan-Guang

2017-01-01

Exercise is essential in regulating energy metabolism. Exercise activates cellular, molecular, and biochemical pathways with regulatory roles in training response adaptation. Among them, endurance/strength training of an individual has been shown to activate its respective signal transduction pathways in skeletal muscle. This was further studied from the viewpoint of quantitative difference (QD). For the mean values, [Formula: see text], of two sets of data, their QD is defined as [Formula: see text] ([Formula: see text]). The function-specific homeostasis (FSH) of a function of a biosystem is a negative-feedback response of the biosystem to maintain the function-specific conditions inside the biosystem so that the function is perfectly performed. A function in/far from its FSH is called a normal/dysfunctional function. A cellular normal function can resist the activation of other signal transduction pathways so that there are normal function-specific signal transduction pathways which full activation maintains the normal function. An acute endurance/strength training may be dysfunctional, but its regular training may be normal. The normal endurance/strength training of an individual may resist the activation of other signal transduction pathways in skeletal muscle so that there may be normal endurance/strength training-specific signal transduction pathways (NEPs/NSPs) in skeletal muscle. The endurance/strength training may activate NSPs/NEPs, but the QD from the control is smaller than 0.80. The simultaneous activation of both NSPs and NEPs may enhance their respective activation, and the QD from the control is larger than 0.80. The low level laser irradiation pretreatment of rats may promote the activation of NSPs in endurance training skeletal muscle. There may be NEPs/NSPs in skeletal muscle trained by normal endurance/strength training.

In vivo quantitative visualization of hypochlorous acid in the liver using a novel selective two-photon fluorescent probe

NASA Astrophysics Data System (ADS)

Wang, Haolu; Jayachandran, Aparna; Gravot, Germain; Liang, Xiaowen; Thorling, Camilla A.; Zhang, Run; Liu, Xin; Roberts, Michael S.

2016-11-01

Hypochlorous acid (HOCl) plays a vital role in physiological events and diseases. During hepatic ischemia-reperfusion (I/R) injury, HOCl is generated by neutrophils and diffuses into hepatocytes, causing oxidant stress-mediated injury. Although many probes have been developed to detect HOCl, most were difficult to be distinguished from endogenous fluorophores in intravital imaging and only can be employed under one-photon microscopy. A novel iridium(III) complex-based ferrocene dual-signaling chemosensor (Ir-Fc) was designed and synthesized. Ir-Fc exhibited a strong positive fluorescent response only in the presence of HOCl, whereas negligible fluorescent signals were observed upon the additions of other reactive oxygen/nitrogen species and metal ions. There was a good linear relationship between probe responsive fluorescent intensity and HOCl concentration. Ir-Fc was then intravenously injected into BALB/c mice at the final concentration of 50 μM and the mouse livers were imaged using multiphoton microscopy (MPM). In the I/R liver, reduced autofluorescence was detected by MPM, indicating the hepatocyte necrosis. Remarkable enhancement of red fluorescence was observed in hepatocytes with decreased autofluorescence, indicating the reaction of Ir-Fc with endogenous HOCl molecules. The cellular concentration of HOCl was first calculated based on the intensity of MPM images. No obvious toxic effects were observed in histological examination of major organs after Ir-Fc injection. In summary, Ir-Fc has low cytotoxicity, high specificity to HOCl, and rapid "off-on" fluorescence. It is suitable for dynamic quantitatively monitoring HOCl generation using MPM at the cellular level. This technique can be readily extended to examination of liver diseases and injury.

Identification of a flavonoid C-glycoside as potent antioxidant.

PubMed

Wen, Lingrong; Zhao, Yupeng; Jiang, Yueming; Yu, Limei; Zeng, Xiaofang; Yang, Jiali; Tian, Miaomiao; Liu, Huiling; Yang, Bao

2017-09-01

Flavonoids have been documented to have good antioxidant activities in vitro. However, reports on the cellular antioxidant activities of flavonoid C-glycosides are very limited. In this work, an apigenin C-glycoside was purified from Artocarpus heterophyllus by column chromatography and was identified to be 2″-O-β-D-xylosylvitexin by nuclear magnetic resonance spectroscopy. The cellular antioxidant activity and anticancer activity of 2″-O-β-D-xylosylvitexin were evaluated for the first time. The quantitative structure-activity relationship was analysed by molecular modeling. Apigenin presented an unexpected cellular antioxidation behaviour. It had an antioxidant activity at low concentration and a prooxidant activity at high concentration, whereas 2″-O-β-D-xylosylvitexin showed a dose-dependent cellular antioxidant activity. It indicated that C-glycosidation improved the cellular antioxidation performance of apigenin and eliminated the prooxidant effect. The ortho-dihydroxyl at C-3'/C-4' and C-3 hydroxyl in the flavonoid skeleton play important roles in the antioxidation behaviour. The cell proliferation assay revealed a low cytotoxicity of 2″-O-β-D-xylosylvitexin. Copyright © 2017 Elsevier Inc. All rights reserved.

Thiol Specific and Mitochondria Selective Fluorogenic Benzofurazan Sulfide for Live Cell Nonprotein Thiol Imaging and Quantification in Mitochondria.

PubMed

Wang, Shenggang; Yin, Huihui; Huang, Yue; Guan, Xiangming

2018-06-11

Cellular thiols are divided into two major categories: nonprotein thiols (NPSH) and protein thiols (PSH). Thiols are unevenly distributed inside the cell and compartmentalized in subcellular structures. Most of our knowledge on functions/dysfunctions of cellular/subcellular thiols is based on the quantification of cellular/subcellular thiols through homogenization of cellular/subcellular structures followed by a thiol quantification method. We would like to report a thiol-specific mitochondria-selective fluorogenic benzofurazan sulfide {7,7'-thiobis( N-rhodamine-benzo[c][1,2,5]oxadiazole-4-sulfonamide) (TBROS)} that can effectively image and quantify live cell NPSH in mitochondria through fluorescence intensity. Limited methods are available for imaging thiols in mitochondria in live cells especially in a quantitative manner. The thiol specificity of TBROS was demonstrated by its ability to react with thiols and inability to react with biologically relevant nucleophilic functional groups other than thiols. TBROS, with minimal fluorescence, formed strong fluorescent thiol adducts (λ ex = 550 nm, λ em = 580 nm) when reacting with NPSH confirming its fluorogenicity. TBROS failed to react with PSH from bovine serum albumin and cell homogenate proteins. The high mitochondrial thiol selectivity of TBROS was achieved by its mitochondria targeting structure and its higher reaction rate with NPSH at mitochondrial pH. Imaging of mitochondrial NPSH in live cells was confirmed by two colocalization methods and use of a thiol-depleting reagent. TBROS effectively imaged NPSH changes in a quantitative manner in mitochondria in live cells. The reagent will be a useful tool in exploring physiological and pathological roles of mitochondrial thiols.

Monitoring Intracellular Oxygen Concentration: Implications for Hypoxia Studies and Real-Time Oxygen Monitoring.

PubMed

Potter, Michelle; Badder, Luned; Hoade, Yvette; Johnston, Iain G; Morten, Karl J

2016-01-01

The metabolic properties of cancer cells have been widely accepted as a hallmark of cancer for a number of years and have shown to be of critical importance in tumour development. It is generally accepted that tumour cells exhibit a more glycolytic phenotype than normal cells. In this study, we investigate the bioenergetic phenotype of two widely used cancer cell lines, RD and U87MG, by monitoring intracellular oxygen concentrations using phosphorescent Pt-porphyrin based intracellular probes. Our study demonstrates that cancer cell lines do not always exhibit an exclusively glycolytic phenotype. RD demonstrates a reliance on oxidative phosphorylation whilst U87MG display a more glycolytic phenotype. Using the intracellular oxygen sensing probe we generate an immediate readout of intracellular oxygen levels, with the glycolytic lines reflecting the oxygen concentration of the environment, and cells with an oxidative phenotype having significantly lower levels of intracellular oxygen. Inhibition of oxygen consumption in lines with high oxygen consumption increases intracellular oxygen levels towards environmental levels. We conclude that the use of intracellular oxygen probes provides a quantitative assessment of intracellular oxygen levels, allowing the manipulation of cellular bioenergetics to be studied in real time.

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

PubMed Central

Dethier, Julie; Drion, Guillaume; Franci, Alessio

2015-01-01

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. PMID:26311181

Altered cellular and humoral immunity to varicella-zoster virus in patients with autoimmune diseases.

PubMed

Rondaan, Christien; de Haan, Aalzen; Horst, Gerda; Hempel, J Cordelia; van Leer, Coretta; Bos, Nicolaas A; van Assen, Sander; Bijl, Marc; Westra, Johanna

2014-11-01

Patients with autoimmune diseases such as systemic lupus erythematosus (SLE) and granulomatosis with polyangiitis (Wegener's) (GPA) have a 3-20-fold increased risk of herpes zoster compared to the general population. The aim of this study was to evaluate if susceptibility is due to decreased levels of cellular and/or humoral immunity to the varicella-zoster virus (VZV). A cross-sectional study of VZV-specific immunity was performed in 38 SLE patients, 33 GPA patients, and 51 healthy controls. Levels of IgG and IgM antibodies to VZV were measured using an in-house glycoprotein enzyme-linked immunosorbent assay (ELISA). Cellular responses to VZV were determined by interferon-γ (IFNγ) enzyme-linked immunospot (ELISpot) assay and carboxyfluorescein succinimidyl ester (CFSE) dye dilution proliferation assay. Levels of IgG antibodies to VZV were increased in SLE patients as compared to healthy controls, but levels of IgM antibodies to VZV were not. Antibody levels in GPA patients did not differ significantly from levels in healthy controls. In response to stimulation with VZV, decreased numbers of IFNγ spot-forming cells were found among SLE patients (although not GPA patients) as compared to healthy controls. Proliferation of CD4+ T cells in response to stimulation with VZV was decreased in SLE patients but not GPA patients. SLE patients have increased levels of IgG antibodies against VZV, while cellular immunity is decreased. In GPA patients, antibody levels as well as cellular responses to VZV were comparable to those in healthy controls. These data suggest that increased prevalence of herpes zoster in SLE patients is due to a poor cellular response. Vaccination strategies should aim to boost cellular immunity against VZV. Copyright © 2014 by the American College of Rheumatology.

The effect of growth phase and medium on the use of the firefly adenosine triphosphate (ATP) assay for the quantitation of bacteria

NASA Technical Reports Server (NTRS)

Bush, V. N.; Picciolo, G. L.; Chappelle, E. W.

1975-01-01

Luciferase assay for adenosine triphosphate (ATP) was used as a rapid method to determine the number of bacteria in a urine sample after nonbacterial components were removed. Accurate cellular ATP values, determined when bacteria were grown in an environment similar to that in which they were found, were necessary for the calculation of bacterial titer in urine. Cellular ATP values vary depending on the extraction method, the cell growth phase, and cell growth conditions. ATP per cell values of stationary E. coli grown in urine were two times greater than ATP per cell values of cells grown in trypticase soy broth. Glucose and urea were examined as possible components responsible for the cellular ATP variation.

Red blood cell dynamics: from cell deformation to ATP release.

PubMed

Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

2011-10-01

The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release. This journal is © The Royal Society of Chemistry 2011

Cellular interface morphologies in directional solidification. III - The effects of heat transfer and solid diffusivity

NASA Technical Reports Server (NTRS)

Ungar, Lyle H.; Bennett, Mark J.; Brown, Robert A.

1985-01-01

The shape and stability of two-dimensional finite-amplitude cellular interfaces arising during directional solidification are compared for several solidification models that account differently for latent heat released at the interface, unequal thermal conductivities of melt and solid, and solute diffusivity in the solid. Finite-element analysis and computer-implemented perturbation methods are used to analyze the families of steadily growing cellular forms that evolve from the planar state. In all models a secondary bifurcation between different families of finite-amplitude cells exists that halves the spatial wavelength of the stable interface. The quantitative location of this transition is very dependent on the details of the model. Large amounts of solute diffusion in the solid retard the growth of large-amplitude cells.

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

Mass spectrometry as a quantitative tool in plant metabolomics

PubMed Central

Jorge, Tiago F.; Mata, Ana T.

2016-01-01

Metabolomics is a research field used to acquire comprehensive information on the composition of a metabolite pool to provide a functional screen of the cellular state. Studies of the plant metabolome include the analysis of a wide range of chemical species with very diverse physico-chemical properties, and therefore powerful analytical tools are required for the separation, characterization and quantification of this vast compound diversity present in plant matrices. In this review, challenges in the use of mass spectrometry (MS) as a quantitative tool in plant metabolomics experiments are discussed, and important criteria for the development and validation of MS-based analytical methods provided. This article is part of the themed issue ‘Quantitative mass spectrometry’. PMID:27644967

Quantitation of Met tyrosine phosphorylation using MRM-MS.

PubMed

Meng, Zhaojing; Srivastava, Apurva K; Zhou, Ming; Veenstra, Timothy

2013-01-01

Phosphorylation has long been accepted as a key cellular regulator of cell signaling pathways. The recent development of multiple-reaction monitoring mass spectrometry (MRM-MS) provides a useful tool for measuring the absolute quantity of phosphorylation occupancy at pivotal sites within signaling proteins, even when the phosphorylation sites are in close proximity. Here, we described a targeted quantitation approach to measure the absolute phosphorylation occupancy at Y1234 and Y1235 of Met. The approach is utilized to obtain absolute occupancy of the two phosphorylation sites in the full-length recombinant Met. It is further applied to quantitate the phosphorylation state of these two sites in SNU-5 cells treated with a Met inhibitor.

Qualitative and quantitative mass spectrometry imaging of drugs and metabolites.

PubMed

Lietz, Christopher B; Gemperline, Erin; Li, Lingjun

2013-07-01

Mass spectrometric imaging (MSI) has rapidly increased its presence in the pharmaceutical sciences. While quantitative whole-body autoradiography and microautoradiography are the traditional techniques for molecular imaging of drug delivery and metabolism, MSI provides advantageous specificity that can distinguish the parent drug from metabolites and modified endogenous molecules. This review begins with the fundamentals of MSI sample preparation/ionization, and then moves on to both qualitative and quantitative applications with special emphasis on drug discovery and delivery. Cutting-edge investigations on sub-cellular imaging and endogenous signaling peptides are also highlighted, followed by perspectives on emerging technology and the path for MSI to become a routine analysis technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Qualitative and quantitative mass spectrometry imaging of drugs and metabolites

PubMed Central

Lietz, Christopher B.; Gemperline, Erin; Li, Lingjun

2013-01-01

Mass spectrometric imaging (MSI) has rapidly increased its presence in the pharmaceutical sciences. While quantitative whole-body autoradiography and microautoradiography are the traditional techniques for molecular imaging of drug delivery and metabolism, MSI provides advantageous specificity that can distinguish the parent drug from metabolites and modified endogenous molecules. This review begins with the fundamentals of MSI sample preparation/ionization, and then moves on to both qualitative and quantitative applications with special emphasis on drug discovery and delivery. Cutting-edge investigations on sub-cellular imaging and endogenous signaling peptides are also highlighted, followed by perspectives on emerging technology and the path for MSI to become a routine analysis technique. PMID:23603211

Quantitative phase microscopy for cellular dynamics based on transport of intensity equation.

PubMed

Li, Ying; Di, Jianglei; Ma, Chaojie; Zhang, Jiwei; Zhong, Jinzhan; Wang, Kaiqiang; Xi, Teli; Zhao, Jianlin

2018-01-08

We demonstrate a simple method for quantitative phase imaging of tiny transparent objects such as living cells based on the transport of intensity equation. The experiments are performed using an inverted bright field microscope upgraded with a flipping imaging module, which enables to simultaneously create two laterally separated images with unequal defocus distances. This add-on module does not include any lenses or gratings and is cost-effective and easy-to-alignment. The validity of this method is confirmed by the measurement of microlens array and human osteoblastic cells in culture, indicating its potential in the applications of dynamically measuring living cells and other transparent specimens in a quantitative, non-invasive and label-free manner.

Plants in silico: why, why now and what?--an integrative platform for plant systems biology research.

PubMed

Zhu, Xin-Guang; Lynch, Jonathan P; LeBauer, David S; Millar, Andrew J; Stitt, Mark; Long, Stephen P

2016-05-01

A paradigm shift is needed and timely in moving plant modelling from largely isolated efforts to a connected community endeavour that can take full advantage of advances in computer science and in mechanistic understanding of plant processes. Plants in silico (Psi) envisions a digital representation of layered dynamic modules, linking from gene networks and metabolic pathways through to cellular organization, tissue, organ and whole plant development, together with resource capture and use efficiency in dynamic competitive environments, ultimately allowing a mechanistically rich simulation of the plant or of a community of plants in silico. The concept is to integrate models or modules from different layers of organization spanning from genome to phenome to ecosystem in a modular framework allowing the use of modules of varying mechanistic detail representing the same biological process. Developments in high-performance computing, functional knowledge of plants, the internet and open-source version controlled software make achieving the concept realistic. Open source will enhance collaboration and move towards testing and consensus on quantitative theoretical frameworks. Importantly, Psi provides a quantitative knowledge framework where the implications of a discovery at one level, for example, single gene function or developmental response, can be examined at the whole plant or even crop and natural ecosystem levels. © 2015 The Authors Plant, Cell & Environment Published by John Wiley & Sons Ltd.

Characterizing differences between MSCs and TM cells: Toward autologous stem cell therapies for the glaucomatous trabecular meshwork.

PubMed

Snider, Eric J; Vannatta, R Taylor; Schildmeyer, Lisa; Stamer, W Daniel; Ethier, C Ross

2018-03-01

Glaucoma, a leading cause of blindness, is characterized by an increase in intraocular pressure, which is largely determined by resistance to aqueous humour outflow through the trabecular meshwork (TM). In glaucoma, the cellularity of the TM is decreased, and, as a result, stem cell therapies for the TM represent a potential therapeutic option for restoring TM function and treating glaucoma patients. We here focus on adipose derived mesenchymal stem cells (MSCs) as a potential autologous cell source for TM regenerative medicine applications and describe characterization techniques at the messenger (reverse transcription-quantitative polymerase chain reaction), protein (western blotting, flow cytometry), and functional (contractility, phagocytosis) levels to distinguish MSCs from TM cells. We present a panel of 12 transcripts to allow: (a) suitable normalization of reverse transcription-quantitative polymerase chain reaction results across cell types and after exposure to potential differentiation stimuli; (b) distinguishing MSCs from TM cells; (c) distinguishing subtypes of TM cells; and (d) distinguishing TM cells from those in neighbouring tissue. At the protein level, dexamethasone induction of myocilin was a robust discriminating factor between MSCs and TM cells and was complemented by other protein markers. Finally, we show that contractility and phagocytosis differ between MSCs and TM cells. These methods are recommended for use in future differentiation studies to fully define if a functional TM-like phenotype is being achieved. Copyright © 2017 John Wiley & Sons, Ltd.

iTRAQ-Based Quantitative Proteomic Analysis of Spirulina platensis in Response to Low Temperature Stress

PubMed Central

Li, Qingye; Chang, Rong; Sun, Yijun; Li, Bosheng

2016-01-01

Low temperature (LT) is one of the most important abiotic stresses that can significantly reduce crop yield. To gain insight into how Spirulina responds to LT stress, comprehensive physiological and proteomic analyses were conducted in this study. Significant decreases in growth and pigment levels as well as excessive accumulation of compatible osmolytes were observed in response to LT stress. An isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach was used to identify changes in protein abundance in Spirulina under LT. A total of 3,782 proteins were identified, of which 1,062 showed differential expression. Bioinformatics analysis indicated that differentially expressed proteins that were enriched in photosynthesis, carbohydrate metabolism, amino acid biosynthesis, and translation are important for the maintenance of cellular homeostasis and metabolic balance in Spirulina when subjected to LT stress. The up-regulation of proteins involved in gluconeogenesis, starch and sucrose metabolism, and amino acid biosynthesis served as coping mechanisms of Spirulina in response to LT stress. Moreover, the down-regulated expression of proteins involved in glycolysis, TCA cycle, pentose phosphate pathway, photosynthesis, and translation were associated with reduced energy consumption. The findings of the present study allow a better understanding of the response of Spirulina to LT stress and may facilitate in the elucidation of mechanisms underlying LT tolerance. PMID:27902743

Correlative Instrumental Neutron Activation Analysis, Light Microscopy, Transmission Electron Microscopy, and X-ray Microanalysis for Qualitative and Quantitative Detection of Colloidal Gold Spheres in Biological Specimens

NASA Astrophysics Data System (ADS)

Hillyer, Julián F.; Albrecht, Ralph M.

1998-10-01

: Colloidal gold, conjugated to ligands or antibodies, is routinely used as a label for the detection of cell structures by light (LM) and electron microscopy (EM). To date, several methods to count the number of colloidal gold labels have been employed with limited success. Instrumental neutron activation analysis (INAA), a physical method for the analysis of the elemental composition of materials, can be used to provide a quantitative index of gold accumulation in bulk specimens. Given that gold is not naturally found in biological specimens in any substantial amount and that colloidal gold and ligand conjugates can be prepared to yield uniform bead sizes, the amount of label can be calculated in bulk biological samples by INAA. Here we describe the use of INAA, LM, transmission EM, and X-ray microanalysis (EDX) in a model to determine both distribution (localization) and amount of colloidal gold at the organ, tissue, cellular, and ultrastructural levels in whole animal systems following administration. In addition, the sensitivity for gold in biological specimens by INAA is compared with that of inductively coupled plasma mass spectrometry (ICP-MS). The correlative use of INAA, LM, TEM, and EDX can be useful, for example, in the quantitative and qualitative tracking of various labeled molecular species following administration in vivo.

iTRAQ-Based Quantitative Proteomic Analysis of Spirulina platensis in Response to Low Temperature Stress.

PubMed

Li, Qingye; Chang, Rong; Sun, Yijun; Li, Bosheng

2016-01-01

Low temperature (LT) is one of the most important abiotic stresses that can significantly reduce crop yield. To gain insight into how Spirulina responds to LT stress, comprehensive physiological and proteomic analyses were conducted in this study. Significant decreases in growth and pigment levels as well as excessive accumulation of compatible osmolytes were observed in response to LT stress. An isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomics approach was used to identify changes in protein abundance in Spirulina under LT. A total of 3,782 proteins were identified, of which 1,062 showed differential expression. Bioinformatics analysis indicated that differentially expressed proteins that were enriched in photosynthesis, carbohydrate metabolism, amino acid biosynthesis, and translation are important for the maintenance of cellular homeostasis and metabolic balance in Spirulina when subjected to LT stress. The up-regulation of proteins involved in gluconeogenesis, starch and sucrose metabolism, and amino acid biosynthesis served as coping mechanisms of Spirulina in response to LT stress. Moreover, the down-regulated expression of proteins involved in glycolysis, TCA cycle, pentose phosphate pathway, photosynthesis, and translation were associated with reduced energy consumption. The findings of the present study allow a better understanding of the response of Spirulina to LT stress and may facilitate in the elucidation of mechanisms underlying LT tolerance.

Alcoholic liver disease: The gut microbiome and liver crosstalk

PubMed Central

Hartmann, Phillipp; Seebauer, Caroline T.; Schnabl, Bernd

2015-01-01

Alcoholic liver disease is a leading cause of morbidity and mortality worldwide. Alcoholic fatty liver disease can progress to steatohepatitis, alcoholic hepatitis, fibrosis, and cirrhosis. Patients with alcohol abuse show quantitative and qualitative changes in the composition of the intestinal microbiome. Furthermore, patients with alcoholic liver disease have increased intestinal permeability and elevated systemic levels of gut-derived microbial products. Maintaining eubiosis, stabilizing the mucosal gut barrier or preventing cellular responses to microbial products protect from experimental alcoholic liver disease. Therefore, intestinal dysbiosis and pathological bacterial translocation appear fundamental for the pathogenesis of alcoholic liver disease. This review highlights causes for intestinal dysbiosis and pathological bacterial translocation, their relationship and consequences for alcoholic liver disease. We also discuss how the liver affects the intestinal microbiota. PMID:25872593

SILAC-Based Quantitative Proteomic Analysis of Human Lung Cell Response to Copper Oxide Nanoparticles

PubMed Central

Edelmann, Mariola J.; Shack, Leslie A.; Naske, Caitlin D.; Walters, Keisha B.; Nanduri, Bindu

2014-01-01

Copper (II) oxide (CuO) nanoparticles (NP) are widely used in industry and medicine. In our study we evaluated the response of BEAS-2B human lung cells to CuO NP, using Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics and phosphoproteomics. Pathway modeling of the protein differential expression showed that CuO NP affect proteins relevant in cellular function and maintenance, protein synthesis, cell death and survival, cell cycle and cell morphology. Some of the signaling pathways represented by BEAS-2B proteins responsive to the NP included mTOR signaling, protein ubiquitination pathway, actin cytoskeleton signaling and epithelial adherens junction signaling. Follow-up experiments showed that CuO NP altered actin cytoskeleton, protein phosphorylation and protein ubiquitination level. PMID:25470785

Functional magnetic resonance imaging in oncology: state of the art.

PubMed

Guimaraes, Marcos Duarte; Schuch, Alice; Hochhegger, Bruno; Gross, Jefferson Luiz; Chojniak, Rubens; Marchiori, Edson

2014-01-01

In the investigation of tumors with conventional magnetic resonance imaging, both quantitative characteristics, such as size, edema, necrosis, and presence of metastases, and qualitative characteristics, such as contrast enhancement degree, are taken into consideration. However, changes in cell metabolism and tissue physiology which precede morphological changes cannot be detected by the conventional technique. The development of new magnetic resonance imaging techniques has enabled the functional assessment of the structures in order to obtain information on the different physiological processes of the tumor microenvironment, such as oxygenation levels, cellularity and vascularity. The detailed morphological study in association with the new functional imaging techniques allows for an appropriate approach to cancer patients, including the phases of diagnosis, staging, response evaluation and follow-up, with a positive impact on their quality of life and survival rate.

Non-invasive molecular imaging for preclinical cancer therapeutic development

PubMed Central

O'Farrell, AC; Shnyder, SD; Marston, G; Coletta, PL; Gill, JH

2013-01-01

Molecular and non-invasive imaging are rapidly emerging fields in preclinical cancer drug discovery. This is driven by the need to develop more efficacious and safer treatments, the advent of molecular-targeted therapeutics, and the requirements to reduce and refine current preclinical in vivo models. Such bioimaging strategies include MRI, PET, single positron emission computed tomography, ultrasound, and optical approaches such as bioluminescence and fluorescence imaging. These molecular imaging modalities have several advantages over traditional screening methods, not least the ability to quantitatively monitor pharmacodynamic changes at the cellular and molecular level in living animals non-invasively in real time. This review aims to provide an overview of non-invasive molecular imaging techniques, highlighting the strengths, limitations and versatility of these approaches in preclinical cancer drug discovery and development. PMID:23488622

Mechanical regulation of cardiac development

PubMed Central

Lindsey, Stephanie E.; Butcher, Jonathan T.; Yalcin, Huseyin C.

2014-01-01

Mechanical forces are essential contributors to and unavoidable components of cardiac formation, both inducing and orchestrating local and global molecular and cellular changes. Experimental animal studies have contributed substantially to understanding the mechanobiology of heart development. More recent integration of high-resolution imaging modalities with computational modeling has greatly improved our quantitative understanding of hemodynamic flow in heart development. Merging these latest experimental technologies with molecular and genetic signaling analysis will accelerate our understanding of the relationships integrating mechanical and biological signaling for proper cardiac formation. These advances will likely be essential for clinically translatable guidance for targeted interventions to rescue malforming hearts and/or reconfigure malformed circulations for optimal performance. This review summarizes our current understanding on the levels of mechanical signaling in the heart and their roles in orchestrating cardiac development. PMID:25191277

Third-Kind Encounters in Biomedicine: Immunology Meets Mathematics and Informatics to Become Quantitative and Predictive.

PubMed

Eberhardt, Martin; Lai, Xin; Tomar, Namrata; Gupta, Shailendra; Schmeck, Bernd; Steinkasserer, Alexander; Schuler, Gerold; Vera, Julio

2016-01-01

The understanding of the immune response is right now at the center of biomedical research. There are growing expectations that immune-based interventions will in the midterm provide new, personalized, and targeted therapeutic options for many severe and highly prevalent diseases, from aggressive cancers to infectious and autoimmune diseases. To this end, immunology should surpass its current descriptive and phenomenological nature, and become quantitative, and thereby predictive.Immunology is an ideal field for deploying the tools, methodologies, and philosophy of systems biology, an approach that combines quantitative experimental data, computational biology, and mathematical modeling. This is because, from an organism-wide perspective, the immunity is a biological system of systems, a paradigmatic instance of a multi-scale system. At the molecular scale, the critical phenotypic responses of immune cells are governed by large biochemical networks, enriched in nested regulatory motifs such as feedback and feedforward loops. This network complexity confers them the ability of highly nonlinear behavior, including remarkable examples of homeostasis, ultra-sensitivity, hysteresis, and bistability. Moving from the cellular level, different immune cell populations communicate with each other by direct physical contact or receiving and secreting signaling molecules such as cytokines. Moreover, the interaction of the immune system with its potential targets (e.g., pathogens or tumor cells) is far from simple, as it involves a number of attack and counterattack mechanisms that ultimately constitute a tightly regulated multi-feedback loop system. From a more practical perspective, this leads to the consequence that today's immunologists are facing an ever-increasing challenge of integrating massive quantities from multi-platforms.In this chapter, we support the idea that the analysis of the immune system demands the use of systems-level approaches to ensure the success in the search for more effective and personalized immune-based therapies.

An affinity-structure database of helix-turn-helix: DNA complexes with a universal coordinate system.

PubMed

AlQuraishi, Mohammed; Tang, Shengdong; Xia, Xide

2015-11-19

Molecular interactions between proteins and DNA molecules underlie many cellular processes, including transcriptional regulation, chromosome replication, and nucleosome positioning. Computational analyses of protein-DNA interactions rely on experimental data characterizing known protein-DNA interactions structurally and biochemically. While many databases exist that contain either structural or biochemical data, few integrate these two data sources in a unified fashion. Such integration is becoming increasingly critical with the rapid growth of structural and biochemical data, and the emergence of algorithms that rely on the synthesis of multiple data types to derive computational models of molecular interactions. We have developed an integrated affinity-structure database in which the experimental and quantitative DNA binding affinities of helix-turn-helix proteins are mapped onto the crystal structures of the corresponding protein-DNA complexes. This database provides access to: (i) protein-DNA structures, (ii) quantitative summaries of protein-DNA binding affinities using position weight matrices, and (iii) raw experimental data of protein-DNA binding instances. Critically, this database establishes a correspondence between experimental structural data and quantitative binding affinity data at the single basepair level. Furthermore, we present a novel alignment algorithm that structurally aligns the protein-DNA complexes in the database and creates a unified residue-level coordinate system for comparing the physico-chemical environments at the interface between complexes. Using this unified coordinate system, we compute the statistics of atomic interactions at the protein-DNA interface of helix-turn-helix proteins. We provide an interactive website for visualization, querying, and analyzing this database, and a downloadable version to facilitate programmatic analysis. This database will facilitate the analysis of protein-DNA interactions and the development of programmatic computational methods that capitalize on integration of structural and biochemical datasets. The database can be accessed at http://ProteinDNA.hms.harvard.edu.

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

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

Mu, Jun; Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing; Chongqing Key Laboratory of Neurobiology, Chongqing

Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology andmore » proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives insights into the pathogenesis of TBM.« less

Quantitative Proteomic Analysis of the Hfq-Regulon in Sinorhizobium meliloti 2011

PubMed Central

Sobrero, Patricio; Schlüter, Jan-Philip; Lanner, Ulrike; Schlosser, Andreas; Becker, Anke; Valverde, Claudio

2012-01-01

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with 15N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti. PMID:23119037

Quantitative proteomic analysis of the Hfq-regulon in Sinorhizobium meliloti 2011.

PubMed

Sobrero, Patricio; Schlüter, Jan-Philip; Lanner, Ulrike; Schlosser, Andreas; Becker, Anke; Valverde, Claudio

2012-01-01

Riboregulation stands for RNA-based control of gene expression. In bacteria, small non-coding RNAs (sRNAs) are a major class of riboregulatory elements, most of which act at the post-transcriptional level by base-pairing target mRNA genes. The RNA chaperone Hfq facilitates antisense interactions between target mRNAs and regulatory sRNAs, thus influencing mRNA stability and/or translation rate. In the α-proteobacterium Sinorhizobium meliloti strain 2011, the identification and detection of multiple sRNAs genes and the broadly pleitropic phenotype associated to the absence of a functional Hfq protein both support the existence of riboregulatory circuits controlling gene expression to ensure the fitness of this bacterium in both free living and symbiotic conditions. In order to identify target mRNAs subject to Hfq-dependent riboregulation, we have compared the proteome of an hfq mutant and the wild type S. meliloti by quantitative proteomics following protein labelling with (15)N. Among 2139 univocally identified proteins, a total of 195 proteins showed a differential abundance between the Hfq mutant and the wild type strain; 65 proteins accumulated ≥2-fold whereas 130 were downregulated (≤0.5-fold) in the absence of Hfq. This profound proteomic impact implies a major role for Hfq on regulation of diverse physiological processes in S. meliloti, from transport of small molecules to homeostasis of iron and nitrogen. Changes in the cellular levels of proteins involved in transport of nucleotides, peptides and amino acids, and in iron homeostasis, were confirmed with phenotypic assays. These results represent the first quantitative proteomic analysis in S. meliloti. The comparative analysis of the hfq mutant proteome allowed identification of novel strongly Hfq-regulated genes in S. meliloti.

A Computational Model of Liver Iron Metabolism

PubMed Central

Mitchell, Simon; Mendes, Pedro

2013-01-01

Iron is essential for all known life due to its redox properties; however, these same properties can also lead to its toxicity in overload through the production of reactive oxygen species. Robust systemic and cellular control are required to maintain safe levels of iron, and the liver seems to be where this regulation is mainly located. Iron misregulation is implicated in many diseases, and as our understanding of iron metabolism improves, the list of iron-related disorders grows. Recent developments have resulted in greater knowledge of the fate of iron in the body and have led to a detailed map of its metabolism; however, a quantitative understanding at the systems level of how its components interact to produce tight regulation remains elusive. A mechanistic computational model of human liver iron metabolism, which includes the core regulatory components, is presented here. It was constructed based on known mechanisms of regulation and on their kinetic properties, obtained from several publications. The model was then quantitatively validated by comparing its results with previously published physiological data, and it is able to reproduce multiple experimental findings. A time course simulation following an oral dose of iron was compared to a clinical time course study and the simulation was found to recreate the dynamics and time scale of the systems response to iron challenge. A disease state simulation of haemochromatosis was created by altering a single reaction parameter that mimics a human haemochromatosis gene (HFE) mutation. The simulation provides a quantitative understanding of the liver iron overload that arises in this disease. This model supports and supplements understanding of the role of the liver as an iron sensor and provides a framework for further modelling, including simulations to identify valuable drug targets and design of experiments to improve further our knowledge of this system. PMID:24244122

On complexity and homogeneity measures in predicting biological aggressiveness of prostate cancer; Implication of the cellular automata model of tumor growth.

PubMed

Tanase, Mihai; Waliszewski, Przemyslaw

2015-12-01

We propose a novel approach for the quantitative evaluation of aggressiveness in prostate carcinomas. The spatial distribution of cancer cell nuclei was characterized by the global spatial fractal dimensions D0, D1, and D2. Two hundred eighteen prostate carcinomas were stratified into the classes of equivalence using results of ROC analysis. A simulation of the cellular automata mix defined a theoretical frame for a specific geometric representation of the cell nuclei distribution called a local structure correlation diagram (LSCD). The LSCD and dispersion Hd were computed for each carcinoma. Data mining generated some quantitative criteria describing tumor aggressiveness. Alterations in tumor architecture along progression were associated with some changes in both shape and the quantitative characteristics of the LSCD consistent with those in the automata mix model. Low-grade prostate carcinomas with low complexity and very low biological aggressiveness are defined by the condition D0 < 1.545 and Hd < 38. High-grade carcinomas with high complexity and very high biological aggressiveness are defined by the condition D0 > 1.764 and Hd < 38. The novel homogeneity measure Hd identifies carcinomas with very low aggressiveness within the class of complexity C1 or carcinomas with very high aggressiveness in the class C7. © 2015 Wiley Periodicals, Inc.

Making quantitative morphological variation from basic developmental processes: where are we? The case of the Drosophila wing

PubMed Central

Alexis, Matamoro-Vidal; Isaac, Salazar-Ciudad; David, Houle

2015-01-01

One of the aims of evolutionary developmental biology is to discover the developmental origins of morphological variation. The discipline has mainly focused on qualitative morphological differences (e.g., presence or absence of a structure) between species. Studies addressing subtle, quantitative variation are less common. The Drosophila wing is a model for the study of development and evolution, making it suitable to investigate the developmental mechanisms underlying the subtle quantitative morphological variation observed in nature. Previous reviews have focused on the processes involved in wing differentiation, patterning and growth. Here, we investigate what is known about how the wing achieves its final shape, and what variation in development is capable of generating the variation in wing shape observed in nature. Three major developmental stages need to be considered: larval development, pupariation, and pupal development. The major cellular processes involved in the determination of tissue size and shape are cell proliferation, cell death, oriented cell division and oriented cell intercalation. We review how variation in temporal and spatial distribution of growth and transcription factors affects these cellular mechanisms, which in turn affects wing shape. We then discuss which aspects of the wing morphological variation are predictable on the basis of these mechanisms. PMID:25619644

A Novel Pulse-Chase SILAC Strategy Measures Changes in Protein Decay and Synthesis Rates Induced by Perturbation of Proteostasis with an Hsp90 Inhibitor

PubMed Central

Fierro-Monti, Ivo; Racle, Julien; Hernandez, Celine; Waridel, Patrice; Hatzimanikatis, Vassily; Quadroni, Manfredo

2013-01-01

Standard proteomics methods allow the relative quantitation of levels of thousands of proteins in two or more samples. While such methods are invaluable for defining the variations in protein concentrations which follow the perturbation of a biological system, they do not offer information on the mechanisms underlying such changes. Expanding on previous work [1], we developed a pulse-chase (pc) variant of SILAC (stable isotope labeling by amino acids in cell culture). pcSILAC can quantitate in one experiment and for two conditions the relative levels of proteins newly synthesized in a given time as well as the relative levels of remaining preexisting proteins. We validated the method studying the drug-mediated inhibition of the Hsp90 molecular chaperone, which is known to lead to increased synthesis of stress response proteins as well as the increased decay of Hsp90 “clients”. We showed that pcSILAC can give information on changes in global cellular proteostasis induced by treatment with the inhibitor, which are normally not captured by standard relative quantitation techniques. Furthermore, we have developed a mathematical model and computational framework that uses pcSILAC data to determine degradation constants kd and synthesis rates Vs for proteins in both control and drug-treated cells. The results show that Hsp90 inhibition induced a generalized slowdown of protein synthesis and an increase in protein decay. Treatment with the inhibitor also resulted in widespread protein-specific changes in relative synthesis rates, together with variations in protein decay rates. The latter were more restricted to individual proteins or protein families than the variations in synthesis. Our results establish pcSILAC as a viable workflow for the mechanistic dissection of changes in the proteome which follow perturbations. Data are available via ProteomeXchange with identifier PXD000538. PMID:24312217

Nature and time course of acclimation to aluminum in juvenile brook trout (Salvelinus fontinalis): II. Gill histology

USGS Publications Warehouse

Mueller, M.E.; Sanchez, D.A.; Bergman, H.L.; McDonald, D.G.; Rhem, R.G.; Wood, C.M.

1991-01-01

Gill samples from juvenile brook trout (Salvelinus fontinalis) acclimated to low-level aluminum at pH 5.2 showed severe damage by day 4, with necrosis and fusion of secondary lamellae and hyperplasia and hypertrophy of mucous cells. Over the following 20 d, there was a continual process of repair with proliferation and hypertrophy of mucous cells. Qualitative analysis of gill samples plus physiology and mortality data collected in a companion study indicated progressive development (by day 10 onward) of increasing acclimation to Al. Quantitative analysis of gill samples on day 13 showed that mucous cell volume density had tripled and mucous cell area had doubled in Al-exposed fish compared with control fish. A lamellar fusion index showed evidence of fusion in Al-exposed fish by day 4 with recovery to nearly control levels by day 13. Physiological disturbances appear to be directly related to the histological changes observed in the gill epithelium. At the cellular level, changes in either mucous cell production and secretion or changes in mucus chemistry contribute, in part, to acclimation to Al.

Apical-to-basolateral transepithelial transport of cow's milk caseins by intestinal Caco-2 cell monolayers: MS-based quantitation of cellularly degraded α- and β-casein fragments.

PubMed

Sakurai, Nao; Nishio, Shunsuke; Akiyama, Yuka; Miyata, Shinji; Oshima, Kenzi; Nadano, Daita; Matsuda, Tsukasa

2018-02-27

Casein is the major milk protein to nourish infants but, in certain population, it causes cow's milk allergy, indicating the uptake of antigenic casein and their peptides through the intestinal epithelium. Using human intestinal Caco-2 cell monolayers, the apical-to-basal transepithelial transport of casein was investigated. Confocal microscopy using component-specific antibodies showed that αs1-casein antigens became detectable as punctate signals at the apical-side cytoplasm and reached to the cytoplasm at a tight-junction level within a few hours. Such intracellular casein signals were more remarkable than those of the other antigens, β-lactoglobulin and ovalbumin, colocalized in part with an early endosome marker protein, EEA1, and decreased in the presence of cytochalasin D or sodium azide and also at lowered temperature at 4 °C. LC-MS analysis of the protein fraction in the basal-side medium identified the αs1-casein fragment including the N-terminal region and the αs2-casein fragment containing the central part of polypeptide at 100∼1000 fmol per well levels. Moreover, β-casein C-terminal overlapping peptides were identified in the peptide fraction below 10 kDa of the basal medium. These results suggest that caseins are partially degraded by cellular proteases and/or peptidases and immunologically active casein fragments are transported to basal side of the cell monolayers.

The Reverse Transcription Inhibitor Abacavir Shows Anticancer Activity in Prostate Cancer Cell Lines

PubMed Central

Molinari, Agnese; Parisi, Chiara; Bozzuto, Giuseppina; Toccacieli, Laura; Formisano, Giuseppe; De Orsi, Daniela; Paradisi, Silvia; Grober, OlÌ Maria Victoria; Ravo, Maria; Weisz, Alessandro; Arcieri, Romano; Vella, Stefano; Gaudi, Simona

2010-01-01

Background Transposable Elements (TEs) comprise nearly 45% of the entire genome and are part of sophisticated regulatory network systems that control developmental processes in normal and pathological conditions. The retroviral/retrotransposon gene machinery consists mainly of Long Interspersed Nuclear Elements (LINEs-1) and Human Endogenous Retroviruses (HERVs) that code for their own endogenous reverse transcriptase (RT). Interestingly, RT is typically expressed at high levels in cancer cells. Recent studies report that RT inhibition by non-nucleoside reverse transcriptase inhibitors (NNRTIs) induces growth arrest and cell differentiation in vitro and antagonizes growth of human tumors in animal model. In the present study we analyze the anticancer activity of Abacavir (ABC), a nucleoside reverse transcription inhibitor (NRTI), on PC3 and LNCaP prostate cancer cell lines. Principal Findings ABC significantly reduces cell growth, migration and invasion processes, considerably slows S phase progression, induces senescence and cell death in prostate cancer cells. Consistent with these observations, microarray analysis on PC3 cells shows that ABC induces specific and dose-dependent changes in gene expression, involving multiple cellular pathways. Notably, by quantitative Real-Time PCR we found that LINE-1 ORF1 and ORF2 mRNA levels were significantly up-regulated by ABC treatment. Conclusions Our results demonstrate the potential of ABC as anticancer agent able to induce antiproliferative activity and trigger senescence in prostate cancer cells. Noteworthy, we show that ABC elicits up-regulation of LINE-1 expression, suggesting the involvement of these elements in the observed cellular modifications. PMID:21151977

Current employment status, occupational category, occupational hazard exposure and job stress in relation to telomere length: the Multiethnic Study of Atherosclerosis (MESA).

PubMed

Fujishiro, Kaori; Diez-Roux, Ana V; Landsbergis, Paul A; Jenny, Nancy Swords; Seeman, Teresa

2013-08-01

Telomere length has been proposed as a biomarker of cell senescence, which is associated with a wide array of adverse health outcomes. While work is a major determinant of health, few studies have investigated the association of telomere length with various dimensions of occupation. Accelerated cellular aging could be a common pathway linking occupational exposure to several health outcomes. Leukocyte telomere length was assessed using quantitative PCR in a community-based sample of 981 individuals (age: 45-84 years). Questionnaires were used to collect information on current employment status, current or main occupation before retirement and job strain. The Occupational Resource Network (O*NET) database was linked to the questionnaire data to create five exposure measures: physical activity on the job, physical hazard exposure, interpersonal stressors, job control and job demands. Linear regression was used to estimate associations of occupational characteristics with telomere lengths after adjustment for age, sex, race, socioeconomic position and several behavioural risk factors. There were no mean differences in telomere lengths across current employment status, occupational category, job strain categories or levels of most O*NET exposure measures. There was also no evidence that being in lower status occupational categories or being exposed to higher levels of adverse physical or psychosocial exposures accelerated the association between age and telomere shortening. Cellular aging as reflected by shorter telomeres does not appear to be an important pathway linking occupation to various health outcomes.

Spatiotemporal dynamics of oscillatory cellular patterns in three-dimensional directional solidification.

PubMed

Bergeon, N; Tourret, D; Chen, L; Debierre, J-M; Guérin, R; Ramirez, A; Billia, B; Karma, A; Trivedi, R

2013-05-31

We report results of directional solidification experiments conducted on board the International Space Station and quantitative phase-field modeling of those experiments. The experiments image for the first time in situ the spatially extended dynamics of three-dimensional cellular array patterns formed under microgravity conditions where fluid flow is suppressed. Experiments and phase-field simulations reveal the existence of oscillatory breathing modes with time periods of several 10's of minutes. Oscillating cells are usually noncoherent due to array disorder, with the exception of small areas where the array structure is regular and stable.

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

High-Throughput Method for Automated Colony and Cell Counting by Digital Image Analysis Based on Edge Detection

PubMed Central

Choudhry, Priya

2016-01-01

Counting cells and colonies is an integral part of high-throughput screens and quantitative cellular assays. Due to its subjective and time-intensive nature, manual counting has hindered the adoption of cellular assays such as tumor spheroid formation in high-throughput screens. The objective of this study was to develop an automated method for quick and reliable counting of cells and colonies from digital images. For this purpose, I developed an ImageJ macro Cell Colony Edge and a CellProfiler Pipeline Cell Colony Counting, and compared them to other open-source digital methods and manual counts. The ImageJ macro Cell Colony Edge is valuable in counting cells and colonies, and measuring their area, volume, morphology, and intensity. In this study, I demonstrate that Cell Colony Edge is superior to other open-source methods, in speed, accuracy and applicability to diverse cellular assays. It can fulfill the need to automate colony/cell counting in high-throughput screens, colony forming assays, and cellular assays. PMID:26848849

Nano/microvehicles for efficient delivery and (bio)sensing at the cellular level

PubMed Central

Esteban-Fernández de Ávila, B.; Yáñez-Sedeño, P.

2017-01-01

A perspective review of recent strategies involving the use of nano/microvehicles to address the key challenges associated with delivery and (bio)sensing at the cellular level is presented. The main types and characteristics of the different nano/microvehicles used for these cellular applications are discussed, including fabrication pathways, propulsion (catalytic, magnetic, acoustic or biological) and navigation strategies, and relevant parameters affecting their propulsion performance and sensing and delivery capabilities. Thereafter, selected applications are critically discussed. An emphasis is made on enhancing the extra- and intra-cellular biosensing capabilities, fast cell internalization, rapid inter- or intra-cellular movement, efficient payload delivery and targeted on-demand controlled release in order to greatly improve the monitoring and modulation of cellular processes. A critical discussion of selected breakthrough applications illustrates how these smart multifunctional nano/microdevices operate as nano/microcarriers and sensors at the intra- and extra-cellular levels. These advances allow both the real-time biosensing of relevant targets and processes even at a single cell level, and the delivery of different cargoes (drugs, functional proteins, oligonucleotides and cells) for therapeutics, gene silencing/transfection and assisted fertilization, while overcoming challenges faced by current affinity biosensors and delivery vehicles. Key challenges for the future and the envisioned opportunities and future perspectives of this remarkably exciting field are discussed. PMID:29147499

Oscillatory cellular patterns in three-dimensional directional solidification

NASA Astrophysics Data System (ADS)

Tourret, D.; Debierre, J.-M.; Song, Y.; Mota, F. L.; Bergeon, N.; Guérin, R.; Trivedi, R.; Billia, B.; Karma, A.

2015-10-01

We present a phase-field study of oscillatory breathing modes observed during the solidification of three-dimensional cellular arrays in microgravity. Directional solidification experiments conducted onboard the International Space Station have allowed us to observe spatially extended homogeneous arrays of cells and dendrites while minimizing the amount of gravity-induced convection in the liquid. In situ observations of transparent alloys have revealed the existence, over a narrow range of control parameters, of oscillations in cellular arrays with a period ranging from about 25 to 125 min. Cellular patterns are spatially disordered, and the oscillations of individual cells are spatiotemporally uncorrelated at long distance. However, in regions displaying short-range spatial ordering, groups of cells can synchronize into oscillatory breathing modes. Quantitative phase-field simulations show that the oscillatory behavior of cells in this regime is linked to a stability limit of the spacing in hexagonal cellular array structures. For relatively high cellular front undercooling (i.e., low growth velocity or high thermal gradient), a gap appears in the otherwise continuous range of stable array spacings. Close to this gap, a sustained oscillatory regime appears with a period that compares quantitatively well with experiment. For control parameters where this gap exists, oscillations typically occur for spacings at the edge of the gap. However, after a change of growth conditions, oscillations can also occur for nearby values of control parameters where this gap just closes and a continuous range of spacings exists. In addition, sustained oscillations at to the opening of this stable gap exhibit a slow periodic modulation of the phase-shift among cells with a slower period of several hours. While long-range coherence of breathing modes can be achieved in simulations for a perfect spatial arrangement of cells as initial condition, global disorder is observed in both three-dimensional experiments and simulations from realistic noisy initial conditions. In the latter case, erratic tip-splitting events promoted by large-amplitude oscillations contribute to maintaining the long-range array disorder, unlike in thin-sample experiments where long-range coherence of oscillations is experimentally observable.

A Rapid Method for Quantifying Viable Mycobacterium avium subsp. paratuberculosis in Cellular Infection Assays

PubMed Central

Pooley, Hannah B.; de Silva, Kumudika; Purdie, Auriol C.; Begg, Douglas J.; Whittington, Richard J.

2016-01-01

ABSTRACT Determining the viability of bacteria is a key outcome of in vitro cellular infection assays. Currently, this is done by culture, which is problematic for fastidious slow-growing bacteria such as Mycobacterium avium subsp. paratuberculosis, where it can take up to 4 months to confirm growth. This study aimed to identify an assay that can rapidly quantify the number of viable M. avium subsp. paratuberculosis cells in a cellular sample. Three commercially available bacterial viability assays along with a modified liquid culture method coupled with high-throughput quantitative PCR growth detection were assessed. Criteria for assessment included the ability of each assay to differentiate live and dead M. avium subsp. paratuberculosis organisms and their accuracy at low bacterial concentrations. Using the culture-based method, M. avium subsp. paratuberculosis growth was reliably detected and quantified within 2 weeks. There was a strong linear association between the 2-week growth rate and the initial inoculum concentration. The number of viable M. avium subsp. paratuberculosis cells in an unknown sample was quantified based on the growth rate, by using growth standards. In contrast, none of the commercially available viability assays were suitable for use with samples from in vitro cellular infection assays. IMPORTANCE Rapid quantification of the viability of Mycobacterium avium subsp. paratuberculosis in samples from in vitro cellular infection assays is important, as it allows these assays to be carried out on a large scale. In vitro cellular infection assays can function as a preliminary screening tool, for vaccine development or antimicrobial screening, and also to extend findings derived from experimental animal trials. Currently, by using culture, it takes up to 4 months to obtain quantifiable results regarding M. avium subsp. paratuberculosis viability after an in vitro infection assay; however, with the quantitative PCR and liquid culture method developed, reliable results can be obtained at 2 weeks. This method will be important for vaccine and antimicrobial screening work, as it will allow a greater number of candidates to be screened in the same amount of time, which will increase the likelihood that a favorable candidate will be found to be subjected to further testing. PMID:27371585

Oscillatory cellular patterns in three-dimensional directional solidification

DOE PAGES

Tourret, D.; Debierre, J. -M.; Song, Y.; ...

2015-09-11

We present a phase-field study of oscillatory breathing modes observed during the solidification of three-dimensional cellular arrays in micro-gravity. Directional solidification experiments conducted onboard the International Space Station have allowed for the first time to observe spatially extended homogeneous arrays of cells and dendrites while minimizing the amount of gravity-induced convection in the liquid. In situ observations of transparent alloys have revealed the existence, over a narrow range of control parameters, of oscillations in cellular arrays with a period ranging from about 25 to 125 minutes. Cellular patterns are spatially disordered, and the oscillations of individual cells are spatiotemporally uncorrelatedmore » at long distance. However, in regions displaying short-range spatial ordering, groups of cells can synchronize into oscillatory breathing modes. Quantitative phase-field simulations show that the oscillatory behavior of cells in this regime is linked to a stability limit of the spacing in hexagonal cellular array structures. For relatively high cellular front undercooling (\\ie low growth velocity or high thermal gradient), a gap appears in the otherwise continuous range of stable array spacings. Close to this gap, a sustained oscillatory regime appears with a period that compares quantitatively well with experiment. For control parameters where this gap exist, oscillations typically occur for spacings at the edge of the gap. However, after a change of growth conditions, oscillations can also occur for nearby values of control parameters where this gap just closes and a continuous range of spacings exists. In addition, sustained oscillations at to the opening of this stable gap exhibit a slow periodic modulation of the phase-shift among cells with a slower period of several hours. While long-range coherence of breathing modes can be achieved in simulations for a perfect spatial arrangement of cells as initial condition, global disorder is observed in both three-dimensional experiments and simulations from realistic noisy initial conditions. The, erratic tip splitting events promoted by large amplitude oscillations contribute to maintaining the long-range array disorder, unlike in thin sample experiments where long-range coherence of oscillations is experimentally observable.« less

Oscillatory cellular patterns in three-dimensional directional solidification

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

Tourret, D.; Debierre, J. -M.; Song, Y.

We present a phase-field study of oscillatory breathing modes observed during the solidification of three-dimensional cellular arrays in micro-gravity. Directional solidification experiments conducted onboard the International Space Station have allowed for the first time to observe spatially extended homogeneous arrays of cells and dendrites while minimizing the amount of gravity-induced convection in the liquid. In situ observations of transparent alloys have revealed the existence, over a narrow range of control parameters, of oscillations in cellular arrays with a period ranging from about 25 to 125 minutes. Cellular patterns are spatially disordered, and the oscillations of individual cells are spatiotemporally uncorrelatedmore » at long distance. However, in regions displaying short-range spatial ordering, groups of cells can synchronize into oscillatory breathing modes. Quantitative phase-field simulations show that the oscillatory behavior of cells in this regime is linked to a stability limit of the spacing in hexagonal cellular array structures. For relatively high cellular front undercooling (\\ie low growth velocity or high thermal gradient), a gap appears in the otherwise continuous range of stable array spacings. Close to this gap, a sustained oscillatory regime appears with a period that compares quantitatively well with experiment. For control parameters where this gap exist, oscillations typically occur for spacings at the edge of the gap. However, after a change of growth conditions, oscillations can also occur for nearby values of control parameters where this gap just closes and a continuous range of spacings exists. In addition, sustained oscillations at to the opening of this stable gap exhibit a slow periodic modulation of the phase-shift among cells with a slower period of several hours. While long-range coherence of breathing modes can be achieved in simulations for a perfect spatial arrangement of cells as initial condition, global disorder is observed in both three-dimensional experiments and simulations from realistic noisy initial conditions. The, erratic tip splitting events promoted by large amplitude oscillations contribute to maintaining the long-range array disorder, unlike in thin sample experiments where long-range coherence of oscillations is experimentally observable.« less

Clearance of a persistent Picornavirus infection is associated with enhanced pro-apoptotic and cellular immune responses

USDA-ARS?s Scientific Manuscript database

Immunomodulatory mechanisms associated with clearance versus persistence of foot-and-mouth disease virus (FMDV) in distinct microanatomic compartments of the bovine nasopharynx were investigated using quantitative RT-PCR and whole transcriptome microarray. Analysis of tissue samples obtained during ...

Creating Context for the Use of DNA Adduct Data in Risk Assessment

EPA Science Inventory

Assessments of human cancer risk require the integration of diverse types of data. Advancing technologies for quantitative measurements at the sub-cellular domain raise the critical issue of interpretation and use of DNA adduct data in context with current understanding of cancer...

Global Proteomics Analysis of the Response to Starvation in C. elegans*

PubMed Central

Larance, Mark; Pourkarimi, Ehsan; Wang, Bin; Brenes Murillo, Alejandro; Kent, Robert; Lamond, Angus I.; Gartner, Anton

2015-01-01

Periodic starvation of animals induces large shifts in metabolism but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass-spectrometry-based quantitative proteomics to identify global changes in the Caenorhabditis elegans proteome due to acute starvation of young adult animals. Measuring changes in the abundance of over 5,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin-associated proteins, including specific linker histones, histone variants, and histone posttranslational modifications associated with the epigenetic control of gene expression. To maximize community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/). PMID:25963834