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

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.

Body composition analysis: Cellular level modeling of body component ratios.

PubMed

Wang, Z; Heymsfield, S B; Pi-Sunyer, F X; Gallagher, D; Pierson, R N

2008-01-01

During the past two decades, a major outgrowth of efforts by our research group at St. Luke's-Roosevelt Hospital is the development of body composition models that include cellular level models, models based on body component ratios, total body potassium models, multi-component models, and resting energy expenditure-body composition models. This review summarizes these models with emphasis on component ratios that we believe are fundamental to understanding human body composition during growth and development and in response to disease and treatments. In-vivo measurements reveal that in healthy adults some component ratios show minimal variability and are relatively 'stable', for example total body water/fat-free mass and fat-free mass density. These ratios can be effectively applied for developing body composition methods. In contrast, other ratios, such as total body potassium/fat-free mass, are highly variable in vivo and therefore are less useful for developing body composition models. In order to understand the mechanisms governing the variability of these component ratios, we have developed eight cellular level ratio models and from them we derived simplified models that share as a major determining factor the ratio of extracellular to intracellular water ratio (E/I). The E/I value varies widely among adults. Model analysis reveals that the magnitude and variability of each body component ratio can be predicted by correlating the cellular level model with the E/I value. Our approach thus provides new insights into and improved understanding of body composition ratios in adults.

A SIMPLE CELLULAR AUTOMATON MODEL FOR HIGH-LEVEL VEGETATION DYNAMICS

EPA Science Inventory

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

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

PubMed Central

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

2014-01-01

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

Implementing online quantitative support modules in an intermediate-level course

NASA Astrophysics Data System (ADS)

Daly, J.

2011-12-01

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

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.

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.

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

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.

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

Understanding Cellular Respiration in Terms of Matter & Energy within Ecosystems

ERIC Educational Resources Information Center

White, Joshua S.; Maskiewicz, April C.

2014-01-01

Using a design-based research approach, we developed a data-rich problem (DRP) set to improve student understanding of cellular respiration at the ecosystem level. The problem tasks engage students in data analysis to develop biological explanations. Several of the tasks and their implementation are described. Quantitative results suggest that…

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.

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

Modulation of PICALM Levels Perturbs Cellular Cholesterol Homeostasis

PubMed Central

Mercer, Jacob L.; Argus, Joseph P.; Crabtree, Donna M.; Keenan, Melissa M.; Wilks, Moses Q.; Chi, Jen-Tsan Ashley; Bensinger, Steven J.

2015-01-01

PICALM (Phosphatidyl Inositol Clathrin Assembly Lymphoid Myeloid protein) is a ubiquitously expressed protein that plays a role in clathrin-mediated endocytosis. PICALM also affects the internalization and trafficking of SNAREs and modulates macroautophagy. Chromosomal translocations that result in the fusion of PICALM to heterologous proteins cause leukemias, and genome-wide association studies have linked PICALM Single Nucleotide Polymorphisms (SNPs) to Alzheimer’s disease. To obtain insight into the biological role of PICALM, we performed gene expression studies of PICALM-deficient and PICALM-expressing cells. Pathway analysis demonstrated that PICALM expression influences the expression of genes that encode proteins involved in cholesterol biosynthesis and lipoprotein uptake. Gas Chromatography-Mass Spectrometry (GC-MS) studies indicated that loss of PICALM increases cellular cholesterol pool size. Isotopic labeling studies revealed that loss of PICALM alters increased net scavenging of cholesterol. Flow cytometry analyses confirmed that internalization of the LDL receptor is enhanced in PICALM-deficient cells as a result of higher levels of LDLR expression. These findings suggest that PICALM is required for cellular cholesterol homeostasis and point to a novel mechanism by which PICALM alterations may contribute to disease. PMID:26075887

Species identification of corynebacteria by cellular fatty acid analysis.

PubMed

Van den Velde, Sandra; Lagrou, Katrien; Desmet, Koen; Wauters, Georges; Verhaegen, Jan

2006-02-01

We evaluated the usefulness of cellular fatty acid analysis for the identification of corynebacteria. Therefore, 219 well-characterized strains belonging to 21 Corynebacterium species were analyzed with the Sherlock System of MIDI (Newark, DE). Most Corynebacterium species have a qualitative different fatty acid profile. Corynebacterium coyleae (subgroup 1), Corynebacterium riegelii, Corynebacterium simulans, and Corynebacterium imitans differ only quantitatively. Corynebacterium afermentans afermentans and C. coyleae (subgroup 2) have both a similar qualitative and quantitative profile. The commercially available database (CLIN 40, MIDI) identified only one third of the 219 strains correctly at the species level. We created a new database with these 219 strains. This new database was tested with 34 clinical isolates and could identify 29 strains correctly. Strains that remained unidentified were 2 Corynebacterium aurimucosum (not included in our database), 1 C. afermentans afermentans, and 2 Corynebacterium pseudodiphtheriticum. Cellular fatty acid analysis with a self-created database can be used for the identification and differentiation of corynebacteria.

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

PubMed Central

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

2011-01-01

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

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

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

PubMed

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

2013-06-04

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

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

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.

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

NASA Astrophysics Data System (ADS)

Yang, Ruiguo

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

High performance cellular level agent-based simulation with FLAME for the GPU.

PubMed

Richmond, Paul; Walker, Dawn; Coakley, Simon; Romano, Daniela

2010-05-01

Driven by the availability of experimental data and ability to simulate a biological scale which is of immediate interest, the cellular scale is fast emerging as an ideal candidate for middle-out modelling. As with 'bottom-up' simulation approaches, cellular level simulations demand a high degree of computational power, which in large-scale simulations can only be achieved through parallel computing. The flexible large-scale agent modelling environment (FLAME) is a template driven framework for agent-based modelling (ABM) on parallel architectures ideally suited to the simulation of cellular systems. It is available for both high performance computing clusters (www.flame.ac.uk) and GPU hardware (www.flamegpu.com) and uses a formal specification technique that acts as a universal modelling format. This not only creates an abstraction from the underlying hardware architectures, but avoids the steep learning curve associated with programming them. In benchmarking tests and simulations of advanced cellular systems, FLAME GPU has reported massive improvement in performance over more traditional ABM frameworks. This allows the time spent in the development and testing stages of modelling to be drastically reduced and creates the possibility of real-time visualisation for simple visual face-validation.

A Quantitative Study of Oxygen as a Metabolic Regulator

NASA Technical Reports Server (NTRS)

Radhakrishnan, Krishnan; LaManna, Joseph C.; Cabera, Marco E.

2000-01-01

An acute reduction in oxygen delivery to a tissue is associated with metabolic changes aimed at maintaining ATP homeostasis. However, given the complexity of the human bio-energetic system, it is difficult to determine quantitatively how cellular metabolic processes interact to maintain ATP homeostasis during stress (e.g., hypoxia, ischemia, and exercise). In particular, we are interested in determining mechanisms relating cellular oxygen concentration to observed metabolic responses at the cellular, tissue, organ, and whole body levels and in quantifying how changes in tissue oxygen 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 oxygen as a metabolic regulator. Specifically, the enhancement - sensitivity analysis - permits studying the effects of variations in tissue oxygenation and parameters controlling cellular respiration on glycolysis, lactate production, and pyruvate oxidation. The analysis can distinguish between parameters that must be determined accurately and those that require less precision, based on their effects on model predictions. This capability may prove to be important in optimizing experimental design, thus reducing use of animals.

Cellular chromophores and signaling in low level light therapy

NASA Astrophysics Data System (ADS)

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

2007-02-01

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

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.

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

Cellular burdens and biological effects on tissue level caused by inhaled radon progenies.

PubMed

Madas, B G; Balásházy, I; Farkas, Á; Szoke, I

2011-02-01

In the case of radon exposure, the spatial distribution of deposited radioactive particles is highly inhomogeneous in the central airways. The object of this research is to investigate the consequences of this heterogeneity regarding cellular burdens in the bronchial epithelium and to study the possible biological effects at tissue level. Applying computational fluid and particle dynamics techniques, the deposition distribution of inhaled radon daughters has been determined in a bronchial airway model for 23 min of work in the New Mexico uranium mine corresponding to 0.0129 WLM exposure. A numerical epithelium model based on experimental data has been utilised in order to quantify cellular hits and doses. Finally, a carcinogenesis model considering cell death-induced cell-cycle shortening has been applied to assess the biological responses. Present computations reveal that cellular dose may reach 1.5 Gy, which is several orders of magnitude higher than tissue dose. The results are in agreement with the histological finding that the uneven deposition distribution of radon progenies may lead to inhomogeneous spatial distribution of tumours in the bronchial airways. In addition, at the macroscopic level, the relationship between cancer risk and radiation burden seems to be non-linear.

Cellular-level surgery using nano robots.

PubMed

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

2012-12-01

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

Dynamics of cellular level function and regulation derived from murine expression array data.

PubMed

de Bivort, Benjamin; Huang, Sui; Bar-Yam, Yaneer

2004-12-21

A major open question of systems biology is how genetic and molecular components interact to create phenotypes at the cellular level. Although much recent effort has been dedicated to inferring effective regulatory influences within small networks of genes, the power of microarray bioinformatics has yet to be used to determine functional influences at the cellular level. In all cases of data-driven parameter estimation, the number of model parameters estimable from a set of data is strictly limited by the size of that set. Rather than infer parameters describing the detailed interactions of just a few genes, we chose a larger-scale investigation so that the cumulative effects of all gene interactions could be analyzed to identify the dynamics of cellular-level function. By aggregating genes into large groups with related behaviors (megamodules), we were able to determine the effective aggregate regulatory influences among 12 major gene groups in murine B lymphocytes over a variety of time steps. Intriguing observations about the behavior of cells at this high level of abstraction include: (i) a medium-term critical global transcriptional dependence on ATP-generating genes in the mitochondria, (ii) a longer-term dependence on glycolytic genes, (iii) the dual role of chromatin-reorganizing genes in transcriptional activation and repression, (iv) homeostasis-favoring influences, (v) the indication that, as a group, G protein-mediated signals are not concentration-dependent in their influence on target gene expression, and (vi) short-term-activating/long-term-repressing behavior of the cell-cycle system that reflects its oscillatory behavior.

Regulatory Nexus of Synthesis and Degradation Deciphers Cellular Nrf2 Expression Levels

PubMed Central

Suzuki, Takafumi; Shibata, Tatsuhiro; Takaya, Kai; Shiraishi, Kouya; Kohno, Takashi; Kunitoh, Hideo; Tsuta, Koji; Furuta, Koh; Goto, Koichi; Hosoda, Fumie; Sakamoto, Hiromi; Motohashi, Hozumi

2013-01-01

Transcription factor Nrf2 (NF-E2-related factor 2) is essential for oxidative and electrophilic stress responses. While it has been well characterized that Nrf2 activity is tightly regulated at the protein level through proteasomal degradation via Keap1 (Kelch-like ECH-associated protein 1)-mediated ubiquitination, not much attention has been paid to the supply side of Nrf2, especially regulation of Nrf2 gene transcription. Here we report that manipulation of Nrf2 transcription is effective in changing the final Nrf2 protein level and activity of cellular defense against oxidative stress even in the presence of Keap1 and under efficient Nrf2 degradation, determined using genetically engineered mouse models. In excellent agreement with this finding, we found that minor A/A homozygotes of a single nucleotide polymorphism (SNP) in the human NRF2 upstream promoter region (rs6721961) exhibited significantly diminished NRF2 gene expression and, consequently, an increased risk of lung cancer, especially those who had ever smoked. Our results support the notion that in addition to control over proteasomal degradation and derepression from degradation/repression, the transcriptional level of the Nrf2 gene acts as another important regulatory point to define cellular Nrf2 levels. These results thus verify the critical importance of human SNPs that influence the levels of transcription of the NRF2 gene for future personalized medicine. PMID:23572560

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

NASA Astrophysics Data System (ADS)

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

2005-07-01

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

Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels

PubMed Central

Riento, Kirsi; Zhang, Qifeng; Clark, Jonathan; Begum, Farida; Stephens, Elaine; Wakelam, Michael J.

2018-01-01

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase. Here, we show that the absence of microdomains generated by multimeric assemblies of flotillin proteins results in reduced S1P levels. Cellular phenotypes of flotillin knockout mice, including changes in histone acetylation and expression of Isg15, are recapitulated when S1P synthesis is perturbed. Flotillins bind to Sph in vitro and increase recruitment of Sph to membranes in cells. Ectopic re-localisation of flotillins within the cell causes concomitant redistribution of Sph. The data suggest that flotillins may directly or indirectly regulate cellular sphingolipid distribution and signalling. PMID:29787576

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

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

PubMed Central

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

2005-01-01

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

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

ERIC Educational Resources Information Center

McLauglin, Jacqueline S.; Seaquist, Stephen B.

2008-01-01

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

Computational modelling of cellular level metabolism

NASA Astrophysics Data System (ADS)

Calvetti, D.; Heino, J.; Somersalo, E.

2008-07-01

The steady and stationary state inverse problems consist of estimating the reaction and transport fluxes, blood concentrations and possibly the rates of change of some of the concentrations based on data which are often scarce noisy and sampled over a population. The Bayesian framework provides a natural setting for the solution of this inverse problem, because a priori knowledge about the system itself and the unknown reaction fluxes and transport rates can compensate for the insufficiency of measured data, provided that the computational costs do not become prohibitive. This article identifies the computational challenges which have to be met when analyzing the steady and stationary states of multicompartment model for cellular metabolism and suggest stable and efficient ways to handle the computations. The outline of a computational tool based on the Bayesian paradigm for the simulation and analysis of complex cellular metabolic systems is also presented.

A Conserved Developmental Patterning Network Produces Quantitatively Different Output in Multiple Species of Drosophila

PubMed Central

Meyer, Miriah; Wunderlich, Zeba; Simirenko, Lisa; Luengo Hendriks, Cris L.; Keränen, Soile V. E.; Henriquez, Clara; Knowles, David W.; Biggin, Mark D.; Eisen, Michael B.; DePace, Angela H.

2011-01-01

Differences in the level, timing, or location of gene expression can contribute to alternative phenotypes at the molecular and organismal level. Understanding the origins of expression differences is complicated by the fact that organismal morphology and gene regulatory networks could potentially vary even between closely related species. To assess the scope of such changes, we used high-resolution imaging methods to measure mRNA expression in blastoderm embryos of Drosophila yakuba and Drosophila pseudoobscura and assembled these data into cellular resolution atlases, where expression levels for 13 genes in the segmentation network are averaged into species-specific, cellular resolution morphological frameworks. We demonstrate that the blastoderm embryos of these species differ in their morphology in terms of size, shape, and number of nuclei. We present an approach to compare cellular gene expression patterns between species, while accounting for varying embryo morphology, and apply it to our data and an equivalent dataset for Drosophila melanogaster. Our analysis reveals that all individual genes differ quantitatively in their spatio-temporal expression patterns between these species, primarily in terms of their relative position and dynamics. Despite many small quantitative differences, cellular gene expression profiles for the whole set of genes examined are largely similar. This suggests that cell types at this stage of development are conserved, though they can differ in their relative position by up to 3–4 cell widths and in their relative proportion between species by as much as 5-fold. Quantitative differences in the dynamics and relative level of a subset of genes between corresponding cell types may reflect altered regulatory functions between species. Our results emphasize that transcriptional networks can diverge over short evolutionary timescales and that even small changes can lead to distinct output in terms of the placement and number of

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

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.

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

ERIC Educational Resources Information Center

Steele, Janeé M.; Rawls, Glinda J.

2015-01-01

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

Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy.

PubMed

Giss, Dominic; Kemmerling, Simon; Dandey, Venkata; Stahlberg, Henning; Braun, Thomas

2014-05-20

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.

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

PubMed

Bordonaro, Michael; Ogryzko, Vasily

2013-04-01

Quantum biology is emerging as a new field at the intersection between fundamental physics and biology, promising novel insights into the nature and origin of biological order. We discuss several elements of QBCL (quantum biology at cellular level) - a research program designed to extend the reach of quantum concepts to higher than molecular levels of biological organization. We propose a new general way to address the issue of environmentally induced decoherence and macroscopic superpositions in biological systems, emphasizing the 'basis-dependent' nature of these concepts. We introduce the notion of 'formal superposition' and distinguish it from that of Schroedinger's cat (i.e., a superposition of macroscopically distinct states). Whereas the latter notion presents a genuine foundational problem, the former one contradicts neither common sense nor observation, and may be used to describe cellular 'decision-making' and adaptation. We stress that the interpretation of the notion of 'formal superposition' should involve non-classical correlations between molecular events in a cell. Further, we describe how better understanding of the physics of Life can shed new light on the mechanism driving evolutionary adaptation (viz., 'Basis-Dependent Selection', BDS). Experimental tests of BDS and the potential role of synthetic biology in closing the 'evolvability mechanism' loophole are also discussed. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging

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

Burkhow, Sadie J.; Stephens, Nicole M.; Mei, Yu

Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level. Here, we report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase ( pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of themore » whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal.« less

Characterizing virus-induced gene silencing at the cellular level with in situ multimodal imaging

DOE PAGES

Burkhow, Sadie J.; Stephens, Nicole M.; Mei, Yu; ...

2018-05-25

Reverse genetic strategies, such as virus-induced gene silencing, are powerful techniques to study gene function. Currently, there are few tools to study the spatial dependence of the consequences of gene silencing at the cellular level. Here, we report the use of multimodal Raman and mass spectrometry imaging to study the cellular-level biochemical changes that occur from silencing the phytoene desaturase ( pds) gene using a Foxtail mosaic virus (FoMV) vector in maize leaves. The multimodal imaging method allows the localized carotenoid distribution to be measured and reveals differences lost in the spatial average when analyzing a carotenoid extraction of themore » whole leaf. The nature of the Raman and mass spectrometry signals are complementary: silencing pds reduces the downstream carotenoid Raman signal and increases the phytoene mass spectrometry signal.« less

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.

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.

Comparative studies of cellular viability levels on 2D and 3D in vitro culture matrices.

PubMed

Gargotti, M; Lopez-Gonzalez, U; Byrne, H J; Casey, A

2018-02-01

In this study, the cellular viability and function of immortalized human cervical and dermal cells are monitored and compared in conventional 2D and two commercial 3D membranes, Collagen and Geltrex, of varying working concentration and volume. Viability was monitored with the aid of the Alamar Blue assay, cellular morphology was monitored with confocal microscopy, and cell cycle studies and cell death mechanism studies were performed with flow cytometry. The viability studies showed apparent differences between the 2D and 3D culture systems, the differences attributed in part to the physical transition from 2D to 3D environment causing alterations to effective resazurin concentration, uptake and conversion rates, which was dependent on exposure time, but also due to the effect of the membrane itself on cellular function. These effects were verified by flow cytometry, in which no significant differences in viable cell numbers between 2D and 3D systems were observed after 24 h culture. The results showed the observed effect was different after shorter exposure periods, was also dependent on working concentration of the 3D system and could be mediated by altering the culture vessel size. Cell cycle analysis revealed cellular function could be altered by growth on the 3D substrates and the alterations were noted to be dependent on 3D membrane concentration. The use of 3D culture matrices has been widely interpreted to result in "improved viability levels" or "reduced" toxicity or cellular "resistance" compared to cells cultured on traditional 2D systems. The results of this study show that cellular health and viability levels are not altered by culture in 3D environments, but their normal cycle can be altered as indicated in the cell cycle studies performed and such variations must be accounted for in studies employing 3D membranes for in vitro cellular screening.

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

PubMed

Simon, Kornél; Wittmann, István

2017-03-01

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.

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 influence of risk factors on blood glucose level.

PubMed

Chen, Songjing; Luo, Senlin; Pan, Limin; Zhang, Tiemei; Han, Longfei; Zhao, Haixiu

2014-01-01

The aim of this study is to quantitatively analyze the influence of risk factors on the blood glucose level, and to provide theory basis for understanding the characteristics of blood glucose change and confirming the intervention index for type 2 diabetes. The quantitative method is proposed to analyze the influence of risk factors on blood glucose using back propagation (BP) neural network. Ten risk factors are screened first. Then the cohort is divided into nine groups by gender and age. According to the minimum error principle, nine BP models are trained respectively. The quantitative values of the influence of different risk factors on the blood glucose change can be obtained by sensitivity calculation. The experiment results indicate that weight is the leading cause of blood glucose change (0.2449). The second factors are cholesterol, age and triglyceride. The total ratio of these four factors reaches to 77% of the nine screened risk factors. And the sensitivity sequences can provide judgment method for individual intervention. This method can be applied to risk factors quantitative analysis of other diseases and potentially used for clinical practitioners to identify high risk populations for type 2 diabetes as well as other disease.

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

PubMed

Lee, Irene; Berdis, Anthony J

2016-01-01

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

Toward a systems-level view of dynamic phosphorylation networks

PubMed Central

Newman, Robert H.; Zhang, Jin; Zhu, Heng

2014-01-01

To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks. PMID:25177341

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...

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.

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.

Quantitative phase measurement for wafer-level optics

NASA Astrophysics Data System (ADS)

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

2015-07-01

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

Heterogeneity of Metazoan Cells and Beyond: To Integrative Analysis of Cellular Populations at Single-Cell Level.

PubMed

Barteneva, Natasha S; Vorobjev, Ivan A

2018-01-01

In this paper, we review some of the recent advances in cellular heterogeneity and single-cell analysis methods. In modern research of cellular heterogeneity, there are four major approaches: analysis of pooled samples, single-cell analysis, high-throughput single-cell analysis, and lately integrated analysis of cellular population at a single-cell level. Recently developed high-throughput single-cell genetic analysis methods such as RNA-Seq require purification step and destruction of an analyzed cell often are providing a snapshot of the investigated cell without spatiotemporal context. Correlative analysis of multiparameter morphological, functional, and molecular information is important for differentiation of more uniform groups in the spectrum of different cell types. Simplified distributions (histograms and 2D plots) can underrepresent biologically significant subpopulations. Future directions may include the development of nondestructive methods for dissecting molecular events in intact cells, simultaneous correlative cellular analysis of phenotypic and molecular features by hybrid technologies such as imaging flow cytometry, and further progress in supervised and non-supervised statistical analysis algorithms.

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.

Quantitative determination of testosterone levels with biolayer interferometry.

PubMed

Zhang, Hao; Li, Wei; Luo, Hong; Xiong, Guangming; Yu, Yuanhua

2017-10-01

Natural and synthetic steroid hormones are widely spread in the environment and are considered as pollutants due to their endocrine activities, even at low concentrations, which are harmful to human health. To detect steroid hormones in the environment, a novel biosensor system was developed based on the principle of biolayer interferometry. Detection is based on changes in the interference pattern of white light reflected from the surface of an optical fiber with bound biomolecules. Monitoring interactions between molecules does not require radioactive, enzymatic, or fluorescent labels. Here, 2 double-stranded DNA fragments of operator 1 (OP1) and OP2 containing 10-bp palindromic sequences in chromosomal Comamonas testosteroni DNA (ATCC11996) were surface-immobilized to streptavidin sensors. Interference changes were detected when repressor protein RepA bound the DNA sequences. DNA-protein interactions were characterized and kinetic parameters were obtained. The dissociation constants between the OP1 and OP2 DNA sequences and RepA were 9.865 × 10 -9  M and 2.750 × 10 -8  M, respectively. The reactions showed high specifically and affinity. Because binding of the 10-bp palindromic sequence and RepA was affected by RepA-testosterone binding, the steroid could be quantitatively determined rapidly using the biosensor system. The mechanism of the binding assay was as follows. RepA could bind both OP1 and testosterone. RepA binding to testosterone changed the protein conformation, which influenced the binding between RepA and OP1. The percentage of the signal detected negative correlation with the testosterone concentration. A standard curve was obtained, and the correlation coefficient value was approximately 0.97. We could quantitatively determine testosterone levels between 2.13 and 136.63 ng/ml. Each sample could be quantitatively detected in 17 min. These results suggested that the specific interaction between double-stranded OP1 DNA and the RepA protein

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

NASA Astrophysics Data System (ADS)

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

2016-10-01

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

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.

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

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

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.

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.

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

NASA Technical Reports Server (NTRS)

Johnson, Terry C.; Brady, John N.

1993-01-01

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

Nitrosothiol Formation and Protection against Fenton Chemistry by Nitric Oxide-induced Dinitrosyliron Complex Formation from Anoxia-initiated Cellular Chelatable Iron Increase*

PubMed Central

Li, Qian; Li, Chuanyu; Mahtani, Harry K.; Du, Jian; Patel, Aashka R.; Lancaster, Jack R.

2014-01-01

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with •NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged •NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief •NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1–2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief •NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of •NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of •NO. PMID:24891512

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.

Acoustically detectable cellular-level lung injury induced by fluid mechanical stresses in microfluidic airway systems.

PubMed

Huh, Dongeun; Fujioka, Hideki; Tung, Yi-Chung; Futai, Nobuyuki; Paine, Robert; Grotberg, James B; Takayama, Shuichi

2007-11-27

We describe a microfabricated airway system integrated with computerized air-liquid two-phase microfluidics that enables on-chip engineering of human airway epithelia and precise reproduction of physiologic or pathologic liquid plug flows found in the respiratory system. Using this device, we demonstrate cellular-level lung injury under flow conditions that cause symptoms characteristic of a wide range of pulmonary diseases. Specifically, propagation and rupture of liquid plugs that simulate surfactant-deficient reopening of closed airways lead to significant injury of small airway epithelial cells by generating deleterious fluid mechanical stresses. We also show that the explosive pressure waves produced by plug rupture enable detection of the mechanical cellular injury as crackling sounds.

Crowding in Cellular Environments at an Atomistic Level from Computer Simulations

PubMed Central

2017-01-01

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

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.

Dual transcriptional-translational cascade permits cellular level tuneable expression control

PubMed Central

Morra, Rosa; Shankar, Jayendra; Robinson, Christopher J.; Halliwell, Samantha; Butler, Lisa; Upton, Mathew; Hay, Sam; Micklefield, Jason; Dixon, Neil

2016-01-01

The ability to induce gene expression in a small molecule dependent manner has led to many applications in target discovery, functional elucidation and bio-production. To date these applications have relied on a limited set of protein-based control mechanisms operating at the level of transcription initiation. The discovery, design and reengineering of riboswitches offer an alternative means by which to control gene expression. Here we report the development and characterization of a novel tunable recombinant expression system, termed RiboTite, which operates at both the transcriptional and translational level. Using standard inducible promoters and orthogonal riboswitches, a multi-layered modular genetic control circuit was developed to control the expression of both bacteriophage T7 RNA polymerase and recombinant gene(s) of interest. The system was benchmarked against a number of commonly used E. coli expression systems, and shows tight basal control, precise analogue tunability of gene expression at the cellular level, dose-dependent regulation of protein production rates over extended growth periods and enhanced cell viability. This novel system expands the number of E. coli expression systems for use in recombinant protein production and represents a major performance enhancement over and above the most widely used expression systems. PMID:26405200

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

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

PubMed Central

Dahmani, Hassen-Reda; Schneeberger, Patricia

2009-01-01

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

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.

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.

Nitrosothiol formation and protection against Fenton chemistry by nitric oxide-induced dinitrosyliron complex formation from anoxia-initiated cellular chelatable iron increase.

PubMed

Li, Qian; Li, Chuanyu; Mahtani, Harry K; Du, Jian; Patel, Aashka R; Lancaster, Jack R

2014-07-18

Dinitrosyliron complexes (DNIC) have been found in a variety of pathological settings associated with (•)NO. However, the iron source of cellular DNIC is unknown. Previous studies on this question using prolonged (•)NO exposure could be misleading due to the movement of intracellular iron among different sources. We here report that brief (•)NO exposure results in only barely detectable DNIC, but levels increase dramatically after 1-2 h of anoxia. This increase is similar quantitatively and temporally with increases in the chelatable iron, and brief (•)NO treatment prevents detection of this anoxia-induced increased chelatable iron by deferoxamine. DNIC formation is so rapid that it is limited by the availability of (•)NO and chelatable iron. We utilize this ability to selectively manipulate cellular chelatable iron levels and provide evidence for two cellular functions of endogenous DNIC formation, protection against anoxia-induced reactive oxygen chemistry from the Fenton reaction and formation by transnitrosation of protein nitrosothiols (RSNO). The levels of RSNO under these high chelatable iron levels are comparable with DNIC levels and suggest that under these conditions, both DNIC and RSNO are the most abundant cellular adducts of (•)NO. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Cellular Levels of Oxidative Stress Affect the Response of Cervical Cancer Cells to Chemotherapeutic Agents

PubMed Central

Williams, Vonetta M.; Kokoza, Anatolii; Bashkirova, Svetlana; Duerksen-Hughes, Penelope

2014-01-01

Treatment of advanced and relapsed cervical cancer is frequently ineffective, due in large part to chemoresistance. To examine the pathways responsible, we employed the cervical carcinoma-derived SiHa and CaSki cells as cellular models of resistance and sensitivity, respectively, to treatment with chemotherapeutic agents, doxorubicin, and cisplatin. We compared the proteomic profiles of SiHa and CaSki cells and identified pathways with the potential to contribute to the differential response. We then extended these findings by comparing the expression level of genes involved in reactive oxygen species (ROS) metabolism through the use of a RT-PCR array. The analyses demonstrated that the resistant SiHa cells expressed higher levels of antioxidant enzymes. Decreasing or increasing oxidative stress led to protection or sensitization, respectively, in both cell lines, supporting the idea that cellular levels of oxidative stress affect responsiveness to treatment. Interestingly, doxorubicin and cisplatin induced different profiles of ROS, and these differences appear to contribute to the sensitivity to treatment displayed by cervical cancer cells. Overall, our findings demonstrate that cervical cancer cells display variable profiles with respect to their redox-generating and -adaptive systems, and that these different profiles have the potential to contribute to their responses to treatments with chemotherapy. PMID:25478571

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

Cellular Reflectarray Antenna

NASA Technical Reports Server (NTRS)

Romanofsky, Robert R.

2010-01-01

The cellular reflectarray antenna is intended to replace conventional parabolic reflectors that must be physically aligned with a particular satellite in geostationary orbit. These arrays are designed for specified geographical locations, defined by latitude and longitude, each called a "cell." A particular cell occupies nominally 1,500 square miles (3,885 sq. km), but this varies according to latitude and longitude. The cellular reflectarray antenna designed for a particular cell is simply positioned to align with magnetic North, and the antenna surface is level (parallel to the ground). A given cellular reflectarray antenna will not operate in any other cell.

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.

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.

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

Quantitative Analysis of Intra Urban Growth Modeling using socio economic agents by combining cellular automata model with agent based model

NASA Astrophysics Data System (ADS)

Singh, V. K.; Jha, A. K.; Gupta, K.; Srivastav, S. K.

2017-12-01

Recent studies indicate that there is a significant improvement in the urban land use dynamics through modeling at finer spatial resolutions. Geo-computational models such as cellular automata and agent based model have given evident proof regarding the quantification of the urban growth pattern with urban boundary. In recent studies, socio- economic factors such as demography, education rate, household density, parcel price of the current year, distance to road, school, hospital, commercial centers and police station are considered to the major factors influencing the Land Use Land Cover (LULC) pattern of the city. These factors have unidirectional approach to land use pattern which makes it difficult to analyze the spatial aspects of model results both quantitatively and qualitatively. In this study, cellular automata model is combined with generic model known as Agent Based Model to evaluate the impact of socio economic factors on land use pattern. For this purpose, Dehradun an Indian city is selected as a case study. Socio economic factors were collected from field survey, Census of India, Directorate of economic census, Uttarakhand, India. A 3X3 simulating window is used to consider the impact on LULC. Cellular automata model results are examined for the identification of hot spot areas within the urban area and agent based model will be using logistic based regression approach where it will identify the correlation between each factor on LULC and classify the available area into low density, medium density, high density residential or commercial area. In the modeling phase, transition rule, neighborhood effect, cell change factors are used to improve the representation of built-up classes. Significant improvement is observed in the built-up classes from 84 % to 89 %. However after incorporating agent based model with cellular automata model the accuracy improved from 89 % to 94 % in 3 classes of urban i.e. low density, medium density and commercial classes

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.

Extraction protocol and liquid chromatography/tandem mass spectrometry method for determining micelle-entrapped paclitaxel at the cellular and subcellular levels: Application to a cellular uptake and distribution study.

PubMed

Zheng, Nan; Lian, Bin; Du, Wenwen; Xu, Guobing; Ji, Jiafu

2018-01-01

Paclitaxel-loaded polymeric micelles (PTX-PM) are commonly used as tumor-targeted nanocarriers and display outstanding antitumor features in clinic, but its accumulation and distribution in vitro are lack of investigation. It is probably due to the complex micellar system and its low concentration at the cellular or subcellular levels. In this study, we developed an improved extraction method, which was a combination of mechanical disruption and liquid-liquid extraction (LLE), to extract the total PTX from micelles in the cell lysate and subcellular compartments. An ultra-performance liquid chromatography tandem mass spectroscopy (UPLC-MS/MS) method was optimized to detect the low concentration of PTX at cellular and subcellular levels simultaneously, using docetaxel as internal standard (IS). The method was proved to release PTX totally from micelles (≥95.93%) with a consistent and reproducible extraction recovery (≥75.04%). Good linearity was obtained at concentrations ranging from 0.2 to 20ng/mL. The relative error (RE%) for accuracy varied from 0.68 to 7.56%, and the intra- and inter-precision (relative standard deviation, RSD%) was less than 8.64% and 13.14%, respectively. This method was fully validated and successfully applied to the cellular uptake and distribution study of PTX-loaded PLGA-PEG micelles in human breast cancer cells (MCF-7). Copyright © 2017 Elsevier B.V. All rights reserved.

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

Alzheimer's as a Systems-Level Disease Involving the Interplay of Multiple Cellular Networks.

PubMed

Castrillo, Juan I; Oliver, Stephen G

2016-01-01

Alzheimer's disease (AD), and many neurodegenerative disorders, are multifactorial in nature. They involve a combination of genomic, epigenomic, interactomic and environmental factors. Progress is being made, and these complex diseases are beginning to be understood as having their origin in altered states of biological networks at the cellular level. In the case of AD, genomic susceptibility and mechanisms leading to (or accompanying) the impairment of the central Amyloid Precursor Protein (APP) processing and tau networks are widely accepted as major contributors to the diseased state. The derangement of these networks may result in both the gain and loss of functions, increased generation of toxic species (e.g., toxic soluble oligomers and aggregates) and imbalances, whose effects can propagate to supra-cellular levels. Although well sustained by empirical data and widely accepted, this global perspective often overlooks the essential roles played by the main counteracting homeostatic networks (e.g., protein quality control/proteostasis, unfolded protein response, protein folding chaperone networks, disaggregases, ER-associated degradation/ubiquitin proteasome system, endolysosomal network, autophagy, and other stress-protective and clearance networks), whose relevance to AD is just beginning to be fully realized. In this chapter, an integrative perspective is presented. Alzheimer's disease is characterized to be a result of: (a) intrinsic genomic/epigenomic susceptibility and, (b) a continued dynamic interplay between the deranged networks and the central homeostatic networks of nerve cells. This interplay of networks will underlie both the onset and rate of progression of the disease in each individual. Integrative Systems Biology approaches are required to effect its elucidation. Comprehensive Systems Biology experiments at different 'omics levels in simple model organisms, engineered to recapitulate the basic features of AD may illuminate the onset and

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

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

WE-DE-202-02: Are Track Structure Simulations Truly Needed for Radiobiology at the Cellular and Tissue Levels?

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

Stewart, R.

Radiation therapy for the treatment of cancer has been established as a highly precise and effective way to eradicate a localized region of diseased tissue. To achieve further significant gains in the therapeutic ratio, we need to move towards biologically optimized treatment planning. To achieve this goal, we need to understand how the radiation-type dependent patterns of induced energy depositions within the cell (physics) connect via molecular, cellular and tissue reactions to treatment outcome such as tumor control and undesirable effects on normal tissue. Several computational biology approaches have been developed connecting physics to biology. Monte Carlo simulations are themore » most accurate method to calculate physical dose distributions at the nanometer scale, however simulations at the DNA scale are slow and repair processes are generally not simulated. Alternative models that rely on the random formation of individual DNA lesions within one or two turns of the DNA have been shown to reproduce the clusters of DNA lesions, including single strand breaks (SSBs), double strand breaks (DSBs) without the need for detailed track structure simulations. Efficient computational simulations of initial DNA damage induction facilitate computational modeling of DNA repair and other molecular and cellular processes. Mechanistic, multiscale models provide a useful conceptual framework to test biological hypotheses and help connect fundamental information about track structure and dosimetry at the sub-cellular level to dose-response effects on larger scales. In this symposium we will learn about the current state of the art of computational approaches estimating radiation damage at the cellular and sub-cellular scale. How can understanding the physics interactions at the DNA level be used to predict biological outcome? We will discuss if and how such calculations are relevant to advance our understanding of radiation damage and its repair, or, if the underlying

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.

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

PubMed

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

2016-09-01

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

Mechanisms of Oxygen Toxicity at the Cellular Level.

DTIC Science & Technology

1982-06-30

exposed and measured using glucose as the sole carbon source. Addition of SH containing reducing agents (cysteine, lipoic acid or dithiothreitol) before...of a Few Seconds. Biotechnology and Bioengineering 16:1645-1657 (1974). (28) Brown, O.R. Failure of Lipoic Acid to Protect Against Cellular Oxygen...respiration, and fatty acid synthesis. The interruption of fatty acid synthesis is not the result of inactivation of the fatty acid synthetase enzyme complex

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

PubMed

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

2010-01-01

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

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

PubMed

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

2017-01-22

B.mori nucleopolyhedrovirus (BmNPV), which produces BV and ODV two virion phenotypes in its life cycle, caused the amount of economic loss in sericulture. But the mechanism of its infection was still unclear. In this study we characterized B.mori nuclear hormone receptor 96 (BmNHR96) as a NHR96 family member, which was localized in the nucleus. We also found BmNHR96 over-expression could enhance the entry of BV as well as cellular cholesterol level. Furthermore, we validated that BmNHR96 increased membrane fusion mediated by GP64, which could probably promote BV-infection. In summary, our study suggested that BmNHR96 plays an important role in BV infection and this function probably actualized by affecting cellular cholesterol level, and our results provided insights to the mechanisms of BV-infection of B.mori. Copyright © 2016 Elsevier Inc. All rights reserved.

Multimodal imaging of the human knee down to the cellular level

NASA Astrophysics Data System (ADS)

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

2017-06-01

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

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.

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

PubMed Central

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

2014-01-01

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

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

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.

A non-neuronal cholinergic system regulates cellular ATP levels to maintain cell viability.

PubMed

Oikawa, Shino; Iketani, Mitsue; Kakinuma, Yoshihiko

2014-01-01

We previously suggested that a non-neuronal cholinergic system modulates energy metabolism through the mitochondria. However, the mechanisms responsible for making this system crucial remained undetermined. In this study, we developed a fusion protein expression vector containing a luciferase gene fused to the folic acid receptor-α gene. This protein of the vector was confirmed to target the plasma membrane of transfected HEK293 cells, and vector-derived luciferase activities and ATP levels in viable cells were positively correlated (r = 0.599). Using this luciferase vector, choline acetyltransferase (ChAT)-expressing cells (i.e., cells with an activated non-neuronal cholinergic system) had increased cellular ATP levels. ChAT-expressing cells also had upregulated IGF-1R and Glut-1 protein expressions as well as increased glucose uptake. This activated non-neuronal cholinergic system with efficient glucose metabolism rendered cells resistant to serum depletion-induced cell death. Our results indicate that a non-neuronal cholinergic system is involved in sustaining ATP levels to render cells resistant to a nutrient-deficient environment. © 2014 S. Karger AG, Basel.

Individuals with autism have higher 8-Iso-PGF2α levels than controls, but no correlation with quantitative assay of Paraoxonase 1 serum levels.

PubMed

Pop, Bianca; Niculae, Alexandru-Ștefan; Pop, Tudor Lucian; Răchișan, Andreea Liana

2017-12-01

Autism spectrum disorder (ASD) represents a very large set of neurodevelopmental issues with diverse clinical outcomes. Various hypotheses have been put forth for the etiology of autism spectrum disorder, including issues pertaining to oxidative stress. In this study, we conducted measurements of serum 8-Iso-Prostaglanding F2 α (8-iso-PGF2α, which is the results of non-enzimatically mediated polyunsaturated fatty acid oxidation) in a population of individuals with autism and a control group of age and sex matched controls. A quantitative assay of Paraoxonase 1 (PON1) was conducted. Data regarding comorbidities, structural MRI scans, medication, intelligence quotient (IQ) and Childhood Autism Rating Scale scores (CARS) were also included in our study. Our results show that patients diagnosed with autism have higher levels of 8-iso-PGF2α than their neurotypical counterparts. Levels of this particular metabolite, however, do not correlate with quantitative serum levels of Paraoxonase 1, which has been shown to be altered in individuals with autism. Neither 8-iso-PGF2α nor quantitative levels of PON1 provide any meaningful correlation with clinical or neuroimaging data in this study group. Future research should focus on providing data regarding PON 1 phenotype, in addition to standard quantitative measurements, in relation to 8-iso-PGF2α as well as other clinical and structural brain findings.

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

PubMed

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

2017-06-01

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

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 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

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

PubMed Central

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

2015-01-01

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

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

The human ubiquitin-conjugating enzyme Cdc34 controls cellular proliferation through regulation of p27{sup Kip1} protein levels

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

Butz, Nicole; Ruetz, Stephan; Natt, Francois

2005-02-15

Ubiquitin-mediated degradation of the cyclin-dependent kinase inhibitor p27{sup Kip1} was shown to be required for the activation of key cyclin-dependent kinases, thereby triggering the onset of DNA replication and cell cycle progression. Although the SCF{sup Skp2} ubiquitin ligase has been reported to mediate p27{sup Kip1} degradation, the nature of the human ubiquitin-conjugating enzyme involved in this process has not yet been determined at the cellular level. Here, we show that antisense oligonucleotides targeting the human ubiquitin-conjugating enzyme Cdc34 downregulate its expression, inhibit the degradation of p27{sup Kip1}, and prevent cellular proliferation. Elevation of p27{sup Kip1} protein level is found tomore » be the sole requirement for the inhibition of cellular proliferation induced upon downregulation of Cdc34. Indeed, reducing the expression of p27{sup Kip1} with a specific antisense oligonucleotide is sufficient to reverse the anti-proliferative phenotype elicited by the Cdc34 antisense. Furthermore, downregulation of Cdc34 is found to specifically increase the abundance of the SCF{sup Skp2} ubiquitin ligase substrate p27{sup Kip1}, but has no concomitant effect on the level of IkB{alpha} and {beta}-catenin, which are known substrates of a closely related SCF ligase.« less

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

PubMed Central

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

2015-01-01

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

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

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

Choi, Joon-Seok; Lee, Cheol-Koo, E-mail: cklee2005@korea.ac.kr

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

Quantitative modeling of clinical, cellular, and extracellular matrix variables suggest prognostic indicators in cancer: a model in neuroblastoma.

PubMed

Tadeo, Irene; Piqueras, Marta; Montaner, David; Villamón, Eva; Berbegall, Ana P; Cañete, Adela; Navarro, Samuel; Noguera, Rosa

2014-02-01

Risk classification and treatment stratification for cancer patients is restricted by our incomplete picture of the complex and unknown interactions between the patient's organism and tumor tissues (transformed cells supported by tumor stroma). Moreover, all clinical factors and laboratory studies used to indicate treatment effectiveness and outcomes are by their nature a simplification of the biological system of cancer, and cannot yet incorporate all possible prognostic indicators. A multiparametric analysis on 184 tumor cylinders was performed. To highlight the benefit of integrating digitized medical imaging into this field, we present the results of computational studies carried out on quantitative measurements, taken from stromal and cancer cells and various extracellular matrix fibers interpenetrated by glycosaminoglycans, and eight current approaches to risk stratification systems in patients with primary and nonprimary neuroblastoma. New tumor tissue indicators from both fields, the cellular and the extracellular elements, emerge as reliable prognostic markers for risk stratification and could be used as molecular targets of specific therapies. The key to dealing with personalized therapy lies in the mathematical modeling. The use of bioinformatics in patient-tumor-microenvironment data management allows a predictive model in neuroblastoma.

Modulation in vitro and in vivo of cytotoxicity but not cellular levels of doxorubicin by the calmodulin inhibitor trifluoperazine is dependent on the level of resistance.

PubMed Central

Ganapathi, R.; Schmidt, H.; Grabowski, D.; Melia, M.; Ratliff, N.

1988-01-01

The role of the calmodulin inhibitor trifluoperazine (TFP) in modulating the cellular levels and cytotoxicity in vitro and antitumour effects in vivo of doxorubicin (DOX), was evaluated in progressively DOX-resistant (5- to 40-fold) sublines of B16-BL6 mouse melanoma. In parental-sensitive B16-BL6 cells treated for 3 h, the IC50 of DOX was 0.1 microgram ml-1, and a less than 2-fold enhancement in DOX cell kill in the presence of a noncytotoxic concentration of 5 microM TFP was observed. However, in the DOX-resistant sublines, the IC50 was 0.7 to 5.0 micrograms ml-1 DOX in the absence of 5 microM TFP and 0.3 to 0.7 microgram ml-1 DOX in the presence of 5 microM TFP. The 2- to 7.5-fold decrease in the IC50 of DOX in the presence of 5 microM TFP, was dependent on the level of DOX-resistance in the various sublines. Compared to parental-sensitive cells, a 2-fold decrease in DOX-accumulation was evident only in the 40-fold DOX-resistant subline. Further, maximal enhancement (50%) of cellular DOX accumulation in the presence of 5 microM TFP was observed only in the 40-fold resistant cells treated with 5.0 micrograms ml-1 DOX. Retention of DOX in the 40-fold resistant subline was only 20% lower than similarly treated sensitive cells, and the inclusion of TFP increased DOX retention less than 10-15%. Antitumour studies in mice with experimental pulmonary metastases revealed that although DOX and DOX plus TFP had similar antitumour activity with the parental sensitive B16-BL6 cells, the combination of DOX plus TFP was significantly more effective than DOX alone with the DOX-resistant sublines. No overt toxicity was observed in normal mice treated with doses of TFP, DOX or DOX plus TFP used for in vivo chemotherapy studies. Results from this study suggest that gross cellular DOX levels do not appear to correlate with the magnitude of resistance, and the effects of TFP in modulating DOX resistance is possibly due to mechanisms other than mere alterations in cellular drug

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

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

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

Cellular adverse actions of dibromoacetonitrile, a by-product in water bacterial control, at sublethal levels in rat thymocytes.

PubMed

Kishida, Takumi; Akiyoshi, Kenji; Erdenedalai, Erdenebat; Enhetomuru, Anu; Imai, Shoji; Oyama, Yasuo

2018-09-01

The aim of this study was to investigate the effects of dibromoacetonitrile (DBAN), a by-product in water bacterial control, at sublethal concentrations on rat thymocytes, by using a cytometric technique with appropriate fluorescent dyes. By using this method, the possibility that DBAN induces cellular actions related to oxidative stress was assessed. DBAN reduced the content of cellular nonprotein thiols under Zn 2+ -free conditions. It elevated the intracellular level of Zn 2+ , being independent from external Zn 2+ . DBAN increased cell vulnerability to the cytotoxic action of hydrogen peroxide. These actions of DBAN were likely related to oxidative stress. DBAN is formed by the reaction of bromides and chlorinated oxidants during water disinfection. Hydrolysis of 2,2-dibromo-3-nitrilopropionamide, an antimicrobial used in hydraulic fracturing fluids for production of shale gas and oil, produces DBAN. Therefore, the concern regarding the levels of DBAN in industrial water systems is necessary to avoid the environmental risk to humans and wild mammals. Copyright © 2018 Elsevier Ltd. All rights reserved.

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.

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.

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

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

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.

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

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.

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

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

ERIC Educational Resources Information Center

Kashyap, Upasana; Mathew, Santhosh

2017-01-01

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

Cellular glutathione levels in HL-60 cells during respiratory burst are not correlated with ultra-weak photon emission.

PubMed

Burgos, Rosilene Cristina Rossetto; Zhang, Wei; van Wijk, Eduard P A; Hankemeier, Thomas; Ramautar, Rawi; van der Greef, Jan

2017-10-01

Recently, ultra-weak photon emission (UPE) was developed as a novel tool for measuring oxidative metabolic processes, as its generation is related to reactive oxygen species (ROS). Both an imbalance in ROS or the uncontrolled production of ROS can lead to oxidative stress, which is commonly associated with many diseases. In addition to playing several biological functions, the thiol amino acid glutathione has an important antioxidant function in the body's defense against ROS. Specifically, glutathione is an important endogenous antioxidant that helps maintain oxidant levels. At the cellular level, glutathione is present in its reduced form (GSH) at relatively high concentrations (in the millimolar range) and in its oxidized form (GSSG) at low concentrations (in the micromolar range). Thus, the GSH/GSSG ratio is often used as an indicator of cellular redox state. Here, we used the HL-60 cell line as a model system in order to determine whether UPE is correlated with intracellular GSH and GSSG levels. HL-60 cells were differentiated into neutrophil-like cells and then stimulated to undergo respiratory burst. We then recorded UPE in real time for 9000 seconds and used capillary electrophoresis coupled to mass spectrometry to measure GSH and GSSG levels in cell extracts. We found that although respiratory burst significantly decreased the GSH/GSSG ratio, this change was not significantly correlated with the UPE profile. Copyright © 2017 Elsevier B.V. All rights reserved.

Quantitation of Cellular Metabolic Fluxes of Methionine

PubMed Central

Shlomi, Tomer; Fan, Jing; Tang, Baiqing; Kruger, Warren D.; Rabinowitz, Joshua D.

2014-01-01

Methionine is an essential proteogenic amino acid. In addition, it is a methyl donor for DNA and protein methylation and a propylamine donor for polyamine biosyn-thesis. Both the methyl and propylamine donation pathways involve metabolic cycles, and methods are needed to quantitate these cycles. Here, we describe an analytical approach for quantifying methionine metabolic fluxes that accounts for the mixing of intracellular and extracellular methionine pools. We observe that such mixing prevents isotope tracing experiments from reaching the steady state due to the large size of the media pools and hence precludes the use of standard stationary metabolic flux analysis. Our approach is based on feeding cells with 13C methionine and measuring the isotope-labeling kinetics of both intracellular and extracellular methionine by liquid chromatography−mass spectrometry (LC-MS). We apply this method to quantify methionine metabolism in a human fibrosarcoma cell line and study how methionine salvage pathway enzyme methylthioadenosine phosphorylase (MTAP), frequently deleted in cancer, affects methionine metabolism. We find that both transmethylation and propylamine transfer fluxes amount to roughly 15% of the net methionine uptake, with no major changes due to MTAP deletion. Our method further enables the quantification of flux through the pro-tumorigenic enzyme ornithine decarboxylase, and this flux increases 2-fold following MTAP deletion. The analytical approach used to quantify methionine metabolic fluxes is applicable for other metabolic systems affected by mixing of intracellular and extracellular metabolite pools. PMID:24397525

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

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

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.

Cellular Contraction and Polarization Drive Collective Cellular Motion.

PubMed

Notbohm, Jacob; Banerjee, Shiladitya; Utuje, Kazage J C; Gweon, Bomi; Jang, Hwanseok; Park, Yongdoo; Shin, Jennifer; Butler, James P; Fredberg, Jeffrey J; Marchetti, M Cristina

2016-06-21

Coordinated motions of close-packed multicellular systems typically generate cooperative packs, swirls, and clusters. These cooperative motions are driven by active cellular forces, but the physical nature of these forces and how they generate collective cellular motion remain poorly understood. Here, we study forces and motions in a confined epithelial monolayer and make two experimental observations: 1) the direction of local cellular motion deviates systematically from the direction of the local traction exerted by each cell upon its substrate; and 2) oscillating waves of cellular motion arise spontaneously. Based on these observations, we propose a theory that connects forces and motions using two internal state variables, one of which generates an effective cellular polarization, and the other, through contractile forces, an effective cellular inertia. In agreement with theoretical predictions, drugs that inhibit contractility reduce both the cellular effective elastic modulus and the frequency of oscillations. Together, theory and experiment provide evidence suggesting that collective cellular motion is driven by at least two internal variables that serve to sustain waves and to polarize local cellular traction in a direction that deviates systematically from local cellular velocity. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

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.

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

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

Pericentrin in cellular function and disease

PubMed Central

Delaval, Benedicte

2010-01-01

Pericentrin is an integral component of the centrosome that serves as a multifunctional scaffold for anchoring numerous proteins and protein complexes. Through these interactions, pericentrin contributes to a diversity of fundamental cellular processes. Recent studies link pericentrin to a growing list of human disorders. Studies on pericentrin at the cellular, molecular, and, more recently, organismal level, provide a platform for generating models to elucidate the etiology of these disorders. Although the complexity of phenotypes associated with pericentrin-mediated disorders is somewhat daunting, insights into the cellular basis of disease are beginning to come into focus. In this review, we focus on human conditions associated with loss or elevation of pericentrin and propose cellular and molecular models that might explain them. PMID:19951897

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.

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

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.

Cell Proliferation, Reactive Oxygen and Cellular Glutathione

PubMed Central

Day, Regina M.; Suzuki, Yuichiro J.

2005-01-01

A variety of cellular activities, including metabolism, growth, and death, are regulated and modulated by the redox status of the environment. A biphasic effect has been demonstrated on cellular proliferation with reactive oxygen species (ROS)—especially hydrogen peroxide and superoxide—in which low levels (usually submicromolar concentrations) induce growth but higher concentrations (usually >10–30 micromolar) induce apoptosis or necrosis. This phenomenon has been demonstrated for primary, immortalized and transformed cell types. However, the mechanism of the proliferative response to low levels of ROS is not well understood. Much of the work examining the signal transduction by ROS, including H2O2, has been performed using doses in the lethal range. Although use of higher ROS doses have allowed the identification of important signal transduction pathways, these pathways may be activated by cells only in association with ROS-induced apoptosis and necrosis, and may not utilize the same pathways activated by lower doses of ROS associated with increased cell growth. Recent data has shown that low levels of exogenous H2O2 up-regulate intracellular glutathione and activate the DNA binding activity toward antioxidant response element. The modulation of the cellular redox environment, through the regulation of cellular glutathione levels, may be a part of the hormetic effect shown by ROS on cell growth. PMID:18648617

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.

A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA.

PubMed

Al-Turkmani, M Rabie; Andersson, Charlotte; Alturkmani, Ragheed; Katrangi, Waddah; Cluette-Brown, Joanne E; Freedman, Steven D; Laposata, Michael

2008-09-01

Specific fatty acid alterations have been described in the blood and tissues of cystic fibrosis (CF) patients. The principal alterations include decreased levels of linoleic acid (LA) and docosahexaenoic acid (DHA). We investigated the potential mechanisms of these alterations by studying the cellular uptake of LA and DHA, their distribution among lipid classes, and the metabolism of LA in a human bronchial epithelial cell model of CF. CF (antisense) cells demonstrated decreased levels of LA and DHA compared with wild type (WT, sense) cells expressing normal CFTR. Cellular uptake of LA and DHA was higher in CF cells compared with WT cells at 1 h and 4 h. Subsequent incorporation of LA and DHA into most lipid classes and individual phospholipids was also increased in CF cells. The metabolic conversion of LA to n-6 metabolites, including 18:3n-6 and arachidonic acid, was upregulated in CF cells, indicating increased flux through the n-6 pathway. Supplementing CF cells with DHA inhibited the production of LA metabolites and corrected the n-6 fatty acid defect. In conclusion, the evidence suggests that low LA level in cultured CF cells is due to its increased metabolism, and this increased LA metabolism is corrected by DHA supplementation.

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

NASA Astrophysics Data System (ADS)

Colombani, Mathieu; Forterre, Yoel; GEP Team

2012-11-01

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

Taming the sphinx: Mechanisms of cellular sphingolipid homeostasis.

PubMed

Olson, D K; Fröhlich, F; Farese, R V; Walther, T C

2016-08-01

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon. Copyright © 2015. Published by Elsevier B.V.

Toxicity of pyrolysis gases from some cellular polymers

NASA Technical Reports Server (NTRS)

Hilado, C. J.; Machado, A. M.

1978-01-01

Various samples of cellular polymers were evaluated for toxicity of pyrolysis gases, using the screening test method developed at the University of San Francisco. The cellular polymer samples included polyimide, polymethacrylimide, polybismaleimide, polyurethane, polyisocyanurate, polyethylene, polychloroprene, polyvinyl chloride, polystyrene, polysiloxane, and polyphosphazene. The cellular polymers exhibited varying levels of toxicity under these test conditions. Among the rigid cellular polymers, times to death were shortest with the imide type foams and longest with polyvinyl chloride and polystyrene. Among the flexible cellular polymers, times to death were shortest with polyimide and polyester, and longest with polychloroprene and polysiloxane. Increased char yield was not necessarily associated with reduced toxicity.

Qualitative and quantitative mass spectrometry imaging of drugs and metabolites in tissue at therapeutic levels.

PubMed

Sun, Na; Walch, Axel

2013-08-01

Mass spectrometry imaging (MSI) is a rapidly evolving technology that yields qualitative and quantitative distribution maps of small pharmaceutical-active molecules and their metabolites in tissue sections in situ. The simplicity, high sensitivity and ability to provide comprehensive spatial distribution maps of different classes of biomolecules make MSI a valuable tool to complement histopathology for diagnostics and biomarker discovery. In this review, qualitative and quantitative MSI of drugs and metabolites in tissue at therapeutic levels are discussed and the impact of this technique in drug discovery and clinical research is highlighted.

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

PubMed Central

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

2015-01-01

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

Complex formation and vectorization of a phosphorothioate oligonucleotide with an amphipathic leucine- and lysine-rich peptide: study at molecular and cellular levels.

PubMed

Boukhalfa-Heniche, Fatima-Zohra; Hernández, Belén; Gaillard, Stéphane; Coïc, Yves-Marie; Huynh-Dinh, Tam; Lecouvey, Marc; Seksek, Olivier; Ghomi, Mahmoud

2004-04-15

Optical spectroscopic techniques such as CD, Raman scattering, and fluorescence imaging allowed us to analyze the complex formation and vectorization of a single-stranded 20-mer phosphorothioate oligodeoxynucleotide with a 15-mer amphipathic peptide at molecular and cellular levels. Different solvent mixtures (methanol and water) and molecular ratios of peptide/oligodeoxynucleotide complexes were tested in order to overcome the problems related to solubility. Optimal conditions for both spectroscopic and cellular experiments were obtained with the molecular ratio peptide/oligodeoxynucleotide equal to 21:4, corresponding to a 7:5 ratio for their respective +/- charge ratio. At the molecular level, CD and Raman spectra were consistent with a alpha-helix conformation of the peptide in water or in a methanol-water mixture. The presence of methanol increased considerably the solubility of the peptide without altering its alpha-helix conformation, as evidenced by CD and Raman spectroscopies. UV absorption melting profile of the oligodeoxynucleotide gave rise to a flat melting profile, corresponding to its random structure in solution. Raman spectra of oligodeoxynucleotide/peptide complexes could only be studied in methanol/water mixture solutions. Drastic changes observed in Raman spectra have undoubtedly shown: (a) the perturbation occurred in the peptide secondary structure, and (b) possible interaction between the lysine residues of the peptide and the oligodeoxynucleotide. At the cellular level, the complex was prepared in a mixture of 10% methanol and 90% cell medium. Cellular uptake in optimal conditions for the oligodeoxynucleotide delivery with low cytotoxicity was controlled by fluorescence imaging allowing to specifically locate the compacted oligonucleotide labeled with fluorescein at its 5'-terminus with the peptide into human glioma cells after 1 h of incubation at 37 degrees C. Copyright 2004 Wiley Periodicals, Inc.

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.

Quantitative assessment of glacial fluctuations in the level of Lake Lisan, Dead Sea rift

NASA Astrophysics Data System (ADS)

Rohling, Eelco J.

2013-06-01

A quantitative understanding of climatic variations in the Levant during the last glacial cycle is needed to support archaeologists in assessing the drivers behind hominin migrations and cultural developments in this key region at the intersection between Africa and Europe. It will also foster a better understanding of the region's natural variability as context to projections of modern climate change. Detailed documentation of variations in the level of Lake Lisan - the lake that occupied the Dead Sea rift during the last glacial cycle - provides crucial climatic information for this region. Existing reconstructions suggest that Lake Lisan highstands during cold intervals of the last glacial cycle represent relatively humid conditions in the region, but these interpretations have remained predominantly qualitative. Here, I evaluate realistic ranges of the key climatological parameters that controlled lake level, based on the observed timing and amplitudes of lake-level variability. I infer that a mean precipitation rate over the wider catchment area of about 500 mm y-1, as proposed in the literature, would be consistent with observed lake levels if there was a concomitant 15-50% increase in wind speed during cold glacial stadials. This lends quantitative support to previous inferences of a notable increase in the intensity of Mediterranean (winter) storms during glacial periods, which tracked eastward into the Levant. In contrast to highstands during ‘regular’ stadials, lake level dropped during Heinrich Events. I demonstrate that this likely indicates a further intensification of the winds during those times.

Taming the Sphinx: Mechanisms of Cellular Sphingolipid Homeostasis

PubMed Central

Olson, D. K.; Fröhlich, F.; Farese, R; Walther, T. C.

2016-01-01

Sphingolipids are important structural membrane components of eukaryotic cells, and potent signaling molecules. As such, their levels must be maintained to optimize cellular functions in different cellular membranes. Here, we review the current knowledge of homeostatic sphingolipid regulation. We describe recent studies in Saccharomyces cerevisiae that have provided insights into how cells sense changes in sphingolipid levels in the plasma membrane and acutely regulate sphingolipid biosynthesis by altering signaling pathways. We also discuss how cellular trafficking has emerged as an important determinant of sphingolipid homeostasis. Finally, we highlight areas where work is still needed to elucidate the mechanisms of sphingolipid regulation and the physiological functions of such regulatory networks, especially in mammalian cells. PMID:26747648

Measuring changes in transmission of neglected tropical diseases, malaria, and enteric pathogens from quantitative antibody levels.

PubMed

Arnold, Benjamin F; van der Laan, Mark J; Hubbard, Alan E; Steel, Cathy; Kubofcik, Joseph; Hamlin, Katy L; Moss, Delynn M; Nutman, Thomas B; Priest, Jeffrey W; Lammie, Patrick J

2017-05-01

Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman's rho = 0.75). In

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

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.

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

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

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

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...

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

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

Detection of interferon alpha protein reveals differential levels and cellular sources in disease.

PubMed

Rodero, Mathieu P; Decalf, Jérémie; Bondet, Vincent; Hunt, David; Rice, Gillian I; Werneke, Scott; McGlasson, Sarah L; Alyanakian, Marie-Alexandra; Bader-Meunier, Brigitte; Barnerias, Christine; Bellon, Nathalia; Belot, Alexandre; Bodemer, Christine; Briggs, Tracy A; Desguerre, Isabelle; Frémond, Marie-Louise; Hully, Marie; van den Maagdenberg, Arn M J M; Melki, Isabelle; Meyts, Isabelle; Musset, Lucile; Pelzer, Nadine; Quartier, Pierre; Terwindt, Gisela M; Wardlaw, Joanna; Wiseman, Stewart; Rieux-Laucat, Frédéric; Rose, Yoann; Neven, Bénédicte; Hertel, Christina; Hayday, Adrian; Albert, Matthew L; Rozenberg, Flore; Crow, Yanick J; Duffy, Darragh

2017-05-01

Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation. © 2017 Rodero et al.

Detection of interferon alpha protein reveals differential levels and cellular sources in disease

PubMed Central

Rodero, Mathieu P.; Rice, Gillian I.; Werneke, Scott; Alyanakian, Marie-Alexandra; Barnerias, Christine; Bellon, Nathalia; Belot, Alexandre; Bodemer, Christine; Desguerre, Isabelle; Meyts, Isabelle; Musset, Lucile; Wardlaw, Joanna; Wiseman, Stewart; Rose, Yoann; Neven, Bénédicte; Hertel, Christina; Hayday, Adrian; Albert, Matthew L.; Rozenberg, Flore

2017-01-01

Type I interferons (IFNs) are essential mediators of antiviral responses. These cytokines have been implicated in the pathogenesis of autoimmunity, most notably systemic lupus erythematosus (SLE), diabetes mellitus, and dermatomyositis, as well as monogenic type I interferonopathies. Despite a fundamental role in health and disease, the direct quantification of type I IFNs has been challenging. Using single-molecule array (Simoa) digital ELISA technology, we recorded attomolar concentrations of IFNα in healthy donors, viral infection, and complex and monogenic interferonopathies. IFNα protein correlated well with functional activity and IFN-stimulated gene expression. High circulating IFNα levels were associated with increased clinical severity in SLE patients, and a study of the cellular source of IFNα protein indicated disease-specific mechanisms. Measurement of IFNα attomolar concentrations by digital ELISA will enhance our understanding of IFN biology and potentially improve the diagnosis and stratification of pathologies associated with IFN dysregulation. PMID:28420733

Neurofilament protein levels: quantitative analysis in essential tremor cerebellar cortex.

PubMed

Louis, Elan D; Ma, Karen; Babij, Rachel; Cortés, Etty; Liem, Ronald K; Vonsattel, Jean-Paul G; Faust, Phyllis L

2012-06-14

Essential tremor (ET) is among the most prevalent neurological diseases. A substantial increase in the number of Purkinje cell axonal swellings (torpedoes) has been identified in ET brains. We recently demonstrated that torpedoes in ET contain an over-accumulation of disorganized neurofilament (NF) proteins. This now raises the question whether NF protein composition and/or phosphorylation state in cerebellar tissue might differ between ET cases and controls. We used a Western blot analysis to compare the levels and phosphorylation state of NF proteins and α-internexin in cerebellar tissue from 47 ET cases versus 26 controls (2:1 ratio). Cases and controls did not differ with respect to the cerebellar levels of NF-light (NF-L), NF-medium (NF-M), NF-heavy (NF-H), or α-internexin. However, SMI-31 levels (i.e., phosphorylated NF-H) and SMI-32 levels (i.e., non-phosphorylated NF-H) were significantly higher in ET cases than controls (1.28±0.47 vs. 1.06±0.32, p=0.02; and 1.38±0.75 vs. 1.00±0.42, p=0.006). Whether the abnormal phosphorylation state that we observed is a cause of defective axonal transport and/or function of NFs in ET is not known. NF abnormalities have been demonstrated in several neurodegenerative diseases. Regardless of whether these protein aggregates are the cause or consequence of these diseases, NF abnormalities have been shown to be an important factor in the cellular disruption observed in several neurodegenerative diseases. Therefore, further analyses of these NF abnormalities and their mechanisms are important to enhance our understanding of disease pathogenesis in ET. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

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

NASA Astrophysics Data System (ADS)

Hughes, Dirk D.

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

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

A Study on Cognitive Radio Coexisting with Cellular Systems

NASA Astrophysics Data System (ADS)

Tandai, Tomoya; Horiguchi, Tomoya; Deguchi, Noritaka; Tomizawa, Takeshi; Tomioka, Tazuko

Cognitive Radios (CRs) are expected to perform more significant role in the view of efficient utilization of the spectrum resources in the future wireless communication networks. In this paper, a cognitive radio coexisting with cellular systems is proposed. In the case that a cellular system adopts Frequency Division Duplex (FDD) as a multiplexing scheme, the proposed CR terminals communicate in local area on uplink channels of the cellular system with transmission powers that don't interfere with base stations of the cellular system. Alternatively, in the case that a cellular system adopts Time Division Duplex (TDD), the CR terminals communicate on uplink slots of the cellular system. However if mobile terminals in the cellular system are near the CR network, uplink signals from the mobile terminals may interfere with the CR communications. In order to avoid interference from the mobile terminals, the CR terminal performs carrier sense during a beginning part of uplink slot, and only when the level of detected signal is below a threshold, then the CR terminal transmits a signal during the remained period of the uplink slot. In this paper, both the single carrier CR network that uses one frequency channel of the cellular system and the multicarrier CR network that uses multiple frequency channels of the cellular system are considered. The probabilities of successful CR communications, the average throughputs of the CR communications according to the positions of the CR network, and the interference levels from cognitive radio network to base stations of the cellular system are evaluated in the computer simulation then the effectiveness of the proposed network is clarified.

High Dose Atorvastatin Decreases Cellular Markers of Immune Activation Without Affecting HIV-1 RNA Levels: Results of a Double-Blind Randomized Placebo Controlled Clinical Trial

DTIC Science & Technology

2011-02-15

M A J O R A R T I C L E High Dose Atorvastatin Decreases Cellular Markers of Immune Activation without Affecting HIV-1 RNA Levels: Results of a... atorvastatin on HIV-1 RNA (primary objective) and cellular markers of immune activation (secondary objective). HIV-infected individuals not receiving...antiretroviral therapy were randomized to receive either 8 weeks of atorvastatin (80 mg) or placebo daily. After a 4–6 week washout phase, participants

Quantitating Antibody Uptake In Vivo: Conditional Dependence on Antigen Expression Levels

PubMed Central

Thurber, Greg M.; Weissleder, Ralph

2010-01-01

Purpose Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation. Procedures Using a cell line with high EpCAM expression and moderate EGFR expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high affinity antibodies. Results As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors. Conclusions These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes. PMID:20809210

Quantitating antibody uptake in vivo: conditional dependence on antigen expression levels.

PubMed

Thurber, Greg M; Weissleder, Ralph

2011-08-01

Antibodies form an important class of cancer therapeutics, and there is intense interest in using them for imaging applications in diagnosis and monitoring of cancer treatment. Despite the expanding body of knowledge describing pharmacokinetic and pharmacodynamic interactions of antibodies in vivo, discrepancies remain over the effect of antigen expression level on tumoral uptake with some reports indicating a relationship between uptake and expression and others showing no correlation. Using a cell line with high epithelial cell adhesion molecule expression and moderate epidermal growth factor receptor expression, fluorescent antibodies with similar plasma clearance were imaged in vivo. A mathematical model and mouse xenograft experiments were used to describe the effect of antigen expression on uptake of these high-affinity antibodies. As predicted by the theoretical model, under subsaturating conditions, uptake of the antibodies in such tumors is similar because localization of both probes is limited by delivery from the vasculature. In a separate experiment, when the tumor is saturated, the uptake becomes dependent on the number of available binding sites. In addition, targeting of small micrometastases is shown to be higher than larger vascularized tumors. These results are consistent with the prediction that high affinity antibody uptake is dependent on antigen expression levels for saturating doses and delivery for subsaturating doses. It is imperative for any probe to understand whether quantitative uptake is a measure of biomarker expression or transport to the region of interest. The data provide support for a predictive theoretical model of antibody uptake, enabling it to be used as a starting point for the design of more efficacious therapies and timely quantitative imaging probes.

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.

Measuring changes in transmission of neglected tropical diseases, malaria, and enteric pathogens from quantitative antibody levels

PubMed Central

van der Laan, Mark J.; Hubbard, Alan E.; Steel, Cathy; Kubofcik, Joseph; Hamlin, Katy L.; Moss, Delynn M.; Nutman, Thomas B.; Priest, Jeffrey W.; Lammie, Patrick J.

2017-01-01

Background Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. Methods/Principal findings We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P

Sleep deprivation and activation of morning levels of cellular and genomic markers of inflammation.

PubMed

Irwin, Michael R; Wang, Minge; Campomayor, Capella O; Collado-Hidalgo, Alicia; Cole, Steve

2006-09-18

Inflammation is associated with increased risk of cardiovascular disorders, arthritis, diabetes mellitus, and mortality. The effects of sleep loss on the cellular and genomic mechanisms that contribute to inflammatory cytokine activity are not known. In 30 healthy adults, monocyte intracellular proinflammatory cytokine production was repeatedly assessed during the day across 3 baseline periods and after partial sleep deprivation (awake from 11 pm to 3 am). We analyzed the impact of sleep loss on transcription of proinflammatory cytokine genes and used DNA microarray analyses to characterize candidate transcription-control pathways that might mediate the effects of sleep loss on leukocyte gene expression. In the morning after a night of sleep loss, monocyte production of interleukin 6 and tumor necrosis factor alpha was significantly greater compared with morning levels following uninterrupted sleep. In addition, sleep loss induced a more than 3-fold increase in transcription of interleukin 6 messenger RNA and a 2-fold increase in tumor necrosis factor alpha messenger RNA. Bioinformatics analyses suggested that the inflammatory response was mediated by the nuclear factor kappaB inflammatory signaling system as well as through classic hormone and growth factor response pathways. Sleep loss induces a functional alteration of the monocyte proinflammatory cytokine response. A modest amount of sleep loss also alters molecular processes that drive cellular immune activation and induce inflammatory cytokines; mapping the dynamics of sleep loss on molecular signaling pathways has implications for understanding the role of sleep in altering immune cell physiologic characteristics. Interventions that target sleep might constitute new strategies to constrain inflammation with effects on inflammatory disease risk.

Measurement of cellular copper levels in Bacillus megaterium during exponential growth and sporulation.

PubMed

Krueger, W B; Kolodziej, B J

1976-01-01

Both atomic absorption spectrophotometry (AAS) and neutron activation analysis have been utilized to determine cellular Cu levels in Bacillus megaterium ATCC 19213. Both methods were selected for their sensitivity to detection of nanogram quantities of Cu. Data from both methods demonstrated identical patterms of Cu uptake during exponenetial growth and sporulation. Late exponential phase cells contained less Cu than postexponential t2 cells while t5 cells contained amounts equivalent to exponential cells. The t11 phase-bright forespore containing cells had a higher Cu content than those of earlier time periods, and the free spores had the highest Cu content. Analysis of the culture medium by AAS corroborated these data by showing concomitant Cu uptake during exponential growth and into t2 postexponential phase of sporulation. From t2 to t4, Cu egressed from the cells followed by a secondary uptake during the maturation of phase-dark forespores into phase-bright forespores (t6--t9).

Holocene relative sea-level change in Hiroshima Bay, Japan: A semi-quantitative reconstruction based on ostracodes

USGS Publications Warehouse

Yasuhara, Moriaki; Seto, Koji

2006-01-01

Holocene relative sea-level changes in Hiroshima Bay were reconstructed from fossil ostracodes from a core, using a semi-quantitative method. In Hiroshima Bay, relative sea level rose rapidly (about 25 m) between ca. 9000 cal yr BP and ca. 5800 cal yr BP, after which it gradually fell (about 5 m) to its present level. The peak in relative sea level occurred at ca. 5800 cal yr BP. The sea-level curve for Hiroshima Bay is similar to curves for tectonically stable areas of Japan (e.g., Osaka Bay). ?? by the Palaeontological Society of Japan.

Simulation of Changes in Diffusion Related to Different Pathologies at Cellular Level After Traumatic Brain Injury

PubMed Central

Lin, Mu; He, Hongjian; Schifitto, Giovanni; Zhong, Jianhui

2016-01-01

Purpose The goal of the current study was to investigate tissue pathology at the cellular level in traumatic brain injury (TBI) as revealed by Monte Carlo simulation of diffusion tensor imaging (DTI)-derived parameters and elucidate the possible sources of conflicting findings of DTI abnormalities as reported in the TBI literature. Methods A model with three compartments separated by permeable membranes was employed to represent the diffusion environment of water molecules in brain white matter. The dynamic diffusion process was simulated with a Monte Carlo method using adjustable parameters of intra-axonal diffusivity, axon separation, glial cell volume fraction, and myelin sheath permeability. The effects of tissue pathology on DTI parameters were investigated by adjusting the parameters of the model corresponding to different stages of brain injury. Results The results suggest that the model is appropriate and the DTI-derived parameters simulate the predominant cellular pathology after TBI. Our results further indicate that when edema is not prevalent, axial and radial diffusivity have better sensitivity to axonal injury and demyelination than other DTI parameters. Conclusion DTI is a promising biomarker to detect and stage tissue injury after TBI. The observed inconsistencies among previous studies are likely due to scanning at different stages of tissue injury after TBI. PMID:26256558

Genome-wide Linkage Analysis for Identifying Quantitative Trait Loci Involved in the Regulation of Lipoprotein a (Lpa) Levels

PubMed Central

López, Sonia; Buil, Alfonso; Ordoñez, Jordi; Souto, Juan Carlos; Almasy, Laura; Lathrop, Mark; Blangero, John; Blanco-Vaca, Francisco; Fontcuberta, Jordi; Soria, José Manuel

2009-01-01

Lipoprotein Lp(a) levels are highly heritable and are associated with cardiovascular risk. We performed a genome-wide linkage analysis to delineate the genomic regions that influence the concentration of Lp(a) in families from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. Lp(a) levels were measured in 387 individuals belonging to 21 extended Spanish families. A total of 485 DNA microsatellite markers were genotyped to provide a 7.1 cM genetic map. A variance component linkage method was used to evaluate linkage and to detect quantitative trait loci (QTLs). The main QTL that showed strong evidence of linkage with Lp(a) levels was located at the structural gene for apo(a) on Chromosome 6 (LOD score=13.8). Interestingly, another QTL influencing Lp(a) concentration was located on Chromosome 2 with a LOD score of 2.01. This region contains several candidate genes. One of them is the tissue factor pathway inhibitor (TFPI), which has antithrombotic action and also has the ability to bind lipoproteins. However, quantitative trait association analyses performed with 12 SNPs in TFPI gene revealed no association with Lp(a) levels. Our study confirms previous results on the genetic basis of Lp(a) levels. In addition, we report a new QTL on Chromosome 2 involved in the quantitative variation of Lp(a). These data should serve as the basis for further detection of candidate genes and to elucidate the relationship between the concentration of Lp(a) and cardiovascular risk. PMID:18560444

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.

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

NASA Astrophysics Data System (ADS)

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

2016-04-01

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

Mechanical properties of porous and cellular materials

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

Sieradzki, K.; Green, D.J.; Gibson, L.J.

1991-01-01

This symposium successfully brought scientists together from a wide variety of disciplines to focus on the mechanical behavior of porous and cellular solids composed of metals, ceramics, polymers, or biological materials. For cellular materials, papers ranged from processing techniques through microstructure-mechanical property relationships to design. In an overview talk, Mike Ashby (Cambridge Univ.) showed how porous cellular materials can be more efficient than dense materials in designs that require minimum weight. He indicated that many biological materials have been able to accomplish such efficiency but there exists an opportunity to design even more efficient, manmade materials controlling microstructures at differentmore » scale levels. In the area of processing, James Aubert (Sandia National Laboratories) discussed techiques for manipulating polymersolvent phase equilibria to control the microstructure of microcellular foams. Other papers on processing discussed the production of cellular ceramics by CVD, HIPing and sol- gel techniques. Papers on the mechanical behavior of cellular materials considered various ceramics microcellular polymers, conventional polymer foams and apples. There were also contributions that considered optimum design procedures for cellular materials. Steven Cowin (City Univ. of New York) discussed procedures to match the discrete microstructural aspects of cellular materials with the continuum mechanics approach to their elastic behavior.« less

Quantitative nanoparticle tracking: applications to nanomedicine.

PubMed

Huang, Feiran; Dempsey, Christopher; Chona, Daniela; Suh, Junghae

2011-06-01

Particle tracking is an invaluable technique to extract quantitative and qualitative information regarding the transport of nanomaterials through complex biological environments. This technique can be used to probe the dynamic behavior of nanoparticles as they interact with and navigate through intra- and extra-cellular barriers. In this article, we focus on the recent developments in the application of particle-tracking technology to nanomedicine, including the study of synthetic and virus-based materials designed for gene and drug delivery. Specifically, we cover research where mean square displacements of nanomaterial transport were explicitly determined in order to quantitatively assess the transport of nanoparticles through biological environments. Particle-tracking experiments can provide important insights that may help guide the design of more intelligent and effective diagnostic and therapeutic nanoparticles.

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…

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

NASA Astrophysics Data System (ADS)

Czylwik, Andreas; Dekorsy, Armin

2004-12-01

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

Cellular phone use while driving at night.

PubMed

Vivoda, Jonathon M; Eby, David W; St Louis, Renée M; Kostyniuk, Lidia P

2008-03-01

Use of a cellular phone has been shown to negatively affect one's attention to the driving task, leading to an increase in crash risk. At any given daylight hour, about 6% of US drivers are actively talking on a hand-held cell phone. However, previous surveys have focused only on cell phone use during the day. Driving at night has been shown to be a riskier activity than driving during the day. The purpose of the current study was to assess the rate of hand-held cellular phone use while driving at night, using specialized night vision equipment. In 2006, two statewide direct observation survey waves of nighttime cellular phone use were conducted in Indiana utilizing specialized night vision equipment. Combined results of driver hand-held cellular phone use from both waves are presented in this manuscript. The rates of nighttime cell phone use were similar to results found in previous daytime studies. The overall rate of nighttime hand-held cellular phone use was 5.8 +/- 0.6%. Cellular phone use was highest for females and for younger drivers. In fact, the highest rate observed during the study (of 11.9%) was for 16-to 29-year-old females. The high level of cellular phone use found within the young age group, coupled with the increased crash risk associated with cellular phone use, nighttime driving, and for young drivers in general, suggests that this issue may become an important transportation-related concern.

[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

Discrete dynamic modeling of cellular signaling networks.

PubMed

Albert, Réka; Wang, Rui-Sheng

2009-01-01

Understanding signal transduction in cellular systems is a central issue in systems biology. Numerous experiments from different laboratories generate an abundance of individual components and causal interactions mediating environmental and developmental signals. However, for many signal transduction systems there is insufficient information on the overall structure and the molecular mechanisms involved in the signaling network. Moreover, lack of kinetic and temporal information makes it difficult to construct quantitative models of signal transduction pathways. Discrete dynamic modeling, combined with network analysis, provides an effective way to integrate fragmentary knowledge of regulatory interactions into a predictive mathematical model which is able to describe the time evolution of the system without the requirement for kinetic parameters. This chapter introduces the fundamental concepts of discrete dynamic modeling, particularly focusing on Boolean dynamic models. We describe this method step-by-step in the context of cellular signaling networks. Several variants of Boolean dynamic models including threshold Boolean networks and piecewise linear systems are also covered, followed by two examples of successful application of discrete dynamic modeling in cell biology.

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

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...

Increased cellular levels of spermidine or spermine are required for optimal DNA synthesis in lymphocytes activated by concanavalin A.

PubMed Central

Fillingame, R H; Jorstad, C M; Morris, D R

1975-01-01

There are large increases in cellular levels of the polyamines spermidine and spermine in lymphocytes induced to transform by concanavalin A. The anti-leukemic agent methylglyoxal bis(guanylhydrazone) (MGBG) blocks synthesis of these polyamines by inhibiting S-adenosylmethionine decarboxylase. Previous results showed that when cells are activated in the presence of MGBG the synthesis and processing of RNA, as well as protein synthesis, proceed as in the absence of the drug. In contrast, the incorporation of [methyl-3H]thymidine into DNA and the rate of entry of the cells into mitosis are inhibited by 60% in the presence of MGBG. Several experiments suggest that MGBG inhibits cell proliferation by directly blocking polyamine synthesis and not by an unrelated pharmacological effect: (1) the inhibitory action of MGBG is reversed by exogenously added spermidine or spermine; (2) inhibition of DNA synthesis by MGBG shows the same dose-response curve as does inhibition of spermidine and spermine synthesis; and (3) if MGBG is added to cells which have been allowed to accumulate their maximum complement of polyamines, there is no inhibition of thymidine incorporation. MGBG-treated and control cultures initiate DNA synthesis at the same time and show the same percentage of labeled cells by autoradiography. Therefore, it appears that in the absence of increased cellular levels of polyamines, lymphocytes progress normally from G0 through G1 and into S-phase. Furthermore, these experiments suggest that the increased levels of spermidine and spermine generally seen in rapidly proliferating eukaryotic systems are necessary for enhanced rates of DNA replication. PMID:1060087

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.

Geometric confinement influences cellular mechanical properties I -- adhesion area dependence.

PubMed

Su, Judith; Jiang, Xingyu; Welsch, Roy; Whitesides, George M; So, Peter T C

2007-06-01

Interactions between the cell and the extracellular matrix regulate a variety of cellular properties and functions, including cellular rheology. In the present study of cellular adhesion, area was controlled by confining NIH 3T3 fibroblast cells to circular micropatterned islands of defined size. The shear moduli of cells adhering to islands of well defined geometry, as measured by magnetic microrheometry, was found to have a significantly lower variance than those of cells allowed to spread on unpatterned surfaces. We observe that the area of cellular adhesion influences shear modulus. Rheological measurements further indicate that cellular shear modulus is a biphasic function of cellular adhesion area with stiffness decreasing to a minimum value for intermediate areas of adhesion, and then increasing for cells on larger patterns. We propose a simple hypothesis: that the area of adhesion affects cellular rheological properties by regulating the structure of the actin cytoskeleton. To test this hypothesis, we quantified the volume fraction of polymerized actin in the cytosol by staining with fluorescent phalloidin and imaging using quantitative 3D microscopy. The polymerized actin volume fraction exhibited a similar biphasic dependence on adhesion area. Within the limits of our simplifying hypothesis, our experimental results permit an evaluation of the ability of established, micromechanical models to predict the cellular shear modulus based on polymerized actin volume fraction. We investigated the "tensegrity", "cellular-solids", and "biopolymer physics" models that have, respectively, a linear, quadratic, and 5/2 dependence on polymerized actin volume fraction. All three models predict that a biphasic trend in polymerized actin volume fraction as a function of adhesion area will result in a biphasic behavior in shear modulus. Our data favors a higher-order dependence on polymerized actin volume fraction. Increasingly better experimental agreement is observed for

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

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

Evolutionary layering and the limits to cellular perfection

PubMed Central

Lynch, Michael

2012-01-01

Although observations from biochemistry and cell biology seemingly illustrate hundreds of examples of exquisite molecular adaptations, the fact that experimental manipulation can often result in improvements in cellular infrastructure raises the question as to what ultimately limits the level of molecular perfection achievable by natural selection. Here, it is argued that random genetic drift can impose a strong barrier to the advancement of molecular refinements by adaptive processes. Moreover, although substantial improvements in fitness may sometimes be accomplished via the emergence of novel cellular features that improve on previously established mechanisms, such advances are expected to often be transient, with overall fitness eventually returning to the level before incorporation of the genetic novelty. As a consequence of such changes, increased molecular/cellular complexity can arise by Darwinian processes, while yielding no long-term increase in adaptation and imposing increased energetic and mutational costs. PMID:23115338

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

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

Sulforaphane Restores Cellular Glutathione Levels and Reduces Chronic Periodontitis Neutrophil Hyperactivity In Vitro

PubMed Central

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

2013-01-01

The production of high levels of reactive oxygen species by neutrophils is associated with the local and systemic destructive phenotype found in the chronic inflammatory disease periodontitis. In the present study, we investigated the ability of sulforaphane (SFN) to restore cellular glutathione levels and reduce the hyperactivity of circulating neutrophils associated with chronic periodontitis. Using differentiated HL60 cells as a neutrophil model, here we show that generation of extracellular O2 . - by the nicotinamide adenine dinucleotide (NADPH) oxidase complex is increased by intracellular glutathione depletion. This may be attributed to the upregulation of thiol regulated acid sphingomyelinase driven lipid raft formation. Intracellular glutathione was also lower in primary neutrophils from periodontitis patients and, consistent with our previous findings, patients neutrophils were hyper-reactive to stimuli. The activity of nuclear factor erythroid-2-related factor 2 (Nrf2), a master regulator of the antioxidant response, is impaired in circulating neutrophils from chronic periodontitis patients. Although patients’ neutrophils exhibit a low reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio and a higher total Nrf2 level, the DNA-binding activity of nuclear Nrf2 remained unchanged relative to healthy controls and had reduced expression of glutamate cysteine ligase catalytic (GCLC), and modifier (GCLM) subunit mRNAs, compared to periodontally healthy subjects neutrophils. Pre-treatment with SFN increased expression of GCLC and GCM, improved intracellular GSH/GSSG ratios and reduced agonist-activated extracellular O2 . - production in both dHL60 and primary neutrophils from patients with periodontitis and controls. These findings suggest that a deficiency in Nrf2-dependent pathways may underpin susceptibility to hyper-reactivity in circulating primary neutrophils during chronic periodontitis. PMID:23826097

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

PubMed

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

2013-01-01

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

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

The similia principle: results obtained in a cellular model system.

PubMed

Wiegant, Fred; Van Wijk, Roeland

2010-01-01

This paper describes the results of a research program focused on the beneficial effect of low dose stress conditions that were applied according to the similia principle to cells previously disturbed by more severe stress conditions. In first instance, we discuss criteria for research on the similia principle at the cellular level. Then, the homologous ('isopathic') approach is reviewed, in which the initial (high dose) stress used to disturb cellular physiology and the subsequent (low dose) stress are identical. Beneficial effects of low dose stress are described in terms of increased cellular survival capacity and at the molecular level as an increase in the synthesis of heat shock proteins (hsps). Both phenomena reflect a stimulation of the endogenous cellular self-recovery capacity. Low dose stress conditions applied in a homologous approach stimulate the synthesis of hsps and enhance survival in comparison with stressed cells that were incubated in the absence of low dose stress conditions. Thirdly, the specificity of the low dose stress condition is described where the initial (high dose) stress is different in nature from the subsequently applied (low dose) stress; the heterologous or 'heteropathic' approach. The results support the similia principle at the cellular level and add to understanding of how low dose stress conditions influence the regulatory processes underlying self-recovery. In addition, the phenomenon of 'symptom aggravation' which is also observed at the cellular level, is discussed in the context of self-recovery. Finally, the difference in efficiency between the homologous and the heterologous approach is discussed; a perspective is indicated for further research; and the relationship between studies on the similia principle and the recently introduced concept of 'postconditioning hormesis' is emphasized. Copyright 2009 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

Asymmetric cellular memory in bacteria exposed to antibiotics.

PubMed

Mathis, Roland; Ackermann, Martin

2017-03-09

The ability to form a cellular memory and use it for cellular decision-making could help bacteria to cope with recurrent stress conditions. We analyzed whether bacteria would form a cellular memory specifically if past events are predictive of future conditions. We worked with the asymmetrically dividing bacterium Caulobacter crescentus where past events are expected to only be informative for one of the two cells emerging from division, the sessile cell that remains in the same microenvironment and does not migrate. Time-resolved analysis of individual cells revealed that past exposure to low levels of antibiotics increases tolerance to future exposure for the sessile but not for the motile cell. Using computer simulations, we found that such an asymmetry in cellular memory could be an evolutionary response to situations where the two cells emerging from division will experience different future conditions. Our results raise the question whether bacteria can evolve the ability to form and use cellular memory conditionally in situations where it is beneficial.

Theoretical aspects of cellular decision-making and information-processing.

PubMed

Kobayashi, Tetsuya J; Kamimura, Atsushi

2012-01-01

Microscopic biological processes have extraordinary complexity and variety at the sub-cellular, intra-cellular, and multi-cellular levels. In dealing with such complex phenomena, conceptual and theoretical frameworks are crucial, which enable us to understand seemingly different intra- and inter-cellular phenomena from unified viewpoints. Decision-making is one such concept that has attracted much attention recently. Since a number of cellular behavior can be regarded as processes to make specific actions in response to external stimuli, decision-making can cover and has been used to explain a broad range of different cellular phenomena [Balázsi et al. (Cell 144(6):910, 2011), Zeng et al. (Cell 141(4):682, 2010)]. Decision-making is also closely related to cellular information-processing because appropriate decisions cannot be made without exploiting the information that the external stimuli contain. Efficiency of information transduction and processing by intra-cellular networks determines the amount of information obtained, which in turn limits the efficiency of subsequent decision-making. Furthermore, information-processing itself can serve as another concept that is crucial for understanding of other biological processes than decision-making. In this work, we review recent theoretical developments on cellular decision-making and information-processing by focusing on the relation between these two concepts.

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.

2L-PCA: a two-level principal component analyzer for quantitative drug design and its applications.

PubMed

Du, Qi-Shi; Wang, Shu-Qing; Xie, Neng-Zhong; Wang, Qing-Yan; Huang, Ri-Bo; Chou, Kuo-Chen

2017-09-19

A two-level principal component predictor (2L-PCA) was proposed based on the principal component analysis (PCA) approach. It can be used to quantitatively analyze various compounds and peptides about their functions or potentials to become useful drugs. One level is for dealing with the physicochemical properties of drug molecules, while the other level is for dealing with their structural fragments. The predictor has the self-learning and feedback features to automatically improve its accuracy. It is anticipated that 2L-PCA will become a very useful tool for timely providing various useful clues during the process of drug development.

A cellular system for quantitation of vitamin K cycle activity: structure-activity effects on vitamin K antagonism by warfarin metabolites

PubMed Central

Haque, Jamil A.; McDonald, Matthew G.; Kulman, John D.

2014-01-01

Warfarin and other 4-hydroxycoumarins inhibit vitamin K epoxide reductase (VKOR) by depleting reduced vitamin K that is required for posttranslational modification of vitamin K–dependent clotting factors. In vitro prediction of the in vivo potency of vitamin K antagonists is complicated by the complex multicomponent nature of the vitamin K cycle. Here we describe a sensitive assay that enables quantitative analysis of γ-glutamyl carboxylation and its antagonism in live cells. We engineered a human embryonic kidney (HEK) 293–derived cell line (HEK 293-C3) to express a chimeric protein (F9CH) comprising the Gla domain of factor IX fused to the transmembrane and cytoplasmic regions of proline-rich Gla protein 2. Maximal γ-glutamyl carboxylation of F9CH required vitamin K supplementation, and was dose-dependently inhibited by racemic warfarin at a physiologically relevant concentration. Cellular γ-glutamyl carboxylation also exhibited differential VKOR inhibition by warfarin enantiomers (S > R) consistent with their in vivo potencies. We further analyzed the structure-activity relationship for inhibition of γ-glutamyl carboxylation by warfarin metabolites, observing tolerance to phenolic substitution at the C-5 and especially C-6, but not C-7 or C-8, positions on the 4-hydroxycoumarin nucleus. After correction for in vivo concentration and protein binding, 10-hydroxywarfarin and warfarin alcohols were predicted to be the most potent inhibitory metabolites in vivo. PMID:24297869

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

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.

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 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

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

PubMed Central

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

2011-01-01

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

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 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)

Cellular studies and interaction mechanisms of extremely low frequency fields

NASA Astrophysics Data System (ADS)

Liburdy, Robert P.

1995-01-01

) or the magnetic (B) field, or if combinations of static B and time-varying B fields represent an exposure metric for the cell. This question relates directly to understanding fundamental interaction mechanisms and to the development of a rationale for ELF dose threshold guidelines. The weight of experimental evidence indicates that an induced E field according to Faraday's law of induction during magnetic field exposures elicits cellular effects. An E-field-mediated interaction has interesting consequences for microdosimetry at the cellular level and is mechanistically consistent with an interaction at the cell surface, since the E field does not penetrate beyond the cell membrane. Recently, several studies have suggested that an ELF B field by itself or in combination with a static B field may elicit cellular effects. Thus in addition to E-field-mediated effects, other interaction mechanisms as yet not fully understood may operate at the cellular level; this complexity is in contrast to the case for ionizing radiation. In addition to the question of an exposure field metric, the biological state of the target cell is important in ELF interactions. Biological factors such as cell type, cell cycle, cell activation, age of donor animal, passage number of cell line, presence of specific growth/mitogenic factors, temperature, shape, and cell density/packing during exposures have been shown to play a role in mediating ELF interactions with cells. Most recently, reports of single-cell studies usher in a new direction for research that can be termed microbioelectromagnetics. Single-cell digital microscopy introduces a new approach to answer the above questions with potential for real-time microdosimetry and bioeffects limited only by the spatial resolution of state-of-the-art microscopy, which is approximately 0.1 /μm. Digital imaging microscopy should therefore permit the quantitative assessment of spatial and temporal features of ELF field interactions within living

Emergence of HGF/SF-Induced Coordinated Cellular Motility

PubMed Central

Zaritsky, Assaf; Natan, Sari; Ben-Jacob, Eshel; Tsarfaty, Ilan

2012-01-01

Collective cell migration plays a major role in embryonic morphogenesis, tissue remodeling, wound repair and cancer invasion. Despite many decades of extensive investigations, only few analytical tools have been developed to enhance the biological understanding of this important phenomenon. Here we present a novel quantitative approach to analyze long term kinetics of bright field time-lapse wound healing. Fully-automated spatiotemporal measures and visualization of cells' motility and implicit morphology were proven to be sound, repetitive and highly informative compared to single-cell tracking analysis. We study cellular collective migration induced by tyrosine kinase-growth factor signaling (Met-Hepatocyte Growth Factor/Scatter Factor (HGF/SF)). Our quantitative approach is applied to demonstrate that collective migration of the adenocarcinoma cell lines is characterized by simple morpho-kinetics. HGF/SF induces complex morpho-kinetic coordinated collective migration: cells at the front move faster and are more spread than those further away from the wound edge. As the wound heals, distant cells gradually accelerate and enhance spread and elongation –resembling the epithelial to mesenchymal transition (EMT), and then the cells become more spread and maintain higher velocity than cells located closer to the wound. Finally, upon wound closure, front cells halt, shrink and round up (resembling mesenchymal to epithelial transition (MET) phenotype) while distant cells undergo the same process gradually. Met inhibition experiments further validate that Met signaling dramatically alters the morpho-kinetic dynamics of the healing wound. Machine-learning classification was applied to demonstrate the generalization of our findings, revealing even subtle changes in motility patterns induced by Met-inhibition. It is concluded that activation of Met-signaling induces an elaborated model in which cells lead a coordinated increased motility along with gradual differentiation

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

PubMed

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

2015-08-01

Meta-analysis of genetic data must account for differences among studies including study designs, markers genotyped, and covariates. The effects of genetic variants may differ from population to population, i.e., heterogeneity. Thus, meta-analysis of combining data of multiple studies is difficult. Novel statistical methods for meta-analysis are needed. In this article, functional linear models are developed for meta-analyses that connect genetic data to quantitative traits, adjusting for covariates. The models can be used to analyze rare variants, common variants, or a combination of the two. Both likelihood-ratio test (LRT) and F-distributed statistics are introduced to test association between quantitative traits and multiple variants in one genetic region. Extensive simulations are performed to evaluate empirical type I error rates and power performance of the proposed tests. The proposed LRT and F-distributed statistics control the type I error very well and have higher power than the existing methods of the meta-analysis sequence kernel association test (MetaSKAT). We analyze four blood lipid levels in data from a meta-analysis of eight European studies. The proposed methods detect more significant associations than MetaSKAT and the P-values of the proposed LRT and F-distributed statistics are usually much smaller than those of MetaSKAT. The functional linear models and related test statistics can be useful in whole-genome and whole-exome association studies. Copyright © 2015 by the Genetics Society of America.

A novel method for morphological pleomorphism and heterogeneity quantitative measurement: Named cell feature level co-occurrence matrix.

PubMed

Saito, Akira; Numata, Yasushi; Hamada, Takuya; Horisawa, Tomoyoshi; Cosatto, Eric; Graf, Hans-Peter; Kuroda, Masahiko; Yamamoto, Yoichiro

2016-01-01

Recent developments in molecular pathology and genetic/epigenetic analysis of cancer tissue have resulted in a marked increase in objective and measurable data. In comparison, the traditional morphological analysis approach to pathology diagnosis, which can connect these molecular data and clinical diagnosis, is still mostly subjective. Even though the advent and popularization of digital pathology has provided a boost to computer-aided diagnosis, some important pathological concepts still remain largely non-quantitative and their associated data measurements depend on the pathologist's sense and experience. Such features include pleomorphism and heterogeneity. In this paper, we propose a method for the objective measurement of pleomorphism and heterogeneity, using the cell-level co-occurrence matrix. Our method is based on the widely used Gray-level co-occurrence matrix (GLCM), where relations between neighboring pixel intensity levels are captured into a co-occurrence matrix, followed by the application of analysis functions such as Haralick features. In the pathological tissue image, through image processing techniques, each nucleus can be measured and each nucleus has its own measureable features like nucleus size, roundness, contour length, intra-nucleus texture data (GLCM is one of the methods). In GLCM each nucleus in the tissue image corresponds to one pixel. In this approach the most important point is how to define the neighborhood of each nucleus. We define three types of neighborhoods of a nucleus, then create the co-occurrence matrix and apply Haralick feature functions. In each image pleomorphism and heterogeneity are then determined quantitatively. For our method, one pixel corresponds to one nucleus feature, and we therefore named our method Cell Feature Level Co-occurrence Matrix (CFLCM). We tested this method for several nucleus features. CFLCM is showed as a useful quantitative method for pleomorphism and heterogeneity on histopathological image

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.

Fabrication of cellular materials

NASA Astrophysics Data System (ADS)

Prud'homme, Robert K.; Aksay, Ilhan A.; Garg, Rajeev

1996-02-01

Nature uses cellular materials in applications requiring strength while, simultaneously, minimizing raw materials requirements. Minimizing raw materials is efficient both in terms of the energy expended by the organism to synthesize the structure and in terms of the strength- to-weight ratio of the structure. Wood is the most obvious example of cellular bio-materials, and it is the focus of other presentations in this symposium. The lightweight bone structure of birds is another excellent example where weight is a key criterion. The anchoring foot of the common muscle [Mytilus edulis] whereby it attaches itself to objects is a further example of a biological system that uses a foam to fill space and yet conserve on raw materials. In the case of the muscle the foam is water filled and the foot structure distributes stress over a larger area so that the strength of the byssal thread from which it is suspended is matched to the strength of interfacial attachment of the foot to a substrate. In these examples the synthesis and fabrication of the cellular material is directed by intercellular, genetically coded, biochemical reactions. The resulting cell sizes are microns in scale. Cellular materials at the next larger scale are created by organisms at the next higher level of integration. For example an African tree frog lays her eggs in a gas/fluid foam sack she builds on a branch overhanging a pond. The outside of the foam sack hardens in the sun and prevents water evaporation. The foam structure minimizes the amount of fluid that needs to be incorporated into the sack and minimizes its weight. However, as far as the developing eggs are concerned, they are in an aqueous medium, i.e. the continuous fluid phase of the foam. After precisely six days the eggs hatch, and the solidified outer wall re-liquefies and dumps the emerging tadpoles into the pond below. The bee honeycomb is an example of a cellular material with exquisite periodicity at millimeter length scales. The

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.

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.

Respiratory syncytial virus nonstructural proteins decrease levels of multiple members of the cellular interferon pathways.

PubMed

Swedan, Samer; Musiyenko, Alla; Barik, Sailen

2009-10-01

Viruses of the Paramyxoviridae family, such as the respiratory syncytial virus (RSV), suppress cellular innate immunity represented by type I interferon (IFN) for optimal growth in their hosts. The two unique nonstructural (NS) proteins, NS1 and NS2, of RSV suppress IFN synthesis, as well as IFN function, but their exact targets are still uncharacterized. Here, we investigate if either or both of the NS proteins affect the steady-state levels of key members of the IFN pathway. We found that both NS1 and NS2 decreased the levels of TRAF3, a strategic integrator of multiple IFN-inducing signals, although NS1 was more efficient. Only NS1 reduced IKKepsilon, a key protein kinase that specifically phosphorylates and activates IFN regulatory factor 3. Loss of the TRAF3 and IKKepsilon proteins appeared to involve a nonproteasomal mechanism. Interestingly, NS2 modestly increased IKKepsilon levels. In the IFN response pathway, NS2 decreased the levels of STAT2, the essential transcription factor for IFN-inducible antiviral genes. Preliminary mapping revealed that the C-terminal 10 residues of NS1 were essential for reducing IKKepsilon levels and the C-terminal 10 residues of NS2 were essential for increasing and reducing IKKepsilon and STAT2, respectively. In contrast, deletion of up to 20 residues of the C termini of NS1 and NS2 did not diminish their TRAF3-reducing activity. Coimmunoprecipitation studies revealed that NS1 and NS2 form a heterodimer. Clearly, the NS proteins of RSV, working individually and together, regulate key signaling molecules of both the IFN activation and response pathways.

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

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.

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.

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

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

Gonda, Kohsuke, E-mail: gonda@med.tohoku.ac.jp; Miyashita, Minoru; Watanabe, Mika

2012-09-28

Highlights: Black-Right-Pointing-Pointer Organic fluorescent material-assembled nanoparticles for IHC were prepared. Black-Right-Pointing-Pointer New nanoparticle fluorescent intensity was 10.2-fold greater than Qdot655. Black-Right-Pointing-Pointer Nanoparticle staining analyzed a wide range of ER expression levels in tissue. Black-Right-Pointing-Pointer Nanoparticle staining enhanced the quantitative sensitivity for ER diagnosis. -- Abstract: The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3 Prime -diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature andmore » substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues

How cells explore shape space: a quantitative statistical perspective of cellular morphogenesis.

PubMed

Yin, Zheng; Sailem, Heba; Sero, Julia; Ardy, Rico; Wong, Stephen T C; Bakal, Chris

2014-12-01

Through statistical analysis of datasets describing single cell shape following systematic gene depletion, we have found that the morphological landscapes explored by cells are composed of a small number of attractor states. We propose that the topology of these landscapes is in large part determined by cell-intrinsic factors, such as biophysical constraints on cytoskeletal organization, and reflects different stable signaling and/or transcriptional states. Cell-extrinsic factors act to determine how cells explore these landscapes, and the topology of the landscapes themselves. Informational stimuli primarily drive transitions between stable states by engaging signaling networks, while mechanical stimuli tune, or even radically alter, the topology of these landscapes. As environments fluctuate, the topology of morphological landscapes explored by cells dynamically adapts to these fluctuations. Finally we hypothesize how complex cellular and tissue morphologies can be generated from a limited number of simple cell shapes. © 2014 WILEY Periodicals, Inc.

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.

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.

Atherogenesis and iron: from epidemiology to cellular level

PubMed Central

Vinchi, Francesca; Muckenthaler, Martina U.; Da Silva, Milene C.; Balla, György; Balla, József; Jeney, Viktória

2014-01-01

Iron accumulates in human atherosclerotic lesions but whether it is a cause or simply a downstream consequence of the atheroma formation has been an open question for decades. According to the so called “iron hypothesis,” iron is believed to be detrimental for the cardiovascular system, thus promoting atherosclerosis development and progression. Iron, in its catalytically active form, can participate in the generation of reactive oxygen species and induce lipid-peroxidation, triggering endothelial activation, smooth muscle cell proliferation and macrophage activation; all of these processes are considered to be proatherogenic. On the other hand, the observation that hemochromatotic patients, affected by life-long iron overload, do not show any increased incidence of atherosclerosis is perceived as the most convincing evidence against the “iron hypothesis.” Epidemiological studies and data from animal models provided conflicting evidences about the role of iron in atherogenesis. Therefore, more careful studies are needed in which issues like the source and the compartmentalization of iron will be addressed. This review article summarizes what we have learnt about iron and atherosclerosis from epidemiological studies, animal models and cellular systems and highlights the rather contributory than innocent role of iron in atherogenesis. PMID:24847266

Haloacetic Acid Water Disinfection Byproducts Affect Pyruvate Dehydrogenase Activity and Disrupt Cellular Metabolism.

PubMed

Dad, Azra; Jeong, Clara H; Wagner, Elizabeth D; Plewa, Michael J

2018-02-06

The disinfection of drinking water has been a major public health achievement. However, haloacetic acids (HAAs), generated as byproducts of water disinfection, are cytotoxic, genotoxic, mutagenic, carcinogenic, and teratogenic. Previous studies of monoHAA-induced genotoxicity and cell stress demonstrated that the toxicity was due to inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), leading to disruption of cellular metabolism and energy homeostasis. DiHAAs and triHAAs are also produced during water disinfection, and whether they share mechanisms of action with monoHAAs is unknown. In this study, we evaluated the effects of mono-, di-, and tri-HAAs on cellular GAPDH enzyme kinetics, cellular ATP levels, and pyruvate dehydrogenase complex (PDC) activity. Here, treatments conducted in Chinese hamster ovary (CHO) cells revealed differences among mono-, di-, and triHAAs in their molecular targets. The monoHAAs, iodoacetic acid and bromoacetic acid, were the strongest inhibitors of GAPDH and greatly reduced cellular ATP levels. Chloroacetic acid, diHAAs, and triHAAs were weaker inhibitors of GAPDH and some increased the levels of cellular ATP. HAAs also affected PDC activity, with most HAAs activating PDC. The primary finding of this work is that mono- versus multi-HAAs address different molecular targets, and the results are generally consistent with a model in which monoHAAs activate the PDC through GAPDH inhibition-mediated disruption in cellular metabolites, including altering ATP-to-ADP and NADH-to-NAD ratios. The monoHAA-mediated reduction in cellular metabolites results in accelerated PDC activity by way of metabolite-ratio-dependent PDC regulation. DiHAAs and triHAAs are weaker inhibitors of GAPDH, but many also increase cellular ATP levels, and we suggest that they increase PDC activity by inhibiting pyruvate dehydrogenase kinase.

Agent-Based Modeling of Mitochondria Links Sub-Cellular Dynamics to Cellular Homeostasis and Heterogeneity.

PubMed

Dalmasso, Giovanni; Marin Zapata, Paula Andrea; Brady, Nathan Ryan; Hamacher-Brady, Anne

2017-01-01

Mitochondria are semi-autonomous organelles that supply energy for cellular biochemistry through oxidative phosphorylation. Within a cell, hundreds of mobile mitochondria undergo fusion and fission events to form a dynamic network. These morphological and mobility dynamics are essential for maintaining mitochondrial functional homeostasis, and alterations both impact and reflect cellular stress states. Mitochondrial homeostasis is further dependent on production (biogenesis) and the removal of damaged mitochondria by selective autophagy (mitophagy). While mitochondrial function, dynamics, biogenesis and mitophagy are highly-integrated processes, it is not fully understood how systemic control in the cell is established to maintain homeostasis, or respond to bioenergetic demands. Here we used agent-based modeling (ABM) to integrate molecular and imaging knowledge sets, and simulate population dynamics of mitochondria and their response to environmental energy demand. Using high-dimensional parameter searches we integrated experimentally-measured rates of mitochondrial biogenesis and mitophagy, and using sensitivity analysis we identified parameter influences on population homeostasis. By studying the dynamics of cellular subpopulations with distinct mitochondrial masses, our approach uncovered system properties of mitochondrial populations: (1) mitochondrial fusion and fission activities rapidly establish mitochondrial sub-population homeostasis, and total cellular levels of mitochondria alter fusion and fission activities and subpopulation distributions; (2) restricting the directionality of mitochondrial mobility does not alter morphology subpopulation distributions, but increases network transmission dynamics; and (3) maintaining mitochondrial mass homeostasis and responding to bioenergetic stress requires the integration of mitochondrial dynamics with the cellular bioenergetic state. Finally, (4) our model suggests sources of, and stress conditions amplifying

Agent-Based Modeling of Mitochondria Links Sub-Cellular Dynamics to Cellular Homeostasis and Heterogeneity

PubMed Central

Dalmasso, Giovanni; Marin Zapata, Paula Andrea; Brady, Nathan Ryan; Hamacher-Brady, Anne

2017-01-01

Mitochondria are semi-autonomous organelles that supply energy for cellular biochemistry through oxidative phosphorylation. Within a cell, hundreds of mobile mitochondria undergo fusion and fission events to form a dynamic network. These morphological and mobility dynamics are essential for maintaining mitochondrial functional homeostasis, and alterations both impact and reflect cellular stress states. Mitochondrial homeostasis is further dependent on production (biogenesis) and the removal of damaged mitochondria by selective autophagy (mitophagy). While mitochondrial function, dynamics, biogenesis and mitophagy are highly-integrated processes, it is not fully understood how systemic control in the cell is established to maintain homeostasis, or respond to bioenergetic demands. Here we used agent-based modeling (ABM) to integrate molecular and imaging knowledge sets, and simulate population dynamics of mitochondria and their response to environmental energy demand. Using high-dimensional parameter searches we integrated experimentally-measured rates of mitochondrial biogenesis and mitophagy, and using sensitivity analysis we identified parameter influences on population homeostasis. By studying the dynamics of cellular subpopulations with distinct mitochondrial masses, our approach uncovered system properties of mitochondrial populations: (1) mitochondrial fusion and fission activities rapidly establish mitochondrial sub-population homeostasis, and total cellular levels of mitochondria alter fusion and fission activities and subpopulation distributions; (2) restricting the directionality of mitochondrial mobility does not alter morphology subpopulation distributions, but increases network transmission dynamics; and (3) maintaining mitochondrial mass homeostasis and responding to bioenergetic stress requires the integration of mitochondrial dynamics with the cellular bioenergetic state. Finally, (4) our model suggests sources of, and stress conditions amplifying

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

ERIC Educational Resources Information Center

Dahmani, Hassen-Reda; Schneeberger, Patricia; Kramer, IJsbrand M.

2009-01-01

The number of experimentally derived structures of cellular components is rapidly expanding, and this phenomenon is accompanied by the development of a new semiotic system for teaching. The infographic approach is shifting from a schematic toward a more realistic representation of cellular components. By realistic we mean artist-prepared or…

Coordination of Cellular Dynamics Contributes to Tooth Epithelium Deformations

PubMed Central

Morita, Ritsuko; Kihira, Miho; Nakatsu, Yousuke; Nomoto, Yohei; Ogawa, Miho; Ohashi, Kazumasa; Mizuno, Kensaku; Tachikawa, Tetsuhiko; Ishimoto, Yukitaka; Morishita, Yoshihiro; Tsuji, Takashi

2016-01-01

The morphologies of ectodermal organs are shaped by appropriate combinations of several deformation modes, such as invagination and anisotropic tissue elongation. However, how multicellular dynamics are coordinated during deformation processes remains to be elucidated. Here, we developed a four-dimensional (4D) analysis system for tracking cell movement and division at a single-cell resolution in developing tooth epithelium. The expression patterns of a Fucci probe clarified the region- and stage-specific cell cycle patterns within the tooth germ, which were in good agreement with the pattern of the volume growth rate estimated from tissue-level deformation analysis. Cellular motility was higher in the regions with higher growth rates, while the mitotic orientation was significantly biased along the direction of tissue elongation in the epithelium. Further, these spatio-temporal patterns of cellular dynamics and tissue-level deformation were highly correlated with that of the activity of cofilin, which is an actin depolymerization factor, suggesting that the coordination of cellular dynamics via actin remodeling plays an important role in tooth epithelial morphogenesis. Our system enhances the understanding of how cellular behaviors are coordinated during ectodermal organogenesis, which cannot be observed from histological analyses. PMID:27588418

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.

At a glance: cellular biology for engineers.

PubMed

Khoshmanesh, K; Kouzani, A Z; Nahavandi, S; Baratchi, S; Kanwar, J R

2008-10-01

Engineering contributions have played an important role in the rise and evolution of cellular biology. Engineering technologies have helped biologists to explore the living organisms at cellular and molecular levels, and have created new opportunities to tackle the unsolved biological problems. There is now a growing demand to further expand the role of engineering in cellular biology research. For an engineer to play an effective role in cellular biology, the first essential step is to understand the cells and their components. However, the stumbling block of this step is to comprehend the information given in the cellular biology literature because it best suits the readers with a biological background. This paper aims to overcome this bottleneck by describing the human cell components as micro-plants that form cells as micro-bio-factories. This concept can accelerate the engineers' comprehension of the subject. In this paper, first the structure and function of different cell components are described. In addition, the engineering attempts to mimic various cell components through numerical modelling or physical implementation are highlighted. Next, the interaction of different cell components that facilitate complicated chemical processes, such as energy generation and protein synthesis, are described. These complex interactions are translated into simple flow diagrams, generally used by engineers to represent multi-component processes.

[The experimental evaluation with flow cytofluorimetry technique of the level of cellular immunologic memory in persons vaccinated against plague and anthrax].

PubMed

Bogacheva, N V; Kriuchkov, A V; Darmov, I V; Vorob'ev, K A; Pechenkin, D V; Elagin, G D; Kolesnikiov, D P

2013-11-01

The article deals with experimental evaluation with flow cytofluorimetry technique of the level of cellular immunologic memory in persons vaccinated with plague and anthrax live dry vaccines. It is established that the introduction of plague and anthrax live dry vaccines into organism of vaccinated persons ignites immunologic rearrangement manifested by reliable increase of level of blood concentration of Th1-lymphocytes (immunologic memory cells) against the background of vaccination. The higher correlation coefficient is detected between leucocytes lysis coefficient and stimulation coefficient according blood concentration level of T-lymphocytes predominantly at the expense of Th1-lymphocytes. The values of stimulation coefficient were calculated for corresponding blood cells of vaccinated persons. This data testifies the effectiveness of application of vaccination against plague and anthrax.

Defining the action spectrum of potential PGC-1α activators on a mitochondrial and cellular level in vivo.

PubMed

Hofer, Annette; Noe, Natalie; Tischner, Christin; Kladt, Nikolay; Lellek, Veronika; Schauß, Astrid; Wenz, Tina

2014-05-01

Previous studies have demonstrated a therapeutic benefit of pharmaceutical PGC-1α activation in cellular and murine model of disorders linked to mitochondrial dysfunction. While in some cases, this effect seems to be clearly associated with boosting of mitochondrial function, additional alterations as well as tissue- and cell-type-specific effects might play an important role. We initiated a comprehensive analysis of the effects of potential PGC-1α-activating drugs and pharmaceutically targeted the PPAR (bezafibrate, rosiglitazone), AMPK (AICAR, metformin) and Sirt1 (resveratrol) pathways in HeLa cells, neuronal cells and PGC-1α-deficient MEFs to get insight into cell type specificity and PGC-1α dependence of their working action. We used bezafibrate as a model drug to assess the effect on a tissue-specific level in a murine model. Not all analyzed drugs activate the PGC pathway or alter mitochondrial protein levels. However, they all affect supramolecular assembly of OXPHOS complexes and OXPHOS protein stability. In addition, a clear drug- and cell-type-specific influence on several cellular stress pathways as well as on post-translational modifications could be demonstrated, which might be relevant to fully understand the action of the analyzed drugs in the disease state. Importantly, the effect on the activation of mitochondrial biogenesis and stress response program upon drug treatment is PGC-1α dependent in MEFs demonstrating not only the pleiotropic effects of this molecule but points also to the working mechanism of the analyzed drugs. The definition of the action spectrum of the different drugs forms the basis for a defect-specific compensation strategy and a future personalized therapeutic approach.

Ultratrace level determination and quantitative analysis of kidney injury biomarkers in patient samples attained by zinc oxide nanorods

NASA Astrophysics Data System (ADS)

Singh, Manpreet; Alabanza, Anginelle; Gonzalez, Lorelis E.; Wang, Weiwei; Reeves, W. Brian; Hahm, Jong-In

2016-02-01

Determining ultratrace amounts of protein biomarkers in patient samples in a straightforward and quantitative manner is extremely important for early disease diagnosis and treatment. Here, we successfully demonstrate the novel use of zinc oxide nanorods (ZnO NRs) in the ultrasensitive and quantitative detection of two acute kidney injury (AKI)-related protein biomarkers, tumor necrosis factor (TNF)-α and interleukin (IL)-8, directly from patient samples. We first validate the ZnO NRs-based IL-8 results via comparison with those obtained from using a conventional enzyme-linked immunosorbent method in samples from 38 individuals. We further assess the full detection capability of the ZnO NRs-based technique by quantifying TNF-α, whose levels in human urine are often below the detection limits of conventional methods. Using the ZnO NR platforms, we determine the TNF-α concentrations of all 46 patient samples tested, down to the fg per mL level. Subsequently, we screen for TNF-α levels in approximately 50 additional samples collected from different patient groups in order to demonstrate a potential use of the ZnO NRs-based assay in assessing cytokine levels useful for further clinical monitoring. Our research efforts demonstrate that ZnO NRs can be straightforwardly employed in the rapid, ultrasensitive, quantitative, and simultaneous detection of multiple AKI-related biomarkers directly in patient urine samples, providing an unparalleled detection capability beyond those of conventional analysis methods. Additional key advantages of the ZnO NRs-based approach include a fast detection speed, low-volume assay condition, multiplexing ability, and easy automation/integration capability to existing fluorescence instrumentation. Therefore, we anticipate that our ZnO NRs-based detection method will be highly beneficial for overcoming the frequent challenges in early biomarker development and treatment assessment, pertaining to the facile and ultrasensitive quantification

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.

Quantitation of Phenol Levels in Oil of Wintergreen Using Gas Chromatography-Mass Spectrometry with Selected Ion Monitoring

ERIC Educational Resources Information Center

Sobel, Robert M.; Ballantine, David S.; Ryzhov, Victor

2005-01-01

Industrial application of gas chromatography-mass spectrometry (GC-MS) analysis is a powerful technique that could be used to elucidate components of a complex mixture while offering the benefits of high-precision quantitative analysis. The natural wintergreen oil is examined for its phenol concentration to determine the level of refining…

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.

Altered cellular magnesium responsiveness to hyperglycemia in hypertensive subjects.

PubMed

Barbagallo, M; Dominguez, L J; Bardicef, O; Resnick, L M

2001-09-01

Previous studies by our group have identified ionic aspects of insulin resistance in hypertension, in which cellular responses to insulin were influenced by the basal intracellular ionic environment-the lower the cytosolic free magnesium (Mg(i)), the less Mg(i) increased following insulin stimulation. To investigate whether this ionic insulin resistance represents a more general abnormality of cellular responsiveness in hypertension, we studied Mg(i) responses to nonhormonal signals such as hyperglycemia (15 mmol/L) and used (31)P-nuclear magnetic resonance (NMR) spectroscopy to measure Mg(i) in erythrocytes from normal (NL, n=14) and hypertensive (HTN, n=12) subjects before and 30, 60, 120, and 180 minutes after in vitro glucose incubations. Basal Mg(i) levels were significantly lower in HTN subjects than in NL subjects (169+/-10 versus 205+/-8 micromol.L(-1), P<0.01). In NL cells, hyperglycemia significantly lowered Mg(i), from 205+/-8 micromol.L(-1) (basal, T=0) to 181+/-8, 162+/-6, 152+/-7, and 175+/-9 micromol.L(-1) (T=30, 60, 120, and 180, respectively; P<0.005 versus T=0 at all times). In HTN cells, maximal Mg(i) responses to hyperglycemia were blunted, from 169+/-10 micromol.L(-1) (basal, T=0) to 170+/-11, 179+/-12, 181+/-14, and 173+/-15 micromol.L(-1) (T=30, 60, 120, and 180, respectively; P=NS versus T=0 at all times). For all subjects, Mg(i) responses to hyperglycemia were closely related to basal Mg(i) levels: the higher the Mg(i), the greater the response (n=26, r=0.620, P<0.001). Thus, (1) erythrocytes from hypertensive vis-à-vis normotensive subjects are resistant to the ionic effects of extracellular hyperglycemia on Mg(i) levels, and (2) cellular ionic responses to glucose depend on the basal Mg(i) environment. Altogether, these data support a role for altered extracellular glucose levels in regulating cellular magnesium metabolism and also suggest the importance of ionic factors in determining cellular responsiveness to nonhormonal as well as

Platinum nanozymes recover cellular ROS homeostasis in an oxidative stress-mediated disease model

NASA Astrophysics Data System (ADS)

Moglianetti, Mauro; de Luca, Elisa; Pedone, Deborah; Marotta, Roberto; Catelani, Tiziano; Sartori, Barbara; Amenitsch, Heinz; Retta, Saverio Francesco; Pompa, Pier Paolo

2016-02-01

In recent years, the use of nanomaterials as biomimetic enzymes has attracted great interest. In this work, we show the potential of biocompatible platinum nanoparticles (Pt NPs) as antioxidant nanozymes, which combine abundant cellular internalization and efficient scavenging activity of cellular reactive oxygen species (ROS), thus simultaneously integrating the functions of nanocarriers and antioxidant drugs. Careful toxicity assessment and intracellular tracking of Pt NPs proved their cytocompatibility and high cellular uptake, with compartmentalization within the endo/lysosomal vesicles. We have demonstrated that Pt NPs possess strong and broad antioxidant properties, acting as superoxide dismutase, catalase, and peroxidase enzymes, with similar or even superior performance than natural enzymes, along with higher adaptability to the changes in environmental conditions. We then exploited their potent activity as radical scavenging materials in a cellular model of an oxidative stress-related disorder, namely human Cerebral Cavernous Malformation (CCM) disease, which is associated with a significant increase in intracellular ROS levels. Noteworthily, we found that Pt nanozymes can efficiently reduce ROS levels, completely restoring the cellular physiological homeostasis.In recent years, the use of nanomaterials as biomimetic enzymes has attracted great interest. In this work, we show the potential of biocompatible platinum nanoparticles (Pt NPs) as antioxidant nanozymes, which combine abundant cellular internalization and efficient scavenging activity of cellular reactive oxygen species (ROS), thus simultaneously integrating the functions of nanocarriers and antioxidant drugs. Careful toxicity assessment and intracellular tracking of Pt NPs proved their cytocompatibility and high cellular uptake, with compartmentalization within the endo/lysosomal vesicles. We have demonstrated that Pt NPs possess strong and broad antioxidant properties, acting as superoxide

Quercetin affects glutathione levels and redox ratio in human aortic endothelial cells not through oxidation but formation and cellular export of quercetin-glutathione conjugates and upregulation of glutamate-cysteine ligase.

PubMed

Li, Chuan; Zhang, Wei-Jian; Choi, Jaewoo; Frei, Balz

2016-10-01

Endothelial dysfunction due to vascular inflammation and oxidative stress critically contributes to the etiology of atherosclerosis. The intracellular redox environment plays a key role in regulating endothelial cell function and is intimately linked to cellular thiol status, including and foremost glutathione (GSH). In the present study we investigated whether and how the dietary flavonoid, quercetin, affects GSH status of human aortic endothelial cells (HAEC) and their response to oxidative stress. We found that treating cells with buthionine sulfoximine to deplete cellular GSH levels significantly reduced the capacity of quercetin to inhibit lipopolysaccharide (LPS)-induced oxidant production. Furthermore, incubation of HAEC with quercetin caused a transient decrease and then full recovery of cellular GSH concentrations. The initial decline in GSH was not accompanied by a corresponding increase in glutathione disulfide (GSSG). To the contrary, GSSG levels, which were less than 0.5% of GSH levels at baseline (0.26±0.01 vs. 64.7±1.9nmol/mg protein, respectively), decreased by about 25% during incubation with quercetin. As a result, the GSH: GSSG ratio increased by about 70%, from 253±7 to 372±23. These quercetin-induced changes in GSH and GSSG levels were not affected by treating HAEC with 500µM ascorbic acid phosphate for 24h to increase intracellular ascorbate levels. Incubation of HAEC with quercetin also led to the appearance of extracellular quercetin-glutathione conjugates, which was paralleled by upregulation of the multidrug resistance protein 1 (MRP1). Furthermore, quercetin slightly but significantly increased mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic and modifier subunits. Taken together, our results suggest that quercetin causes loss of GSH in HAEC, not because of oxidation but due to formation and cellular export of quercetin-glutathione conjugates. Induction by quercetin of GCL subsequently restores GSH levels, thereby

Cerebral Metabolic Rate of Oxygen (CMRO2 ) Mapping by Combining Quantitative Susceptibility Mapping (QSM) and Quantitative Blood Oxygenation Level-Dependent Imaging (qBOLD).

PubMed

Cho, Junghun; Kee, Youngwook; Spincemaille, Pascal; Nguyen, Thanh D; Zhang, Jingwei; Gupta, Ajay; Zhang, Shun; Wang, Yi

2018-03-07

To map the cerebral metabolic rate of oxygen (CMRO 2 ) by estimating the oxygen extraction fraction (OEF) from gradient echo imaging (GRE) using phase and magnitude of the GRE data. 3D multi-echo gradient echo imaging and perfusion imaging with arterial spin labeling were performed in 11 healthy subjects. CMRO 2 and OEF maps were reconstructed by joint quantitative susceptibility mapping (QSM) to process GRE phases and quantitative blood oxygen level-dependent (qBOLD) modeling to process GRE magnitudes. Comparisons with QSM and qBOLD alone were performed using ROI analysis, paired t-tests, and Bland-Altman plot. The average CMRO 2 value in cortical gray matter across subjects were 140.4 ± 14.9, 134.1 ± 12.5, and 184.6 ± 17.9 μmol/100 g/min, with corresponding OEFs of 30.9 ± 3.4%, 30.0 ± 1.8%, and 40.9 ± 2.4% for methods based on QSM, qBOLD, and QSM+qBOLD, respectively. QSM+qBOLD provided the highest CMRO 2 contrast between gray and white matter, more uniform OEF than QSM, and less noisy OEF than qBOLD. Quantitative CMRO 2 mapping that fits the entire complex GRE data is feasible by combining QSM analysis of phase and qBOLD analysis of magnitude. © 2018 International Society for Magnetic Resonance in Medicine.

The thorny path linking cellular senescence to organismalaging

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

Patil, Christopher K.; Mian, Saira; Campisi, Judith

2005-08-09

Half a century is fast approaching since Hayflick and colleagues formally described the limited ability of normal human cells to proliferate in culture (Hayflick and Moorhead, 1961). This finding--that normal somatic cells, in contrast to cancer cells, cannot divide indefinitely--challenged the prevailing idea that cells from mortal multicellular organisms were intrinsically ''immortal'' (Carrell, 1912). It also spawned two hypotheses, essential elements of which persist today. The first held that the restricted proliferation of normal cells, now termed cellular senescence, suppresses cancer (Hayflick, 1965; Sager, 1991; Campisi, 2001). The second hypothesis, as explained in the article by Lorenzini et al., suggestedmore » that the limited proliferation of cells in culture recapitulated aspects of organismal aging (Hayflick, 1965; Martin, 1993). How well have these hypotheses weathered the ensuing decades? Before answering this question, we first consider current insights into the causes and consequences of cellular senescence. Like Lorenzini et al., we limit our discussion to mammals. We also focus on fibroblasts, the cell type studied by Lorenzini et al., but consider other types as well. We suggest that replicative capacity in culture is not a straightforward assessment, and that it correlates poorly with both longevity and body mass. We speculate this is due to the malleable and variable nature of replicative capacity, which renders it an indirect metric of qualitative and quantitative differences among cells to undergo senescence, a response that directly alters cellular phenotype and might indirectly alter tissue structure and function.« less

The cellular immunity and oxidative stress markers in early pregnancy loss.

PubMed

Daglar, Korkut; Biberoglu, Ebru; Kirbas, Ayse; Dirican, Aylin Onder; Genc, Metin; Avci, Aslihan; Biberoglu, Kutay

2016-01-01

We investigated whether changes in cellular immunity and oxidative stress in pregnancy have any association with spontaneous miscarriage. Circulating adenosine deaminase (ADA) activity as a marker of cellular immunity and malondialdehyde (MDA) and catalase (CAT), glutathione peroxidase (GPx) as markers of T lymphocyte activation and parameters of oxidative stress and antioxidant defense were compared between 40 women with early pregnancy loss and another 40 women with ungoing healthy pregnancy. Women with miscarriage had higher serum ADA and GPx levels when compared with women with normal pregnancy (p = 0.034 and p < 0.001, respectively). Although serum MDA level was slightly higher in women with miscarriage, the difference was not significant (p = 0.083). CAT levels were alike in both groups. We have demonstrated an increased cellular immunity and perhaps a compensated oxidative stress related to increased antioxidant activation in women with early spontaneous pregnancy loss.

Cellular senescence in the Penna model of aging

NASA Astrophysics Data System (ADS)

Periwal, Avikar

2013-11-01

Cellular senescence is thought to play a major role in age-related diseases, which cause nearly 67% of all human deaths worldwide. Recent research in mice showed that exercising mice had higher levels of telomerase, an enzyme that helps maintain telomere length, than nonexercising mice. A commonly used model for biological aging was proposed by Penna. I propose a modification of the Penna model that incorporates cellular senescence and find an analytical steady-state solution following Coe, Mao, and Cates [Phys. Rev. Lett.PRLTAO0031-900710.1103/PhysRevLett.89.288103 89, 288103 (2002)]. I find that models corresponding to delayed cellular senescence have younger populations that live longer. I fit the model to the United Kingdom's death distribution, which the original Penna model cannot do.

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

Quantitative fluorescence nanoscopy for cancer biomedicine

NASA Astrophysics Data System (ADS)

Huang, Tao; Nickerson, Andrew; Peters, Alec; Nan, Xiaolin

2015-08-01

Cancer is a major health threat worldwide. Options for targeted cancer therapy, however, are often limited, in a large part due to our incomplete understanding of how key processes including oncogenesis and drug response are mediated at the molecular level. New imaging techniques for visualizing biomolecules and their interactions at the nanometer and single molecule scales, collectively named fluorescence nanoscopy, hold the promise to transform biomedical research by providing direct mechanistic insight into cellular processes. We discuss the principles of quantitative single-molecule localization microscopy (SMLM), a subset of fluorescence nanoscopy, and their applications to cancer biomedicine. In particular, we will examine oncogenesis and drug resistance mediated by mutant Ras, which is associated with ~1/3 of all human cancers but has remained an intractable drug target. At ~20 nm spatial and single-molecule stoichiometric resolutions, SMLM clearly showed that mutant Ras must form dimers to activate its effector pathways and drive oncogenesis. SMLM further showed that the Raf kinase, one of the most important effectors of Ras, also forms dimers upon activation by Ras. Moreover, treatment of cells expressing wild type Raf with Raf inhibitors induces Raf dimer formation in a manner dependent on Ras dimerization. Together, these data suggest that Ras dimers mediate oncogenesis and drug resistance in tumors with hyperactive Ras and can potentially be targeted for cancer therapy. We also discuss recent advances in SMLM that enable simultaneous imaging of multiple biomolecules and their interactions at the nanoscale. Our work demonstrates the power of quantitative SMLM in cancer biomedicine.

Point process models for localization and interdependence of punctate cellular structures.

PubMed

Li, Ying; Majarian, Timothy D; Naik, Armaghan W; Johnson, Gregory R; Murphy, Robert F

2016-07-01

Accurate representations of cellular organization for multiple eukaryotic cell types are required for creating predictive models of dynamic cellular function. To this end, we have previously developed the CellOrganizer platform, an open source system for generative modeling of cellular components from microscopy images. CellOrganizer models capture the inherent heterogeneity in the spatial distribution, size, and quantity of different components among a cell population. Furthermore, CellOrganizer can generate quantitatively realistic synthetic images that reflect the underlying cell population. A current focus of the project is to model the complex, interdependent nature of organelle localization. We built upon previous work on developing multiple non-parametric models of organelles or structures that show punctate patterns. The previous models described the relationships between the subcellular localization of puncta and the positions of cell and nuclear membranes and microtubules. We extend these models to consider the relationship to the endoplasmic reticulum (ER), and to consider the relationship between the positions of different puncta of the same type. Our results do not suggest that the punctate patterns we examined are dependent on ER position or inter- and intra-class proximity. With these results, we built classifiers to update previous assignments of proteins to one of 11 patterns in three distinct cell lines. Our generative models demonstrate the ability to construct statistically accurate representations of puncta localization from simple cellular markers in distinct cell types, capturing the complex phenomena of cellular structure interaction with little human input. This protocol represents a novel approach to vesicular protein annotation, a field that is often neglected in high-throughput microscopy. These results suggest that spatial point process models provide useful insight with respect to the spatial dependence between cellular structures. �

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

Monitoring Astronaut Health at the Nanoscale Cellular Level Through the Eye

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

Microfluidics-based in vivo mimetic systems for the study of cellular biology.

PubMed

Kim, Donghyuk; Wu, Xiaojie; Young, Ashlyn T; Haynes, Christy L

2014-04-15

The human body is a complex network of molecules, organelles, cells, tissues, and organs: an uncountable number of interactions and transformations interconnect all the system's components. In addition to these biochemical components, biophysical components, such as pressure, flow, and morphology, and the location of all of these interactions play an important role in the human body. Technical difficulties have frequently limited researchers from observing cellular biology as it occurs within the human body, but some state-of-the-art analytical techniques have revealed distinct cellular behaviors that occur only in the context of the interactions. These types of findings have inspired bioanalytical chemists to provide new tools to better understand these cellular behaviors and interactions. What blocks us from understanding critical biological interactions in the human body? Conventional approaches are often too naïve to provide realistic data and in vivo whole animal studies give complex results that may or may not be relevant for humans. Microfluidics offers an opportunity to bridge these two extremes: while these studies will not model the complexity of the in vivo human system, they can control the complexity so researchers can examine critical factors of interest carefully and quantitatively. In addition, the use of human cells, such as cells isolated from donated blood, captures human-relevant data and limits the use of animals in research. In addition, researchers can adapt these systems easily and cost-effectively to a variety of high-end signal transduction mechanisms, facilitating high-throughput studies that are also spatially, temporally, or chemically resolved. These strengths should allow microfluidic platforms to reveal critical parameters in the human body and provide insights that will help with the translation of pharmacological advances to clinical trials. In this Account, we describe selected microfluidic innovations within the last 5 years

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.

Quantitative Imaging in Cancer Evolution and Ecology

PubMed Central

Grove, Olya; Gillies, Robert J.

2013-01-01

Cancer therapy, even when highly targeted, typically fails because of the remarkable capacity of malignant cells to evolve effective adaptations. These evolutionary dynamics are both a cause and a consequence of cancer system heterogeneity at many scales, ranging from genetic properties of individual cells to large-scale imaging features. Tumors of the same organ and cell type can have remarkably diverse appearances in different patients. Furthermore, even within a single tumor, marked variations in imaging features, such as necrosis or contrast enhancement, are common. Similar spatial variations recently have been reported in genetic profiles. Radiologic heterogeneity within tumors is usually governed by variations in blood flow, whereas genetic heterogeneity is typically ascribed to random mutations. However, evolution within tumors, as in all living systems, is subject to Darwinian principles; thus, it is governed by predictable and reproducible interactions between environmental selection forces and cell phenotype (not genotype). This link between regional variations in environmental properties and cellular adaptive strategies may permit clinical imaging to be used to assess and monitor intratumoral evolution in individual patients. This approach is enabled by new methods that extract, report, and analyze quantitative, reproducible, and mineable clinical imaging data. However, most current quantitative metrics lack spatialness, expressing quantitative radiologic features as a single value for a region of interest encompassing the whole tumor. In contrast, spatially explicit image analysis recognizes that tumors are heterogeneous but not well mixed and defines regionally distinct habitats, some of which appear to harbor tumor populations that are more aggressive and less treatable than others. By identifying regional variations in key environmental selection forces and evidence of cellular adaptation, clinical imaging can enable us to define intratumoral

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.

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.

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

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

Flow cytometric determination of quantitative immunophenotypes

NASA Astrophysics Data System (ADS)

Redelman, Douglas; Ensign, Wayne; Roberts, Don

2001-05-01

Immunofluorescent flow cytometric analysis of peripheral blood leucocytes is most commonly used to identify and enumerate cells defined by one or more clusters of differentiation (CD) antigens. Although less widely employed, quantitative tests that measure the amounts of CD antigens expressed per cell are used in some situations such as the characterization of lymphomas and leukocytes or the measurement of CD38 on CD3plu8pluT cells in HIV infected individuals. The CD antigens used to identify leukocyte populations are functionally important molecules and it is known that under- or over-expression of some CD antigens can affect cellular responses. For example, high or low expression of CD19 on B cells is associated with autoimmune conditions or depressed antibody responses, respectively. In the current studies, the quantitative expression of CD antigens on T cells, B cells and monocytes was determined in a group of age and sex-matched Marines at several times before and after training exercises. There was substantial variation among these individuals in the quantitative expression of CD antigens and in the number of cells in various populations. However, there was relatively little variation within individuals during the two months they were examined. Thus, the number of cells in leukocyte sub-populations and the amount of CD antigens expressed per cell appear to comprise a characteristic quantitative immunophenotype.

Pirin Inhibits Cellular Senescence in Melanocytic Cells

PubMed Central

Licciulli, Silvia; Luise, Chiara; Scafetta, Gaia; Capra, Maria; Giardina, Giuseppina; Nuciforo, Paolo; Bosari, Silvano; Viale, Giuseppe; Mazzarol, Giovanni; Tonelli, Chiara; Lanfrancone, Luisa; Alcalay, Myriam

2011-01-01

Cellular senescence has been widely recognized as a tumor suppressing mechanism that acts as a barrier to cancer development after oncogenic stimuli. A prominent in vivo model of the senescence barrier is represented by nevi, which are composed of melanocytes that, after an initial phase of proliferation induced by activated oncogenes (most commonly BRAF), are blocked in a state of cellular senescence. Transformation to melanoma occurs when genes involved in controlling senescence are mutated or silenced and cells reacquire the capacity to proliferate. Pirin (PIR) is a highly conserved nuclear protein that likely functions as a transcriptional regulator whose expression levels are altered in different types of tumors. We analyzed the expression pattern of PIR in adult human tissues and found that it is expressed in melanocytes and has a complex pattern of regulation in nevi and melanoma: it is rarely detected in mature nevi, but is expressed at high levels in a subset of melanomas. Loss of function and overexpression experiments in normal and transformed melanocytic cells revealed that PIR is involved in the negative control of cellular senescence and that its expression is necessary to overcome the senescence barrier. Our results suggest that PIR may have a relevant role in melanoma progression. PMID:21514450

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

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

Local cellular neighborhood controls proliferation in cell competition

PubMed Central

Bove, Anna; Gradeci, Daniel; Fujita, Yasuyuki; Banerjee, Shiladitya; Charras, Guillaume; Lowe, Alan R.

2017-01-01

Cell competition is a quality-control mechanism through which tissues eliminate unfit cells. Cell competition can result from short-range biochemical inductions or long-range mechanical cues. However, little is known about how cell-scale interactions give rise to population shifts in tissues, due to the lack of experimental and computational tools to efficiently characterize interactions at the single-cell level. Here, we address these challenges by combining long-term automated microscopy with deep-learning image analysis to decipher how single-cell behavior determines tissue makeup during competition. Using our high-throughput analysis pipeline, we show that competitive interactions between MDCK wild-type cells and cells depleted of the polarity protein scribble are governed by differential sensitivity to local density and the cell type of each cell’s neighbors. We find that local density has a dramatic effect on the rate of division and apoptosis under competitive conditions. Strikingly, our analysis reveals that proliferation of the winner cells is up-regulated in neighborhoods mostly populated by loser cells. These data suggest that tissue-scale population shifts are strongly affected by cellular-scale tissue organization. We present a quantitative mathematical model that demonstrates the effect of neighbor cell–type dependence of apoptosis and division in determining the fitness of competing cell lines. PMID:28931601

Historic and current hepatitis B viral DNA and quantitative HBsAg level are not associated with cirrhosis in non-Asian women with chronic hepatitis B.

PubMed

Harkisoen, S; Arends, J E; van den Hoek, J A R; Whelan, J; van Erpecum, K J; Boland, G J; Hoepelman, A I M

2014-12-01

Some studies done in Asian patients have shown that serum levels of hepatitis B virus (HBV) DNA predict the development of cirrhosis. However, it is unclear whether this also applies for non-Asian patients. This study investigated historic and current HBV DNA and quantitative hepatitis B surface antigen (HBsAg) levels as predictors of cirrhosis in non-Asian women with chronic HBV. A retrospective cohort study of non-Asian women with chronic HBV was performed. Among other variables, HBV DNA and quantitative HBsAg levels were measured in stored historic serum samples obtained during pregnancy (period 1990-2004) and current serum samples (period 2011-2012) to determine any association with liver cirrhosis by liver stiffness measurement (LSM). One hundred and nineteen asymptomatic, treatment-naïve non-Asian women were included; the median number of years between the historic sample and the current sample was 17 (interquartile range (IQR) 13-20). The median historic log HBV DNA and quantitative log HBsAg levels were 2.5 (IQR 1.9-3.4) IU/ml and 4.2 (IQR 3.6-4.5) IU/ml, respectively. LSM diagnosed 14 patients (12%) with F3-F4 fibrosis, i.e. stiffness >8.1kPa. No association of cirrhosis was found with historic HBV DNA (relative risk (RR) 0.34, 95% confidence interval (CI) 0.05-2.44) or with the quantitative HBsAg level (HBsAg level >1000 IU/ml, RR 0.35, 95% CI 0.11-1.11). Multivariable analysis identified alcohol consumption (odds ratio (OR) 6.4, 95% CI 1.3-30.1), aspartate aminotransferase >0.5 times the upper limit of normal (OR 15.4, 95% CI 1.9-122.6), and prothrombin time (OR 12.0, 95% CI 1.2-120.4), but not HBV DNA or quantitative HBsAg level, to be independent predictors of the presence of cirrhosis. Neither historic nor current HBV DNA or the quantitative HBsAg level is associated with the development of HBV-related cirrhosis in non-Asian women. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Vascular biology: cellular and molecular profiling.

PubMed

Baird, Alison E; Wright, Violet L

2006-02-01

Our understanding of the mechanisms underlying cerebrovascular atherosclerosis has improved in recent years, but significant gaps remain. New insights into the vascular biological processes that result in ischemic stroke may come from cellular and molecular profiling studies of the peripheral blood. In recent cellular profiling studies, increased levels of a proinflammatory T-cell subset (CD4 (+)CD28 (-)) have been associated with stroke recurrence and death. Expansion of this T-cell subset may occur after ischemic stroke and be a pathogenic mechanism leading to recurrent stroke and death. Increases in certain phenotypes of endothelial cell microparticles have been found in stroke patients relative to controls, possibly indicating a state of increased vascular risk. Molecular profiling approaches include gene expression profiling and proteomic methods that permit large-scale analyses of the transcriptome and the proteome, respectively. Ultimately panels of genes and proteins may be identified that are predictive of stroke risk. Cellular and molecular profiling studies of the peripheral blood and of atherosclerotic plaques may also pave the way for the development of therapeutic agents for primary and secondary stroke prevention.

Quantitative Analysis of High-Quality Officer Selection by Commandants Career-Level Education Board

DTIC Science & Technology

2017-03-01

due to Marines being evaluated before the end of their initial service commitment. Our research utilizes quantitative variables to analyze the...not provide detailed information why. B. LIMITATIONS The photograph analysis in this research is strictly limited to a quantitative analysis in...NAVAL POSTGRADUATE SCHOOL MONTEREY, CALIFORNIA THESIS Approved for public release. Distribution is unlimited. QUANTITATIVE

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

NASA Astrophysics Data System (ADS)

Chakraborty, Jayajit; Green, Donna

2014-04-01

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

Frequent cellular phone use modifies hypothalamic-pituitary-adrenal axis response to a cellular phone call after mental stress in healthy children and adolescents: A pilot study.

PubMed

Geronikolou, Styliani A; Chamakou, Aikaterini; Mantzou, Aimilia; Chrousos, George; KanakaGantenbein, Christina

2015-12-01

The hypothalamic-pituitary-adrenal (HPA) axis is the main "gate-keeper" of the organism's response to every somatic or mental stress. This prospective study aims to investigate the HPA-axis response to a cellular phone call exposure after mental stress in healthy children and adolescents and to assess the possible predictive role of baseline endocrine markers to this response. Two groups of healthy school-age children aged 11-14 (12.5±1.5) years were included in the study, the one comprising those who are occasional users of a cellular phone (Group A) while the second those who do regularly use one (Group B). Blood samples were obtained from all participants at 8.00 am after a 12-hour overnight fasting for thyroid hormone, glucose, insulin, and cortisol levels determination. The participants performed the Trier Social Stress Test for Children (TSST-C) (5 minoral task followed by 5 min arithmetic task). Salivary cortisol samples were obtained at baseline, 10' and 20' min after the TSST-C and 10' and 20' after a 5 minute cellular phone call. Significant changes in the salivary cortisol levels were noted between 10' and 20' mins after the cellular phone call with different responses between the two groups. Baseline thyroid hormone levels seem to predict the cortisol response to mental stress mainly in group A, while HOMA had no impact on salivary cortisol response at any phase of the test, in either group. HPA axis response to cellular phone after mental stress in children and adolescents follow a different pattern in frequent users than in occasional users that seems to be influenced by the baseline thyroid hormone levels. Copyright © 2015 Elsevier B.V. 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

Cellular Sites of Immunologic Unresponsiveness*

PubMed Central

Chiller, Jacques M.; Habicht, Gail S.; Weigle, William O.

1970-01-01

The reconstitution of the immune response of lethally irradiated mice to human γ-globulin is dependent on the synergistic action of bone marrow with thymus cells. Immunologic unresponsiveness appears to involve a functional defect at each of these cellular levels, inasmuch as neither bone marrow nor thymus cells from unresponsive donors are capable of demonstrating synergism in combination with their normal counterpart. PMID:4192271

Cellular Analogs of Operant Behavior.

DTIC Science & Technology

1992-07-31

confirmed at the behavioral level in parallel studies of hippocampal and intravenous self-administration. The results are consistent with the hypothesis...research is to elucidate the cellular reinforcement mechanisms underlying goal-directed or operant behavior. The specific aim here was to study the... studies and behavioral operant conditioning studies . Most of the research reported was initiated and completed in the present project period. Also

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

Cellular Response to Ionizing Radiation: A MicroRNA Story

PubMed Central

Halimi, Mohammad; Asghari, S. Mohsen; Sariri, Reyhaneh; Moslemi, Dariush; Parsian, Hadi

2012-01-01

MicroRNAs (miRNAs) represent a class of small non-coding RNA molecules that regulate gene expression at the post-transcriptional level. They play a crucial role in diverse cellular pathways. Ionizing radiation (IR) is one of the most important treatment protocols for patients that suffer from cancer and affects directly or indirectly cellular integration. Recently it has been discovered that microRNA-mediated gene regulation interferes with radio-related pathways in ionizing radiation. Here, we review the recent discoveries about miRNAs in cellular response to IR. Thoroughly understanding the mechanism of miRNAs in radiation response, it will be possible to design new strategies for improving radiotherapy efficiency and ultimately cancer treatment. PMID:24551775

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.

Enzymatic and cellular responses in relation to body burden of PAHs in bivalve molluscs: a case study with chronic levels of North Sea and Barents Sea dispersed oil.

PubMed

Baussant, T; Bechmann, R K; Taban, I C; Larsen, B K; Tandberg, A H; Bjørnstad, A; Torgrimsen, S; Naevdal, A; Øysaed, K B; Jonsson, G; Sanni, S

2009-12-01

Mytilus edulis and Chlamys islandica were exposed to nominal dispersed crude oil concentrations in the range 0.015-0.25 mg/l for one month. Five biomarkers (enzymatic and cellular responses) were analysed together with bioaccumulation of PAHs at the end of exposure. In both species, PAH tissue residues reflected the exposure concentration measured in the water and lipophilicity determined the bioaccumulation levels. Oil caused biomarker responses in both species but more significant alterations in exposed C. islandica were observed. The relationships between exposure levels and enzymatic responses were apparently complex. The integrated biomarker response related against the exposure levels was U-shaped in both species and no correlation with total PAH body burden was found. For the monitoring of chronic offshore discharges, dose- and time-related events should be evaluated in the selection of biomarkers to apply. From this study, cellular damages appear more fitted than enzymatic responses, transient and more complex to interpret.

Fuzzy cellular automata models in immunology

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

Ahmed, E.

1996-10-01

The self-nonself character of antigens is considered to be fuzzy. The Chowdhury et al. cellular automata model is generalized accordingly. New steady states are found. The first corresponds to a below-normal help and suppression and is proposed to be related to autoimmune diseases. The second corresponds to a below-normal B-cell level.

Identification of cellular MMP substrates using quantitative proteomics: isotope-coded affinity tags (ICAT) and isobaric tags for relative and absolute quantification (iTRAQ).

PubMed

Butler, Georgina S; Dean, Richard A; Morrison, Charlotte J; Overall, Christopher M

2010-01-01

Identification of protease substrates is essential to understand the functional consequences of normal proteolytic processing and dysregulated proteolysis in disease. Quantitative proteomics and mass spectrometry can be used to identify protease substrates in the cellular context. Here we describe the use of two protein labeling techniques, Isotope-Coded Affinity Tags (ICAT and Isobaric Tags for Relative and Absolute Quantification (iTRAQ), which we have used successfully to identify novel matrix metalloproteinase (MMP) substrates in cell culture systems (1-4). ICAT and iTRAQ can label proteins and protease cleavage products of secreted proteins, protein domains shed from the cell membrane or pericellular matrix of protease-transfected cells that have accumulated in conditioned medium, or cell surface proteins in membrane preparations; isotopically distinct labels are used for control cells. Tryptic digestion and tandem mass spectrometry of the generated fragments enable sequencing of differentially labeled but otherwise identical pooled peptides. The isotopic tag, which is unique for each label, identifies the peptides originating from each sample, for instance, protease-transfected or control cells, and comparison of the peak areas enables relative quantification of the peptide in each sample. Thus proteins present in altered amounts between protease-expressing and null cells are implicated as protease substrates and can be further validated as such.

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

PubMed

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

2015-07-07

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

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

NASA Astrophysics Data System (ADS)

Johansson, Erland; Lindh, Ulf

1985-08-01

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

Evaluation of cellular influences of platinum nanoparticles by stable medium dispersion.

PubMed

Horie, Masanori; Kato, Haruhisa; Endoh, Shigehisa; Fujita, Katsuhide; Nishio, Keiko; Komaba, Lilian Kaede; Fukui, Hiroko; Nakamura, Ayako; Miyauchi, Arisa; Nakazato, Tetsuya; Kinugasa, Shinichi; Yoshida, Yasukazu; Hagihara, Yoshihisa; Morimoto, Yasuo; Iwahashi, Hitoshi

2011-11-01

Platinum nanoparticles have industrial application, for example in catalysis, and are used in consumer products such as cosmetics and supplements. Therefore, among the many nanoparticles, platinum is one of the more accessible nanoparticles for consumers. Most platinum nanoparticles that are used in cosmetics and supplements which have an anti-oxidant activity are modified particles. However, the cellular influences of pristine platinum nanoparticles are still unclear, although it has been reported that platinum nanoparticles induce oxidative stress. In this study, we investigated the cellular influences induced by pure pristine platinum nanoparticles. Platinum nanoparticles of 100% purity were dispersed in a cell culture medium and stable medium dispersion was obtained. The platinum nanoparticle medium dispersion was applied to two kinds of cultured cells, A549 and HaCaT cells, and the cellular influences were examined. Cell viability (MTT assay), cell proliferation (clonogenic assay), apoptosis induction (caspase-3 activity), intracellular ROS level (DCFH assay), and lipid peroxidation level (DPPP assay) were measured as markers of cellular influences. Transmission electron microscope observation showed cellular uptake of platinum nanoparticles. However, the platinum nanoparticles did not drive any markers. It is known that some metal oxide nanoparticles such as NiO and CuO show severe cytotoxicity via metal ion release. Compared with these toxic nanoparticles, the platinum nanoparticles used in this study did not release platinum ions into the culture media. These results suggest that the physically and chemically inactive cellular influences of platinum nanoparticles are small.

Dynamics of Cellular Responses to Radiation

PubMed Central

Wodarz, Dominik; Sorace, Ron; Komarova, Natalia L.

2014-01-01

Understanding the consequences of exposure to low dose ionizing radiation is an important public health concern. While the risk of low dose radiation has been estimated by extrapolation from data at higher doses according to the linear non-threshold model, it has become clear that cellular responses can be very different at low compared to high radiation doses. Important phenomena in this respect include radioadaptive responses as well as low-dose hyper-radiosensitivity (HRS) and increased radioresistance (IRR). With radioadaptive responses, low dose exposure can protect against subsequent challenges, and two mechanisms have been suggested: an intracellular mechanism, inducing cellular changes as a result of the priming radiation, and induction of a protected state by inter-cellular communication. We use mathematical models to examine the effect of these mechanisms on cellular responses to low dose radiation. We find that the intracellular mechanism can account for the occurrence of radioadaptive responses. Interestingly, the same mechanism can also explain the existence of the HRS and IRR phenomena, and successfully describe experimentally observed dose-response relationships for a variety of cell types. This indicates that different, seemingly unrelated, low dose phenomena might be connected and driven by common core processes. With respect to the inter-cellular communication mechanism, we find that it can also account for the occurrence of radioadaptive responses, indicating redundancy in this respect. The model, however, also suggests that the communication mechanism can be vital for the long term survival of cell populations that are continuously exposed to relatively low levels of radiation, which cannot be achieved with the intracellular mechanism in our model. Experimental tests to address our model predictions are proposed. PMID:24722167

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.

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

The sub-cellular fate of mercury in the liver of wild mullets (Liza aurata)--Contribution to the understanding of metal-induced cellular toxicity.

PubMed

Araújo, Olinda; Pereira, Patrícia; Cesário, Rute; Pacheco, Mário; Raimundo, Joana

2015-06-15

Mercury is a recognized harmful pollutant in aquatic systems but still little is known about its sub-cellular partitioning in wild fish. Mercury concentrations in liver homogenate (whole organ load) and in six sub-cellular compartments were determined in wild Liza aurata from two areas - contaminated (LAR) and reference. Water and sediment contamination was also assessed. Fish from LAR displayed higher total mercury (tHg) organ load as well as in sub-cellular compartments than those from the reference area, reflecting environmental differences. However, spatial differences in percentage of tHg were only observed for mitochondria (Mit) and lysosomes plus microsomes (Lys+Mic). At LAR, Lys+Mic exhibited higher levels of tHg than the other fractions. Interestingly, tHg in Mit, granules (Gran) and heat-denaturable proteins was linearly correlated with the whole organ. Low tHg concentrations in heat stable proteins and Gran suggests that accumulated levels might be below the physiological threshold to activate those detoxification fractions. Copyright © 2015 Elsevier Ltd. All rights reserved.

HPV integration hijacks and multimerizes a cellular enhancer to generate a viral-cellular super-enhancer that drives high viral oncogene expression

PubMed Central

Redmond, Catherine J.; Dooley, Katharine E.; Fu, Haiqing; Gillison, Maura L.; Akagi, Keiko; Symer, David E.; Aladjem, Mirit I.

2018-01-01

Integration of human papillomavirus (HPV) genomes into cellular chromatin is common in HPV-associated cancers. Integration is random, and each site is unique depending on how and where the virus integrates. We recently showed that tandemly integrated HPV16 could result in the formation of a super-enhancer-like element that drives transcription of the viral oncogenes. Here, we characterize the chromatin landscape and genomic architecture of this integration locus to elucidate the mechanisms that promoted de novo super-enhancer formation. Using next-generation sequencing and molecular combing/fiber-FISH, we show that ~26 copies of HPV16 are integrated into an intergenic region of chromosome 2p23.2, interspersed with 25 kb of amplified, flanking cellular DNA. This interspersed, co-amplified viral-host pattern is frequent in HPV-associated cancers and here we designate it as Type III integration. An abundant viral-cellular fusion transcript encoding the viral E6/E7 oncogenes is expressed from the integration locus and the chromatin encompassing both the viral enhancer and a region in the adjacent amplified cellular sequences is strongly enriched in the super-enhancer markers H3K27ac and Brd4. Notably, the peak in the amplified cellular sequence corresponds to an epithelial-cell-type specific enhancer. Thus, HPV16 integration generated a super-enhancer-like element composed of tandem interspersed copies of the viral upstream regulatory region and a cellular enhancer, to drive high levels of oncogene expression. PMID:29364907

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

47 CFR 22.970 - Unacceptable interference to part 90 non-cellular 800 MHz licensees from cellular radiotelephone...

Code of Federal Regulations, 2011 CFR

2011-10-01

...-cellular 800 MHz licensees from cellular radiotelephone or part 90-800 MHz cellular systems. 22.970 Section... MOBILE SERVICES Cellular Radiotelephone Service § 22.970 Unacceptable interference to part 90 non-cellular 800 MHz licensees from cellular radiotelephone or part 90-800 MHz cellular systems. (a) Definition...

Cellular phones and their hazards: the current evidence.

PubMed

Munshi, Anusheel; Jalali, Rakesh

2002-01-01

The past decade has seen an exponential increase globally in the use of cellular phones (popularly known as mobile or cell phones). These phones are convenient and trendy. Discarding the wire means that the communication is through electromagnetic waves, which could have potential hazards. Alarmist reports in the lay press and high profile lawsuits, particularly in the West, have attracted attention to the possible harmful effects of cellular phones. Adverse effects investigated by various clinical trials include the possible link to increased risk of vehicular accidents, leukaemias, sleep disturbances and the more serious brain tumours. Available level II evidence suggests that the only proven side-effect is an increased risk of vehicular accidents. So far, all studies have consistently negated any association between cellular phones and brain tumours. Yet, the final word remains to be said.

The role of actin networks in cellular mechanosensing

NASA Astrophysics Data System (ADS)

Azatov, Mikheil

Physical processes play an important role in many biological phenomena, such as wound healing, organ development, and tumor metastasis. During these processes, cells constantly interact with and adapt to their environment by exerting forces to mechanically probe the features of their surroundings and generating appropriate biochemical responses. The mechanisms underlying how cells sense the physical properties of their environment are not well understood. In this thesis, I present my studies to investigate cellular responses to the stiffness and topography of the environment. In order to sense the physical properties of their environment, cells dynamically reorganize the structure of their actin cytoskeleton, a dynamic network of biopolymers, altering the shape and spatial distribution of protein assemblies. Several observations suggest that proteins that crosslink actin filaments may play an important role in cellular mechanosensitivity. Palladin is an actin-crosslinking protein that is found in the lamellar actin network, stress fibers and focal adhesions, cellular structures that are critical for mechanosensing of the physical environment. By virtue of its close interactions with these structures in the cell, palladin may play an important role in cell mechanics. However, the role of actin crosslinkers in general, and palladin in particular, in cellular force generation and mechanosensing is not well known. I have investigated the role of palladin in regulating the plasticity of the actin cytoskeleton and cellular force generation in response to alterations in substrate stiffness. I have shown that the expression levels of palladin modulate the forces exerted by cells and their ability to sense substrate stiffness. Perturbation experiments also suggest that palladin levels in cells altered myosin motor activity. These results suggest that the actin crosslinkers, such as palladin, and myosin motors coordinate for optimal cell function and to prevent aberrant

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.

Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii

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

Tian, Shengke; Xie, Ruohan; Wang, Haixin

Sedum alfredii is one of a few plant species known to hyperaccumulate cadmium (Cd). Uptake, localization, and tolerance of Cd at cellular levels in shoots were compared in hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii. X-ray fluorescence images of Cd in stems and leaves showed only a slight Cd signal restricted within vascular bundles in the NHEs, while enhanced localization of Cd, with significant tissue- and age-dependent variations, was detected in HEs. In contrast to the vascular-enriched Cd in young stems, parenchyma cells in leaf mesophyll, stem pith and cortex tissues served as terminal storage sites for Cdmore » sequestration in HEs. Kinetics of Cd transport into individual leaf protoplasts of the two ecotypes showed little difference in Cd accumulation. However, far more efficient storage of Cd in vacuoles was apparent in HEs. Subsequent analysis of cell viability and hydrogen peroxide levels suggested that HE protoplasts exhibited higher resistance to Cd than those of NHE protoplasts. These results suggest that efficient sequestration into vacuoles, as opposed to rapid transport into parenchyma cells, is a pivotal process in Cd accumulation and homeostasis in shoots of HE S. alfredii. This is in addition to its efficient root-to-shoot translocation of Cd.« less

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.

Review of cellular mechanotransduction

NASA Astrophysics Data System (ADS)

Wang, Ning

2017-06-01

Living cells and tissues experience physical forces and chemical stimuli in the human body. The process of converting mechanical forces into biochemical activities and gene expression is mechanochemical transduction or mechanotransduction. Significant advances have been made in understanding mechanotransduction at the cellular and molecular levels over the last two decades. However, major challenges remain in elucidating how a living cell integrates signals from mechanotransduction with chemical signals to regulate gene expression and to generate coherent biological responses in living tissues in physiological conditions and diseases.

Cellular Imaging With MRI.

PubMed

Makela, Ashley V; Murrell, Donna H; Parkins, Katie M; Kara, Jenna; Gaudet, Jeffrey M; Foster, Paula J

2016-10-01

Cellular magnetic resonance imaging (MRI) is an evolving field of imaging with strong translational and research potential. The ability to detect, track, and quantify cells in vivo and over time allows for studying cellular events related to disease processes and may be used as a biomarker for decisions about treatments and for monitoring responses to treatments. In this review, we discuss methods for labeling cells, various applications for cellular MRI, the existing limitations, strategies to address these shortcomings, and clinical cellular MRI.

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.

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.

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.

Cellular fatty acid composition of Erysipelothrix rhusiopathiae and Erysipelothrix tonsillarum.

PubMed

Takahashi, T; Tamura, Y; Endoh, Y S; Hara, N

1994-04-01

The cellular fatty acid compositions in 6 strains of Erysipelothrix rhusiopathiae and 7 strains of Erysipelothrix tonsillarum were determined by gas chromatography. Fatty acids, ranging from C10 to C18, were detected in the test strains. The fatty acid profile was characterized by very high percentages of 18:1 (cis-9) (cis-9-octadecenoic acid; 72.4 to 82.1%) and 16:1 (hexadecenoic acid; 8.7 to 13.7%). The profiles of the E. rhusiopathiae and E. tonsillarum strains resembled each other, indicating that discrimination between E. rhusiopathiae and E. tonsillarum from qualitative or quantitative fatty acid differences is difficult.

Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast

PubMed Central

Pang, Wei; Coghill, George M.

2015-01-01

In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. PMID:25864377

Determining the Extremes of the Cellular NAD(H) Level by Using an Escherichia coli NAD+-Auxotrophic Mutant ▿

PubMed Central

Zhou, Yongjin; Wang, Lei; Yang, Fan; Lin, Xinping; Zhang, Sufang; Zhao, Zongbao K.

2011-01-01

NAD (NAD+) and its reduced form (NADH) are omnipresent cofactors in biological systems. However, it is difficult to determine the extremes of the cellular NAD(H) level in live cells because the NAD+ level is tightly controlled by a biosynthesis regulation mechanism. Here, we developed a strategy to determine the extreme NAD(H) levels in Escherichia coli cells that were genetically engineered to be NAD+ auxotrophic. First, we expressed the ntt4 gene encoding the NAD(H) transporter in the E. coli mutant YJE001, which had a deletion of the nadC gene responsible for NAD+ de novo biosynthesis, and we showed NTT4 conferred on the mutant strain better growth in the presence of exogenous NAD+. We then constructed the NAD+-auxotrophic mutant YJE003 by disrupting the essential gene nadE, which is responsible for the last step of NAD+ biosynthesis in cells harboring the ntt4 gene. The minimal NAD+ level was determined in M9 medium in proliferating YJE003 cells that were preloaded with NAD+, while the maximal NAD(H) level was determined by exposing the cells to high concentrations of exogenous NAD(H). Compared with supplementation of NADH, cells grew faster and had a higher intracellular NAD(H) level when NAD+ was fed. The intracellular NAD(H) level increased with the increase of exogenous NAD+ concentration, until it reached a plateau. Thus, a minimal NAD(H) level of 0.039 mM and a maximum of 8.49 mM were determined, which were 0.044× and 9.6× those of wild-type cells, respectively. Finally, the potential application of this strategy in biotechnology is briefly discussed. PMID:21742902

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.

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.

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.

Disruption of Hox9,10,11 function results in cellular level lineage infidelity in the kidney.

PubMed

Drake, Keri A; Adam, Mike; Mahoney, Robert; Potter, S Steven

2018-04-20

Hox genes are important regulators of development. The 39 mammalian Hox genes have considerable functional overlap, greatly confounding their study. In this report, we generated mice with multiple combinations of paralogous and flanking Abd-B Hox gene mutations to investigate functional redundancies in kidney development. The resulting mice developed a number of kidney abnormalities, including hypoplasia, agenesis, and severe cysts, with distinct Hox functions observed in early metanephric kidney formation and nephron progenitor maintenance. Most surprising, however, was that extensive removal of Hox shared function in these kidneys resulted in cellular level lineage infidelity. Strikingly, mutant nephron tubules consisted of intermixed cells with proximal tubule, loop of Henle, and collecting duct identities, with some single cells expressing markers associated with more than one nephron segment. These results indicate that Hox genes are required for proper lineage selection/maintenance and full repression of genes involved in cell fate restriction in the developing kidney.

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

PubMed

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

2017-05-15

Infections and autoimmune disorders are more frequent in Down syndrome, suggesting abnormality of adaptive immunity. Although the role of B cells and antibodies is well characterized, knowledge regarding T cells is limited. Lymphocyte subpopulations of 40 children and adolescents with Down syndrome and 51 controls were quantified, and phenotype and functionality of antigen-specific effector T cells were analyzed with flow cytometry after polyclonal and pathogen-specific stimulation (with varicella-zoster virus [VZV] and cytomegalovirus [CMV]). Results were correlated with immunoglobulin (Ig) G responses. Apart from general alterations in the percentage of lymphocytes, regulatory T cells, and T-helper 1 and 17 cells, all major T-cell subpopulations showed higher expression of the inhibitory receptor PD-1. Polyclonally stimulated effector CD4+ T-cell frequencies were significantly higher in subjects with Down syndrome, whereas their inhibitory receptor expression (programmed cell death 1 [PD-1] and cytotoxic T-lymphocyte antigen 4 [CTLA-4]) was similar to that of controls and cytokine expression profiles were only marginally altered. Pathogen-specific immunity showed age-appropriate levels of endemic infection, with correlation of CMV-specific cellular and humoral immunity in all subjects. Among VZV IgG-positive individuals, a higher percentage of VZV-specific T-cell-positive subjects was seen in those with Down syndrome. Despite alterations in lymphocyte subpopulations, individuals with Down syndrome can mount effector T-cell responses with similar phenotype and functionality as controls but may require higher effector T-cell frequencies to ensure pathogen control. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Cotton Defense Induction Patterns Under Spatially, Temporally and Quantitatively Varying Herbivory Levels.

PubMed

Eisenring, Michael; Meissle, Michael; Hagenbucher, Steffen; Naranjo, Steven E; Wettstein, Felix; Romeis, Jörg

2017-01-01

In its defense against herbivores, cotton ( Gossypium sp.) relies in part on the production of a set of inducible, non-volatile terpenoids. Under uniform damage levels, in planta allocation of induced cotton terpenoids has been found to be highest in youngest leaves, supporting assumptions of the optimal defense theory (ODT) which predicts that plants allocate defense compounds to tissues depending on their value and the likelihood of herbivore attack. However, our knowledge is limited on how varying, and thus more realistic, damage levels might affect cotton defense organization. We hypothesized that the allocation of terpenoids and densities of terpenoid-storing glands in leaves aligns with assumptions of the ODT, even when plants are subjected to temporally, spatially and quantitatively varying caterpillar ( Heliothis virescens ) damage. As expected, cotton plants allocated most of their defenses to their youngest leaves regardless of damage location. However, defense induction in older leaves varied with damage location. For at least 14 days after damage treatments ended, plants reallocated defense resources from previously young leaves to newly developed leaves. Furthermore, we observed a positive hyperbolic relationship between leaf damage area and both terpenoid concentrations and gland densities, indicating that cotton plants can fine-tune defense allocation. Although it appears that factors like vascular constraints and chemical properties of individual defense compounds can affect defense levels, our results overall demonstrate that induced defense organization of cotton subjected to varying damage treatments is in alignment with key assumptions of the ODT.

Cotton Defense Induction Patterns Under Spatially, Temporally and Quantitatively Varying Herbivory Levels

PubMed Central

Eisenring, Michael; Meissle, Michael; Hagenbucher, Steffen; Naranjo, Steven E.; Wettstein, Felix; Romeis, Jörg

2017-01-01

In its defense against herbivores, cotton (Gossypium sp.) relies in part on the production of a set of inducible, non-volatile terpenoids. Under uniform damage levels, in planta allocation of induced cotton terpenoids has been found to be highest in youngest leaves, supporting assumptions of the optimal defense theory (ODT) which predicts that plants allocate defense compounds to tissues depending on their value and the likelihood of herbivore attack. However, our knowledge is limited on how varying, and thus more realistic, damage levels might affect cotton defense organization. We hypothesized that the allocation of terpenoids and densities of terpenoid-storing glands in leaves aligns with assumptions of the ODT, even when plants are subjected to temporally, spatially and quantitatively varying caterpillar (Heliothis virescens) damage. As expected, cotton plants allocated most of their defenses to their youngest leaves regardless of damage location. However, defense induction in older leaves varied with damage location. For at least 14 days after damage treatments ended, plants reallocated defense resources from previously young leaves to newly developed leaves. Furthermore, we observed a positive hyperbolic relationship between leaf damage area and both terpenoid concentrations and gland densities, indicating that cotton plants can fine-tune defense allocation. Although it appears that factors like vascular constraints and chemical properties of individual defense compounds can affect defense levels, our results overall demonstrate that induced defense organization of cotton subjected to varying damage treatments is in alignment with key assumptions of the ODT. PMID:28270830

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.

The laforin-malin complex negatively regulates glycogen synthesis by modulating cellular glucose uptake via glucose transporters.

PubMed

Singh, Pankaj Kumar; Singh, Sweta; Ganesh, Subramaniam

2012-02-01

Lafora disease (LD), an inherited and fatal neurodegenerative disorder, is characterized by increased cellular glycogen content and the formation of abnormally branched glycogen inclusions, called Lafora bodies, in the affected tissues, including neurons. Therefore, laforin phosphatase and malin ubiquitin E3 ligase, the two proteins that are defective in LD, are thought to regulate glycogen synthesis through an unknown mechanism, the defects in which are likely to underlie some of the symptoms of LD. We show here that laforin's subcellular localization is dependent on the cellular glycogen content and that the stability of laforin is determined by the cellular ATP level, the activity of 5'-AMP-activated protein kinase, and the affinity of malin toward laforin. By using cell and animal models, we further show that the laforin-malin complex regulates cellular glucose uptake by modulating the subcellular localization of glucose transporters; loss of malin or laforin resulted in an increased abundance of glucose transporters in the plasma membrane and therefore excessive glucose uptake. Loss of laforin or malin, however, did not affect glycogen catabolism. Thus, the excessive cellular glucose level appears to be the primary trigger for the abnormally higher levels of cellular glycogen seen in LD.

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

Matrix and Backstage: Cellular Substrates for Viral Vaccines

PubMed Central

Jordan, Ingo; Sandig, Volker

2014-01-01

Vaccines are complex products that are manufactured in highly dynamic processes. Cellular substrates are one critical component that can have an enormous impact on reactogenicity of the final preparation, level of attenuation of a live virus, yield of infectious units or antigens, and cost per vaccine dose. Such parameters contribute to feasibility and affordability of vaccine programs both in industrialized countries and developing regions. This review summarizes the diversity of cellular substrates for propagation of viral vaccines from primary tissue explants and embryonated chicken eggs to designed continuous cell lines of human and avian origin. PMID:24732259

Microfluidics-Based in Vivo Mimetic Systems for the Study of Cellular Biology

PubMed Central

2015-01-01

Conspectus The human body is a complex network of molecules, organelles, cells, tissues, and organs: an uncountable number of interactions and transformations interconnect all the system’s components. In addition to these biochemical components, biophysical components, such as pressure, flow, and morphology, and the location of all of these interactions play an important role in the human body. Technical difficulties have frequently limited researchers from observing cellular biology as it occurs within the human body, but some state-of-the-art analytical techniques have revealed distinct cellular behaviors that occur only in the context of the interactions. These types of findings have inspired bioanalytical chemists to provide new tools to better understand these cellular behaviors and interactions. What blocks us from understanding critical biological interactions in the human body? Conventional approaches are often too naïve to provide realistic data and in vivo whole animal studies give complex results that may or may not be relevant for humans. Microfluidics offers an opportunity to bridge these two extremes: while these studies will not model the complexity of the in vivo human system, they can control the complexity so researchers can examine critical factors of interest carefully and quantitatively. In addition, the use of human cells, such as cells isolated from donated blood, captures human-relevant data and limits the use of animals in research. In addition, researchers can adapt these systems easily and cost-effectively to a variety of high-end signal transduction mechanisms, facilitating high-throughput studies that are also spatially, temporally, or chemically resolved. These strengths should allow microfluidic platforms to reveal critical parameters in the human body and provide insights that will help with the translation of pharmacological advances to clinical trials. In this Account, we describe selected microfluidic innovations within the

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

PubMed

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

2015-02-01

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

Analysis of cellular and extracellular DNA in fingerprints

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

Button, Julie M.

It has been previously shown that DNA can be recovered from latent fingerprints left on various surfaces [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. However, the source of the DNA, extracellular versus cellular origin, is difficult to determine. If the DNA is cellular, it is believed to belong to skin cells while extracellular DNA is believed to originate from body fluids such as sweat [D. J. Daly et. al, Forensic Sci. Int. Genet. 6, 41-46 (2012); V. V. Vlassov et. al, BioEssays 29, 654-667 (2007)]. The origin of the DNA in fingerprints has implicationsmore » for processing and interpretation of forensic evidence. The determination of the origin of DNA in fingerprints is further complicated by the fact that the DNA in fingerprints tends to be at a very low quantity [R. A. H. van Oorschot and M. K. Jones, Nature 387, 767 (1997)]. This study examined fingerprints from five volunteers left on sterilized glass slides and plastic pens. Three fingerprints were left on each glass slide (thumb, index, and middle fingers) while the pens were held as if one was writing with them. The DNA was collected from the objects using the wet swabbing technique (TE buffer). Following collection, the cellular and extracellular components of each sample were separated using centrifugation and an acoustofluidics system. Centrifugation is still the primary separation technique utilized in forensics laboratories, while acoustic focusing uses sound waves to focus large particles (cells) into low pressure nodes, separating them from the rest of the sample matrix. After separation, all samples were quantified using real-time quantitative PCR (qPCR). The overall trend is that there is more DNA in the extracellular fractions than cellular fractions for both centrifugation and acoustofluidic processing. Additionally, more DNA was generally collected from the pen samples than the samples left on glass slides.« less

Cellular interactions with tissue-engineered microenvironments and nanoparticles

NASA Astrophysics Data System (ADS)

Pan, Zhi

Tissue-engineered hydrogels composed of intermolecularlly crosslinked hyaluronan (HA-DTPH) and fibronectin functional domains (FNfds) were applied as a physiological relevant ECM mimic with controlled mechanical and biochemical properties. Cellular interactions with this tissue-engineered environment, especially physical interactions (cellular traction forces), were quantitatively measured by using the digital image speckle correlation (DISC) technique and finite element method (FEM). By correlating with other cell functions such as cell morphology and migration, a comprehensive structure-function relationship between cells and their environments was identified. Furthermore, spatiotemporal redistribution of cellular traction stresses was time-lapse measured during cell migration to better understand the dynamics of cell mobility. The results suggest that the reinforcement of the traction stresses around the nucleus, as well as the relaxation of nuclear deformation, are critical steps during cell migration, serving as a speed regulator, which must be considered in any dynamic molecular reconstruction model of tissue cell migration. Besides single cell migration, en masse cell migration was studied by using agarose droplet migration assay. Cell density was demonstrated to be another important parameter to influence cell behaviors besides substrate properties. Findings from these studies will provide fundamental design criteria to develop novel and effective tissue-engineered constructs. Cellular interactions with rutile and anatase TiO2 nanoparticles were also studied. These particles can penetrate easily through the cell membrane and impair cell function, with the latter being more damaging. The exposure to nanoparticles was found to decrease cell area, cell proliferation, motility, and contractility. To prevent this, a dense grafted polymer brush coating was applied onto the nanoparticle surface. These modified nanoparticles failed to adhere to and penetrate

Haemoglobin function in vertebrates: evolutionary changes in cellular regulation in hypoxia.

PubMed

Nikinmaa, M

2001-11-15

The evolution of erythrocytic hypoxia responses is reviewed by comparing the cellular control of haemoglobin-oxygen affinity in agnathans, teleost fish and terrestrial vertebrates. The most ancient response to hypoxic conditions appears to be an increase in cell volume, which increases the haemoglobin-oxygen affinity in lampreys. In teleost fish, an increase of cell volume in hypoxic conditions is also evident. The volume increase is coupled to an increase in erythrocyte pH. These changes are caused by an adrenergic activation of sodium/proton exchange across the erythrocyte membrane. The mechanism is important in acute hypoxia and is followed by a decrease in cellular adenosine triphosphate (ATP) and guanosine triphosphate (GTP) concentrations in continued hypoxia. In hypoxic bird embryos, the ATP levels are also reduced. The mechanisms by which hypoxia decreases cellular ATP and GTP concentrations remains unknown, although at least in bird embryos cAMP-dependent mechanisms have been implicated. In mammals, hypoxia responses appear to occur mainly via modulation of cellular organic phosphate concentrations. In moderate hypoxia, 2,3-diphosphoglycerate levels are increased as a result of alkalosis caused by increased ventilation.

A novel image encryption algorithm using chaos and reversible cellular automata

NASA Astrophysics Data System (ADS)

Wang, Xingyuan; Luan, Dapeng

2013-11-01

In this paper, a novel image encryption scheme is proposed based on reversible cellular automata (RCA) combining chaos. In this algorithm, an intertwining logistic map with complex behavior and periodic boundary reversible cellular automata are used. We split each pixel of image into units of 4 bits, then adopt pseudorandom key stream generated by the intertwining logistic map to permute these units in confusion stage. And in diffusion stage, two-dimensional reversible cellular automata which are discrete dynamical systems are applied to iterate many rounds to achieve diffusion on bit-level, in which we only consider the higher 4 bits in a pixel because the higher 4 bits carry almost the information of an image. Theoretical analysis and experimental results demonstrate the proposed algorithm achieves a high security level and processes good performance against common attacks like differential attack and statistical attack. This algorithm belongs to the class of symmetric systems.

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

NASA Astrophysics Data System (ADS)

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

2016-08-01

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

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.

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

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.

Qualitative, semi-quantitative, and quantitative simulation of the osmoregulation system in yeast.

PubMed

Pang, Wei; Coghill, George M

2015-05-01

In this paper we demonstrate how Morven, a computational framework which can perform qualitative, semi-quantitative, and quantitative simulation of dynamical systems using the same model formalism, is applied to study the osmotic stress response pathway in yeast. First the Morven framework itself is briefly introduced in terms of the model formalism employed and output format. We then built a qualitative model for the biophysical process of the osmoregulation in yeast, and a global qualitative-level picture was obtained through qualitative simulation of this model. Furthermore, we constructed a Morven model based on existing quantitative model of the osmoregulation system. This model was then simulated qualitatively, semi-quantitatively, and quantitatively. The obtained simulation results are presented with an analysis. Finally the future development of the Morven framework for modelling the dynamic biological systems is discussed. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Study of cellular oncometabolism via multidimensional protein identification technology.

PubMed

Aukim-Hastie, Claire; Garbis, Spiros D

2014-01-01

Cellular proteomics is becoming a widespread clinical application, matching the definition of bench-to-bedside translation. Among various fields of investigation, this approach can be applied to the study of the metabolic alterations that accompany oncogenesis and tumor progression, which are globally referred to as oncometabolism. Here, we describe a multidimensional protein identification technology (MuDPIT)-based strategy that can be employed to study the cellular proteome of malignant cells and tissues. This method has previously been shown to be compatible with the reproducible, in-depth analysis of up to a thousand proteins in clinical samples. The possibility to employ this technique to study clinical specimens demonstrates its robustness. MuDPIT is advantageous as compared to other approaches because it is direct, highly sensitive, and reproducible, it provides high resolution with ultra-high mass accuracy, it allows for relative quantifications, and it is compatible with multiplexing (thus limiting costs).This method enables the direct assessment of the proteomic profile of neoplastic cells and tissues and could be employed in the near future as a high-throughput, rapid, quantitative, and cost-effective screening platform for clinical samples. © 2014 Elsevier Inc. All rights reserved.

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.

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.

Driver hand-held cellular phone use: a four-year analysis.

PubMed

Eby, David W; Vivoda, Jonathon M; St Louis, Renée M

2006-01-01

The use of hand-held cellular (mobile) phones while driving has stirred more debate, passion, and research than perhaps any other traffic safety issue in the past several years. There is ample research showing that the use of either hand-held or hands-free cellular phones can lead to unsafe driving patterns. Whether or not these performance deficits increase the risk of crash is difficult to establish, but recent studies are beginning to suggest that cellular phone use elevates crash risk. The purpose of this study was to assess changes in the rate of hand-held cellular phone use by motor-vehicle drivers on a statewide level in Michigan. This study presents the results of 13 statewide surveys of cellular phone use over a 4-year period. Hand-held cellular phone use data were collected through direct observation while vehicles were stopped at intersections and freeway exit ramps. Data were weighted to be representative of all drivers traveling during daylight hours in Michigan. The study found that driver hand-held cellular phone use has more than doubled between 2001 and 2005, from 2.7% to 5.8%. This change represents an average increase of 0.78 percentage points per year. The 5.8% use rate observed in 2005 means that at any given daylight hour, around 36,550 drivers were conversing on cellular phones while driving on Michigan roadways. The trend line fitted to these data predicts that by the year 2010, driver hand-held cellular phone use will be around 8.6%, or 55,000 drivers at any given daylight hour. These results make it clear that cellular phone use while driving will continue to be an important traffic safety issue, and highlight the importance of continued attempts to generate new ways of alleviating this potential hazard.

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

ERIC Educational Resources Information Center

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

2010-01-01

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

Biomechanics of cellular solids.

PubMed

Gibson, Lorna J

2005-03-01

Materials with a cellular structure are widespread in nature and include wood, cork, plant parenchyma and trabecular bone. Natural cellular materials are often mechanically efficient: the honeycomb-like microstructure of wood, for instance, gives it an exceptionally high performance index for resisting bending and buckling. Here we review the mechanics of a wide range of natural cellular materials and examine their role in lightweight natural sandwich structures (e.g. iris leaves) and natural tubular structures (e.g. plant stems or animal quills). We also describe two examples of engineered biomaterials with a cellular structure, designed to replace or regenerate tissue in the body.

Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

PubMed

Nagayasu, Yuko; Morita, Shin-Ya; Hayashi, Hideki; Miura, Yutaka; Yokoyama, Kazuki; Michikawa, Makoto; Ito, Jin-Ichi

2014-05-14

We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress

Correlates of Protective Cellular Immunity Revealed by Analysis of Population-Level Immune Escape Pathways in HIV-1

PubMed Central

Brumme, Chanson J.; Martin, Eric; Listgarten, Jennifer; Brockman, Mark A.; Le, Anh Q.; Chui, Celia K. S.; Cotton, Laura A.; Knapp, David J. H. F.; Riddler, Sharon A.; Haubrich, Richard; Nelson, George; Pfeifer, Nico; DeZiel, Charles E.; Heckerman, David; Apps, Richard; Carrington, Mary; Mallal, Simon; Harrigan, P. Richard; John, Mina

2012-01-01

HLA class I-associated polymorphisms identified at the population level mark viral sites under immune pressure by individual HLA alleles. As such, analysis of their distribution, frequency, location, statistical strength, sequence conservation, and other properties offers a unique perspective from which to identify correlates of protective cellular immunity. We analyzed HLA-associated HIV-1 subtype B polymorphisms in 1,888 treatment-naïve, chronically infected individuals using phylogenetically informed methods and identified characteristics of HLA-associated immune pressures that differentiate protective and nonprotective alleles. Over 2,100 HLA-associated HIV-1 polymorphisms were identified, approximately one-third of which occurred inside or within 3 residues of an optimally defined cytotoxic T-lymphocyte (CTL) epitope. Differential CTL escape patterns between closely related HLA alleles were common and increased with greater evolutionary distance between allele group members. Among 9-mer epitopes, mutations at HLA-specific anchor residues represented the most frequently detected escape type: these occurred nearly 2-fold more frequently than expected by chance and were computationally predicted to reduce peptide-HLA binding nearly 10-fold on average. Characteristics associated with protective HLA alleles (defined using hazard ratios for progression to AIDS from natural history cohorts) included the potential to mount broad immune selection pressures across all HIV-1 proteins except Nef, the tendency to drive multisite and/or anchor residue escape mutations within known CTL epitopes, and the ability to strongly select mutations in conserved regions within HIV's structural and functional proteins. Thus, the factors defining protective cellular immune responses may be more complex than simply targeting conserved viral regions. The results provide new information to guide vaccine design and immunogenicity studies. PMID:23055555

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.

Impact of ionic current variability on human ventricular cellular electrophysiology.

PubMed

Romero, Lucía; Pueyo, Esther; Fink, Martin; Rodríguez, Blanca

2009-10-01

Abnormalities in repolarization and its rate dependence are known to be related to increased proarrhythmic risk. A number of repolarization-related electrophysiological properties are commonly used as preclinical biomarkers of arrhythmic risk. However, the variability and complexity of repolarization mechanisms make the use of cellular biomarkers to predict arrhythmic risk preclinically challenging. Our goal is to investigate the role of ionic current properties and their variability in modulating cellular biomarkers of arrhythmic risk to improve risk stratification and identification in humans. A systematic investigation into the sensitivity of the main preclinical biomarkers of arrhythmic risk to changes in ionic current conductances and kinetics was performed using computer simulations. Four stimulation protocols were applied to the ten Tusscher and Panfilov human ventricular model to quantify the impact of +/-15 and +/-30% variations in key model parameters on action potential (AP) properties, Ca(2+) and Na(+) dynamics, and their rate dependence. Simulations show that, in humans, AP duration is moderately sensitive to changes in all repolarization current conductances and in L-type Ca(2+) current (I(CaL)) and slow component of the delayed rectifier current (I(Ks)) inactivation kinetics. AP triangulation, however, is strongly dependent only on inward rectifier K(+) current (I(K1)) and delayed rectifier current (I(Kr)) conductances. Furthermore, AP rate dependence (i.e., AP duration rate adaptation and restitution properties) and intracellular Ca(2+) and Na(+) levels are highly sensitive to both I(CaL) and Na(+)/K(+) pump current (I(NaK)) properties. This study provides quantitative insights into the sensitivity of preclinical biomarkers of arrhythmic risk to variations in ionic current properties in humans. The results show the importance of sensitivity analysis as a powerful method for the in-depth validation of mathematical models in cardiac electrophysiology.

Low-level lasers alter mRNA levels from traditional reference genes used in breast cancer cells

NASA Astrophysics Data System (ADS)

Teixeira, A. F.; Canuto, K. S.; Rodrigues, J. A.; Fonseca, A. S.; Mencalha, A. L.

2017-07-01

Cancer is among the leading causes of mortality worldwide, increasing the importance of treatment development. Low-level lasers are used in several diseases, but some concerns remains on cancers. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a technique used to understand cellular behavior through quantification of mRNA levels. Output data from target genes are commonly relative to a reference that cannot vary according to treatment. This study evaluated reference genes levels from MDA-MB-231 cells exposed to red or infrared lasers at different fluences. Cultures were exposed to red and infrared lasers, incubated (4 h, 37 °C), total RNA was extracted and cDNA synthesis was performed to evaluate mRNA levels from ACTB, GUSB and TRFC genes by RT-qPCR. Specific amplification was verified by melting curves and agarose gel electrophoresis. RefFinder enabled data analysis by geNorm, NormFinder and BestKeeper. Specific amplifications were obtained and, although mRNA levels from ACTB, GUSB or TRFC genes presented no significant variation through traditional statistical analysis, Excel-based tools revealed that the use of these reference genes are dependent of laser characteristics. Our data showed that exposure to low-level red and infrared lasers at different fluences alter the mRNA levels from ACTB, GUSB and TRFC in MDA-MB-231 cells.

Relative Contributions of Various Cellular Mechanisms to Loss of Algae during Cnidarian Bleaching.

PubMed

Bieri, Tamaki; Onishi, Masayuki; Xiang, Tingting; Grossman, Arthur R; Pringle, John R

2016-01-01

When exposed to stress such as high seawater temperature, corals and other cnidarians can bleach due to loss of symbiotic algae from the host tissue and/or loss of pigments from the algae. Although the environmental conditions that trigger bleaching are reasonably well known, its cellular and molecular mechanisms are not well understood. Previous studies have reported the occurrence of at least four different cellular mechanisms for the loss of symbiotic algae from the host tissue: in situ degradation of algae, exocytic release of algae from the host, detachment of host cells containing algae, and death of host cells containing algae. The relative contributions of these several mechanisms to bleaching remain unclear, and it is also not known whether these relative contributions change in animals subjected to different types and/or durations of stresses. In this study, we used a clonal population of the small sea anemone Aiptasia, exposed individuals to various precisely controlled stress conditions, and quantitatively assessed the several possible bleaching mechanisms in parallel. Under all stress conditions tested, except for acute cold shock at 4°C, expulsion of intact algae from the host cells appeared to be by far the predominant mechanism of bleaching. During acute cold shock, in situ degradation of algae and host-cell detachment also became quantitatively significant, and the algae released under these conditions appeared to be severely damaged.

Relative Contributions of Various Cellular Mechanisms to Loss of Algae during Cnidarian Bleaching

PubMed Central

Bieri, Tamaki; Onishi, Masayuki; Xiang, Tingting; Grossman, Arthur R.; Pringle, John R

2016-01-01

When exposed to stress such as high seawater temperature, corals and other cnidarians can bleach due to loss of symbiotic algae from the host tissue and/or loss of pigments from the algae. Although the environmental conditions that trigger bleaching are reasonably well known, its cellular and molecular mechanisms are not well understood. Previous studies have reported the occurrence of at least four different cellular mechanisms for the loss of symbiotic algae from the host tissue: in situ degradation of algae, exocytic release of algae from the host, detachment of host cells containing algae, and death of host cells containing algae. The relative contributions of these several mechanisms to bleaching remain unclear, and it is also not known whether these relative contributions change in animals subjected to different types and/or durations of stresses. In this study, we used a clonal population of the small sea anemone Aiptasia, exposed individuals to various precisely controlled stress conditions, and quantitatively assessed the several possible bleaching mechanisms in parallel. Under all stress conditions tested, except for acute cold shock at 4°C, expulsion of intact algae from the host cells appeared to be by far the predominant mechanism of bleaching. During acute cold shock, in situ degradation of algae and host-cell detachment also became quantitatively significant, and the algae released under these conditions appeared to be severely damaged. PMID:27119147

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

PubMed

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

2016-01-01

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

Electromagnetic cellular interactions.

PubMed

Cifra, Michal; Fields, Jeremy Z; Farhadi, Ashkan

2011-05-01

Chemical and electrical interaction within and between cells is well established. Just the opposite is true about cellular interactions via other physical fields. The most probable candidate for an other form of cellular interaction is the electromagnetic field. We review theories and experiments on how cells can generate and detect electromagnetic fields generally, and if the cell-generated electromagnetic field can mediate cellular interactions. We do not limit here ourselves to specialized electro-excitable cells. Rather we describe physical processes that are of a more general nature and probably present in almost every type of living cell. The spectral range included is broad; from kHz to the visible part of the electromagnetic spectrum. We show that there is a rather large number of theories on how cells can generate and detect electromagnetic fields and discuss experimental evidence on electromagnetic cellular interactions in the modern scientific literature. Although small, it is continuously accumulating. Copyright © 2010 Elsevier Ltd. All rights reserved.

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.

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.

The cellular transducer in bone: What is it?

PubMed

Taylor, David; Hazenberg, Jan; Lee, T Clive

2006-01-01

Bone is able to detect its strain environment and respond accordingly. In particular it is able to adapt to over-use and under-use by bone deposition or resorption. How can bone sense strain? Various physical mechanisms have been proposed for the so-called cellular transducer, but there is no conclusive proof for any one of them. This paper examines the theories and evidence, with particular reference to a new theory proposed by the authors, involving damage to cellular processes by microcracks. Experiments on bone samples ex-vivo showed that cracks cannot fracture osteocytes, but that cellular processes which span the crack can be broken. A theoretical model was developed for predicting the number of broken processes as a function of crack size and applied stress. This showed that signals emitted by fractured processes could be used to detect cracks which needed repairing and to provide information on the overall level of damage which could be used to initiate repair and adaptation responses.

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

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

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.

Determining absolute protein numbers by quantitative fluorescence microscopy.

PubMed

Verdaasdonk, Jolien Suzanne; Lawrimore, Josh; Bloom, Kerry

2014-01-01

Biological questions are increasingly being addressed using a wide range of quantitative analytical tools to examine protein complex composition. Knowledge of the absolute number of proteins present provides insights into organization, function, and maintenance and is used in mathematical modeling of complex cellular dynamics. In this chapter, we outline and describe three microscopy-based methods for determining absolute protein numbers--fluorescence correlation spectroscopy, stepwise photobleaching, and ratiometric comparison of fluorescence intensity to known standards. In addition, we discuss the various fluorescently labeled proteins that have been used as standards for both stepwise photobleaching and ratiometric comparison analysis. A detailed procedure for determining absolute protein number by ratiometric comparison is outlined in the second half of this chapter. Counting proteins by quantitative microscopy is a relatively simple yet very powerful analytical tool that will increase our understanding of protein complex composition. © 2014 Elsevier Inc. All rights reserved.

Interconnectivity of human cellular metabolism and disease prevalence

NASA Astrophysics Data System (ADS)

Lee, Deok-Sun

2010-12-01

Fluctuations of metabolic reaction fluxes may cause abnormal concentrations of toxic or essential metabolites, possibly leading to metabolic diseases. The mutual binding of enzymatic proteins and ones involving common metabolites enforces distinct coupled reactions, by which local perturbations may spread through the cellular network. Such network effects at the molecular interaction level in human cellular metabolism can reappear in the patterns of disease occurrence. Here we construct the enzyme-reaction network and the metabolite-reaction network, capturing the flux coupling of metabolic reactions caused by the interacting enzymes and the shared metabolites, respectively. Diseases potentially caused by the failure of individual metabolic reactions can be identified by using the known disease-gene association, which allows us to derive the probability of an inactivated reaction causing diseases from the disease records at the population level. We find that the greater the number of proteins that catalyze a reaction, the higher the mean prevalence of its associated diseases. Moreover, the number of connected reactions and the mean size of the avalanches in the networks constructed are also shown to be positively correlated with the disease prevalence. These findings illuminate the impact of the cellular network topology on disease development, suggesting that the global organization of the molecular interaction network should be understood to assist in disease diagnosis, treatment, and drug discovery.

Hydrogen Peroxide Probes Directed to Different Cellular Compartments

PubMed Central

Malinouski, Mikalai; Zhou, You; Belousov, Vsevolod V.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

Background Controlled generation and removal of hydrogen peroxide play important roles in cellular redox homeostasis and signaling. We used a hydrogen peroxide biosensor HyPer, targeted to different compartments, to examine these processes in mammalian cells. Principal Findings Reversible responses were observed to various redox perturbations and signaling events. HyPer expressed in HEK 293 cells was found to sense low micromolar levels of hydrogen peroxide. When targeted to various cellular compartments, HyPer occurred in the reduced state in the nucleus, cytosol, peroxisomes, mitochondrial intermembrane space and mitochondrial matrix, but low levels of the oxidized form of the biosensor were also observed in each of these compartments, consistent with a low peroxide tone in mammalian cells. In contrast, HyPer was mostly oxidized in the endoplasmic reticulum. Using this system, we characterized control of hydrogen peroxide in various cell systems, such as cells deficient in thioredoxin reductase, sulfhydryl oxidases or subjected to selenium deficiency. Generation of hydrogen peroxide could also be monitored in various compartments following signaling events. Conclusions We found that HyPer can be used as a valuable tool to monitor hydrogen peroxide generated in different cellular compartments. The data also show that hydrogen peroxide generated in one compartment could translocate to other compartments. Our data provide information on compartmentalization, dynamics and homeostatic control of hydrogen peroxide in mammalian cells. PMID:21283738

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.

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.

Gene, Immune and Cellular Responses to Single and Combined Space Flight Conditions-B (TripleLux-B):

NASA Image and Video Library

2015-03-31

ISS043E070945 (03/31/2015) --- ESA (European Space Agency) astronaut Samantha Cristoforetti, Expedition 43 flight engineer aboard the International Space Station, is seen working on a science experiment that includes photographic documentation of Cellular Responses to Single and Combined Space Flight Conditions. Some effects of the space environment level appear to act at the cellular level and it is important to understand the underlying mechanisms of these effects. This science project uses invertebrate hemocytes to focus on two aspects of cellular function which may have medical importance. The synergy between the effects of the space radiation environment and microgravity on cellular function is the goal of this experiment along with studying the impairment of immune functions under spaceflight conditions.

Kinetic theory approach to modeling of cellular repair mechanisms under genome stress.

PubMed

Qi, Jinpeng; Ding, Yongsheng; Zhu, Ying; Wu, Yizhi

2011-01-01

Under acute perturbations from outer environment, a normal cell can trigger cellular self-defense mechanism in response to genome stress. To investigate the kinetics of cellular self-repair process at single cell level further, a model of DNA damage generating and repair is proposed under acute Ion Radiation (IR) by using mathematical framework of kinetic theory of active particles (KTAP). Firstly, we focus on illustrating the profile of Cellular Repair System (CRS) instituted by two sub-populations, each of which is made up of the active particles with different discrete states. Then, we implement the mathematical framework of cellular self-repair mechanism, and illustrate the dynamic processes of Double Strand Breaks (DSBs) and Repair Protein (RP) generating, DSB-protein complexes (DSBCs) synthesizing, and toxins accumulating. Finally, we roughly analyze the capability of cellular self-repair mechanism, cellular activity of transferring DNA damage, and genome stability, especially the different fates of a certain cell before and after the time thresholds of IR perturbations that a cell can tolerate maximally under different IR perturbation circumstances.

Synthesizing qualitative and quantitative evidence on non-financial access barriers: implications for assessment at the district level.

PubMed

O'Connell, Thomas S; Bedford, K Juliet A; Thiede, Michael; McIntyre, Di

2015-06-09

A key element of the global drive to universal health coverage is ensuring access to needed health services for everyone, and to pursue this goal in an equitable way. This requires concerted efforts to reduce disparities in access through understanding and acting on barriers facing communities with the lowest utilisation levels. Financial barriers dominate the empirical literature on health service access. Unless the full range of access barriers are investigated, efforts to promote equitable access to health care are unlikely to succeed. This paper therefore focuses on exploring the nature and extent of non-financial access barriers. We draw upon two structured literature reviews on barriers to access and utilization of maternal, newborn and child health services in Ghana, Bangladesh, Vietnam and Rwanda. One review analyses access barriers identified in published literature using qualitative research methods; the other in published literature using quantitative analysis of household survey data. We then synthesised the key qualitative and quantitative findings through a conjoint iterative analysis. Five dominant themes on non-financial access barriers were identified: ethnicity; religion; physical accessibility; decision-making, gender and autonomy; and knowledge, information and education. The analysis highlighted that non-financial factors pose considerable barriers to access, many of which relate to the acceptability dimension of access and are challenging to address. Another key finding is that quantitative research methods, while yielding important findings, are inadequate for understanding non-financial access barriers in sufficient detail to develop effective responses. Qualitative research is critical in filling this gap. The analysis also indicates that the nature of non-financial access barriers vary considerably, not only between countries but also between different communities within individual countries. To adequately understand access barriers as a

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.

Effects of high CO₂ levels on fermentation, peroxidation, and cellular water stress in Fragaria vesca stored at low temperature in conditions of unlimited O₂.

PubMed

Blanch, Maria; Rosales, Raquel; Mateos, Raquel; Perez-Gago, María B; Sanchez-Ballesta, Maria T; Escribano, María I; Merodio, Carmen

2015-01-28

To better understand the tolerance of strawberries (Fragaria vesca L.) to high CO2 in storage atmospheres, fermentation and cellular damage were investigated. Fruits were stored for 3 and 6 days at 0 °C in the presence of different CO2 levels (0, 20, or 40%) with 20% O2. Changes in pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) gene expression and in fermentative metabolites, as well as in bound water and malondialdehyde (MDA) concentrations, were analyzed. In strawberries stored without added CO2, up-regulation of PDC and ADH was not associated with an increase in fermentative metabolites. By contrast, moderate ethanol fermentation in fruits exposed to 20% CO2 seems to be essential to maintain fruit metabolism, reducing both lipid peroxidation and cellular water stress. However, if the CO2 concentration increases (40%), the excess acetaldehyde and ethanol produced were closely correlated with a decrease in bound water and production of MDA.

Cellular Particle Dynamics simulation of biomechanical relaxation processes of multi-cellular systems

NASA Astrophysics Data System (ADS)

McCune, Matthew; Kosztin, Ioan

2013-03-01

Cellular Particle Dynamics (CPD) is a theoretical-computational-experimental framework for describing and predicting the time evolution of biomechanical relaxation processes of multi-cellular systems, such as fusion, sorting and compression. 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 numerical integration of their equations of motion. Here we present CPD simulation results for the fusion of both spherical and cylindrical multi-cellular aggregates. First, we calibrate the relevant CPD model parameters for a given cell type by comparing the CPD simulation results for the fusion of two spherical aggregates to the corresponding experimental results. Next, CPD simulations are used to predict the time evolution of the fusion of cylindrical aggregates. The latter is relevant for the formation of tubular multi-cellular structures (i.e., primitive blood vessels) created by the novel bioprinting technology. Work supported by NSF [PHY-0957914]. Computer time provided by the University of Missouri Bioinformatics Consortium.

High-throughput dual-colour precision imaging for brain-wide connectome with cytoarchitectonic landmarks at the cellular level

PubMed Central

Gong, Hui; Xu, Dongli; Yuan, Jing; Li, Xiangning; Guo, Congdi; Peng, Jie; Li, Yuxin; Schwarz, Lindsay A.; Li, Anan; Hu, Bihe; Xiong, Benyi; Sun, Qingtao; Zhang, Yalun; Liu, Jiepeng; Zhong, Qiuyuan; Xu, Tonghui; Zeng, Shaoqun; Luo, Qingming

2016-01-01

The precise annotation and accurate identification of neural structures are prerequisites for studying mammalian brain function. The orientation of neurons and neural circuits is usually determined by mapping brain images to coarse axial-sampling planar reference atlases. However, individual differences at the cellular level likely lead to position errors and an inability to orient neural projections at single-cell resolution. Here, we present a high-throughput precision imaging method that can acquire a co-localized brain-wide data set of both fluorescent-labelled neurons and counterstained cell bodies at a voxel size of 0.32 × 0.32 × 2.0 μm in 3 days for a single mouse brain. We acquire mouse whole-brain imaging data sets of multiple types of neurons and projections with anatomical annotation at single-neuron resolution. The results show that the simultaneous acquisition of labelled neural structures and cytoarchitecture reference in the same brain greatly facilitates precise tracing of long-range projections and accurate locating of nuclei. PMID:27374071

Cellular reprogramming through mitogen-activated protein kinases.

PubMed

Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

2015-01-01

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression-including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly-Diagnosed Patients in Europe

PubMed Central

Demetriou, Victoria L.; van de Vijver, David A. M. C.; Kousiappa, Ioanna; Balotta, Claudia; Clotet, Bonaventura; Grossman, Zehava; Jørgensen, Louise B.; Lepej, Snjezana Z.; Levy, Itzchak; Nielsen, Claus; Paraskevis, Dimitrios; Poljak, Mario; Roman, Francois; Ruiz, Lidia; Schmidt, Jean-Claude; Vandamme, Anne-Mieke; Van Laethem, Kristel; Vercauteren, Jurgen; Kostrikis, Leondios G.

2010-01-01

Background HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load. Methodology/Principal Findings Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log10 HIV-1 copies/106 PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4+ T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two. Conclusions/Significance An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furthermore, a correlation found between RNA- and DNA

A medaka model of cancer allowing direct observation of transplanted tumor cells in vivo at a cellular-level resolution.

PubMed

Hasegawa, Sumitaka; Maruyama, Kouichi; Takenaka, Hikaru; Furukawa, Takako; Saga, Tsuneo

2009-08-18

The recent success with small fish as an animal model of cancer with the aid of fluorescence technique has attracted cancer modelers' attention because it would be possible to directly visualize tumor cells in vivo in real time. Here, we report a medaka model capable of allowing the observation of various cell behaviors of transplanted tumor cells, such as cell proliferation and metastasis, which were visualized easily in vivo. We established medaka melanoma (MM) cells stably expressing GFP and transplanted them into nonirradiated and irradiated medaka. The tumor cells were grown at the injection sites in medaka, and the spatiotemporal changes were visualized under a fluorescence stereoscopic microscope at a cellular-level resolution, and even at a single-cell level. Tumor dormancy and metastasis were also observed. Interestingly, in irradiated medaka, accelerated tumor growth and metastasis of the transplanted tumor cells were directly visualized. Our medaka model provides an opportunity to visualize in vivo tumor cells "as seen in a culture dish" and would be useful for in vivo tumor cell biology.

Waning and aging of cellular immunity to Bordetella pertussis.

PubMed

van Twillert, Inonge; Han, Wanda G H; van Els, Cécile A C M

2015-11-01

While it is clear that the maintenance of Bordetella pertussis-specific immunity evoked both after vaccination and infection is insufficient, it is unknown at which pace waning occurs and which threshold levels of sustained functional memory B and T cells are required to provide long-term protection. Longevity of human cellular immunity to B. pertussis has been studied less extensively than serology, but is suggested to be key for the observed differences between the duration of protection induced by acellular vaccination and whole cell vaccination or infection. The induction and maintenance of levels of protective memory B and T cells may alter with age, associated with changes of the immune system throughout life and with accumulating exposures to circulating B. pertussis or vaccine doses. This is relevant since pertussis affects all age groups. This review summarizes current knowledge on the waning patterns of human cellular immune responses to B. pertussis as addressed in diverse vaccination and infection settings and in various age groups. Knowledge on the effectiveness and flaws in human B. pertussis-specific cellular immunity ultimately will advance the improvement of pertussis vaccination strategies. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

PubMed

Gizak, Agnieszka; Rakus, Dariusz

2016-01-11

Molecular and cellular biology methodology is traditionally based on the reasoning called "the mechanistic explanation". In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems' complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites), and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

Cellular functions of the microprocessor.

PubMed

Macias, Sara; Cordiner, Ross A; Cáceres, Javier F

2013-08-01

The microprocessor is a complex comprising the RNase III enzyme Drosha and the double-stranded RNA-binding protein DGCR8 (DiGeorge syndrome critical region 8 gene) that catalyses the nuclear step of miRNA (microRNA) biogenesis. DGCR8 recognizes the RNA substrate, whereas Drosha functions as an endonuclease. Recent global analyses of microprocessor and Dicer proteins have suggested novel functions for these components independent of their role in miRNA biogenesis. A HITS-CLIP (high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation) experiment designed to identify novel substrates of the microprocessor revealed that this complex binds and regulates a large variety of cellular RNAs. The microprocessor-mediated cleavage of several classes of RNAs not only regulates transcript levels, but also modulates alternative splicing events, independently of miRNA function. Importantly, DGCR8 can also associate with other nucleases, suggesting the existence of alternative DGCR8 complexes that may regulate the fate of a subset of cellular RNAs. The aim of the present review is to provide an overview of the diverse functional roles of the microprocessor.

Continuum-level modelling of cellular adhesion and matrix production in aggregates.

PubMed

Geris, Liesbet; Ashbourn, Joanna M A; Clarke, Tim

2011-05-01

Key regulators in tissue-engineering processes such as cell culture and cellular organisation are the cell-cell and cell-matrix interactions. As mathematical models are increasingly applied to investigate biological phenomena in the biomedical field, it is important, for some applications, that these models incorporate an adequate description of cell adhesion. This study describes the development of a continuum model that represents a cell-in-gel culture system used in bone-tissue engineering, namely that of a cell aggregate embedded in a hydrogel. Cell adhesion is modelled through the use of non-local (integral) terms in the partial differential equations. The simulation results demonstrate that the effects of cell-cell and cell-matrix adhesion are particularly important for the survival and growth of the cell population and the production of extracellular matrix by the cells, concurring with experimental observations in the literature.

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

Dynamic behavior of cellular materials and cellular structures: Experiments and modeling

NASA Astrophysics Data System (ADS)

Gao, Ziyang

Cellular solids, including cellular materials and cellular structures (CMS), have attracted people's great interests because of their low densities and novel physical, mechanical, thermal, electrical and acoustic properties. They offer potential for lightweight structures, energy absorption, thermal management, etc. Therefore, the studies of cellular solids have become one of the hottest research fields nowadays. From energy absorption point of view, any plastically deformed structures can be divided into two types (called type I and type II), and the basic cells of the CMS may take the configurations of these two types of structures. Accordingly, separated discussions are presented in this thesis. First, a modified 1-D model is proposed and numerically solved for a typical type II structure. Good agreement is achieved with the previous experimental data, hence is used to simulate the dynamic behavior of a type II chain. Resulted from different load speeds, interesting collapse modes are observed, and the parameters which govern the cell's post-collapse behavior are identified through a comprehensive non-dimensional analysis on general cellular chains. Secondly, the MHS specimens are chosen as an example of type I foam materials because of their good uniformity of the cell geometry. An extensive experimental study was carried out, where more attention was paid to their responses to dynamic loadings. Great enhancement of the stress-strain curve was observed in dynamic cases, and the energy absorption capacity is found to be several times higher than that of the commercial metal foams. Based on the experimental study, finite elemental simulations and theoretical modeling are also conducted, achieving good agreements and demonstrating the validities of those models. It is believed that the experimental, numerical and analytical results obtained in the present study will certainly deepen the understanding of the unsolved fundamental issues on the mechanical behavior of

Multi-Cellular Logistics of Collective Cell Migration

PubMed Central

Yamao, Masataka; Naoki, Honda; Ishii, Shin

2011-01-01

During development, the formation of biological networks (such as organs and neuronal networks) is controlled by multicellular transportation phenomena based on cell migration. In multi-cellular systems, cellular locomotion is restricted by physical interactions with other cells in a crowded space, similar to passengers pushing others out of their way on a packed train. The motion of individual cells is intrinsically stochastic and may be viewed as a type of random walk. However, this walk takes place in a noisy environment because the cell interacts with its randomly moving neighbors. Despite this randomness and complexity, development is highly orchestrated and precisely regulated, following genetic (and even epigenetic) blueprints. Although individual cell migration has long been studied, the manner in which stochasticity affects multi-cellular transportation within the precisely controlled process of development remains largely unknown. To explore the general principles underlying multicellular migration, we focus on the migration of neural crest cells, which migrate collectively and form streams. We introduce a mechanical model of multi-cellular migration. Simulations based on the model show that the migration mode depends on the relative strengths of the noise from migratory and non-migratory cells. Strong noise from migratory cells and weak noise from surrounding cells causes “collective migration,” whereas strong noise from non-migratory cells causes “dispersive migration.” Moreover, our theoretical analyses reveal that migratory cells attract each other over long distances, even without direct mechanical contacts. This effective interaction depends on the stochasticity of the migratory and non-migratory cells. On the basis of these findings, we propose that stochastic behavior at the single-cell level works effectively and precisely to achieve collective migration in multi-cellular systems. PMID:22205934

Cellular stress associated with the differentiation of Plasmodium berghei ookinetes.

PubMed

Duran-Bedolla, Josefina; Téllez-Sosa, Juan; Valdovinos-Torres, Humberto; Pavón, Natalia; Buelna-Chontal, Mabel; Tello-López, Angel T; Argotte-Ramos, Rocio; Rodríguez, Mario Henry; Rodríguez, María Carmen

2017-04-01

For malaria transmission, Plasmodium parasites must develop in the mosquito vector. Oxidative stress in the insect midgut, triggered by environmental changes (e.g., pH and temperature), influences the cellular signaling involved in differentiation from gametocytes to mobile ookinetes for the purpose of parasite survival. Oxidative stress activates the homeostatic response to stress characterized by the phosphorylation eIF2α, the attenuation of protein synthesis, and the transcription of genes participating in the unfolded protein response and antioxidant processes, forming a part of an integrated stress response (ISR). We hypothesized that ISR operates during the differentiation of gametocytes to ookinetes to assure Plasmodium survival. Using in-vitro conditions resembling the mosquito midgut conditions, we cultured Plasmodium berghei gametocytes to ookinetes and evaluated the redox balance by detecting reactive oxygen species and superoxide dismutase activity. Additionally, we evaluated the phosphorylation of eIF2α, the attenuation of the global protein synthesis, and the gene expression of cellular stress markers (e.g., endoplasmic reticulum chaperones and antioxidant molecules, measured by reverse-transcription quantitative polymerase chain reaction), finding that these processes were all taking place, probably to improve survival during the differentiation of Plasmodium berghei ookinetes.

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

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.

MSAT and cellular hybrid networking

NASA Astrophysics Data System (ADS)

Baranowsky, Patrick W., II

Westinghouse Electric Corporation is developing both the Communications Ground Segment and the Series 1000 Mobile Phone for American Mobile Satellite Corporation's (AMSC's) Mobile Satellite (MSAT) system. The success of the voice services portion of this system depends, to some extent, upon the interoperability of the cellular network and the satellite communication circuit switched communication channels. This paper will describe the set of user-selectable cellular interoperable modes (cellular first/satellite second, etc.) provided by the Mobile Phone and described how they are implemented with the ground segment. Topics including roaming registration and cellular-to-satellite 'seamless' call handoff will be discussed, along with the relevant Interim Standard IS-41 Revision B Cellular Radiotelecommunications Intersystem Operations and IOS-553 Mobile Station - Land Station Compatibility Specification.

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

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

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.

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

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

PubMed

Khor, S C; Mohd Yusof, Y A; Wan Ngah, W Z; Makpol, S

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.

Raised protein levels and altered cellular expression of factor VII activating protease (FSAP) in the lungs of patients with acute respiratory distress syndrome (ARDS)

PubMed Central

Wygrecka, Malgorzata; Markart, Philipp; Fink, Ludger; Guenther, Andreas; Preissner, Klaus T

2007-01-01

Background The acute respiratory distress syndrome (ARDS) is characterised by inflammation of the lung parenchyma and changes in alveolar haemostasis with extravascular fibrin deposition. Factor VII activating protease (FSAP) is a recently described serine protease in plasma and tissues known to be involved in haemostasis, cell proliferation and migration. Methods The level of FSAP protein expression was examined by western blotting/ELISA/immunohistochemistry and its activity was investigated by coagulation/fibrinolysis assays in plasma, bronchoalveolar lavage (BAL) fluid and lung tissue of mechanically ventilated patients with early ARDS and compared with patients with cardiogenic pulmonary oedema and healthy controls. Cell culture experiments were performed to assess the influence of different inflammatory stimuli on FSAP expression by various cell populations of the lung. Results FSAP protein level and activity were markedly increased in the plasma and BAL fluid of patients with ARDS with a significant contribution to the increased alveolar procoagulant activity. Immunoreactivity for FSAP was observed in alveolar macrophages, bronchial epithelial and endothelial cells of lungs of patients with ARDS, while in controls the immunoreactivity for FSAP was restricted to alveolar macrophages. Only a low basal level of FSAP expression was detected in these cell populations. However, FSAP‐specific mRNA expression was induced by lipopolysaccharide and interleukin‐8 in human lung microvascular endothelial cells and in bronchial epithelial cells. FSAP was also found to be taken up by alveolar macrophages and degraded within the lysosomal compartment. Conclusions Increased levels of FSAP and an altered cellular expression pattern are found in the lungs of patients with ARDS. This may represent a novel pathological mechanism which contributes to pulmonary extravascular fibrin deposition and may also modulate inflammation in the acutely injured lung via haemostasis�

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.

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

PubMed

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

2015-01-01

We present the nanosurgery on the cytoskeleton of live cells using AFM based nanorobotics to achieve adhesiolysis and mimic the effect of pathophysiological modulation of intercellular adhesion. Nanosurgery successfully severs the intermediate filament bundles and disrupts cell-cell adhesion similar to the desmosomal protein disassembly in autoimmune disease, or the cationic modulation of desmosome formation. Our nanomechanical analysis revealed that adhesion loss results in a decrease in cellular stiffness in both cases of biochemical modulation of the desmosome junctions and mechanical disruption of intercellular adhesion, supporting the notion that intercellular adhesion through intermediate filaments anchors the cell structure as focal adhesion does and that intermediate filaments are integral components in cell mechanical integrity. The surgical process could potentially help reveal the mechanism of autoimmune pathology-induced cell-cell adhesion loss as well as its related pathways that lead to cell apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Organochlorine pesticide, endosulfan induced cellular and organismal response in Drosophila melanogaster.

PubMed

Sharma, Anurag; Mishra, M; Shukla, A K; Kumar, R; Abdin, M Z; Chowdhuri, D Kar

2012-06-30

The effect of endosulfan (0.02-2.0μgmL(-1)) to Drosophila melanogaster (Oregon R(+)) at the cellular and organismal levels was examined. Third instar larvae of D. melanogaster and the strains transgenic for hsp70, hsp83 and hsp26 were exposed to endosulfan through food for 12-48h to examine the heat shock proteins (hsps), reactive oxygen species (ROS) generation, anti-oxidant stress markers and xenobiotic metabolism enzymes. We observed a concentration- and time-dependent significant induction of only small hsps (hsp23>hsp22) in the exposed organism in concurrence with a significant induction of ROS generation, oxidative stress and xenobiotic metabolism markers. Sub-organismal response was to be propagated towards organismal response, i.e., delay in the emergence of flies and decreased locomotor behaviour. Organisms with diminished locomotion also exhibited significantly lowered acetylcholinesterase activity. A significant positive correlation observed among ROS generation and different cellular endpoints (small hsps, oxidative stress markers, cytochrome P450 activities) in the exposed organism indicate a modulatory role of ROS in endosulfan-mediated cellular toxicity. The study thus suggests that the adverse effects of endosulfan in exposed Drosophila are manifested both at cellular and organismal levels and recommends Drosophila as an alternative animal model for screening the risk caused by environmental chemicals. Copyright © 2012 Elsevier B.V. All rights reserved.

Abscisic-acid-induced cellular apoptosis and differentiation in glioma via the retinoid acid signaling pathway.

PubMed

Zhou, Nan; Yao, Yu; Ye, Hongxing; Zhu, Wei; Chen, Liang; Mao, Ying

2016-04-15

Retinoid acid (RA) plays critical roles in regulating differentiation and apoptosis in a variety of cancer cells. Abscisic acid (ABA) and RA are direct derivatives of carotenoids and share structural similarities. Here we proposed that ABA may also play a role in cellular differentiation and apoptosis by sharing a similar signaling pathway with RA that may be involved in glioma pathogenesis. We reported for the first time that the ABA levels were twofold higher in low-grade gliomas compared with high-grade gliomas. In glioma tissues, there was a positive correlation between the ABA levels and the transcription of cellular retinoic acid-binding protein 2 (CRABP2) and a negative correlation between the ABA levels and transcription of fatty acid-binding protein 5 (FABP5). ABA treatment induced a significant increase in the expression of CRABP2 and a decrease in the expression of peroxisome proliferator-activated receptor (PPAR) in glioblastoma cells. Remarkably, both cellular apoptosis and differentiation were increased in the glioblastoma cells after ABA treatment. ABA-induced cellular apoptosis and differentiation were significantly reduced by selectively silencing RAR-α, while RAR-α overexpression exaggerated the ABA-induced effects. These results suggest that ABA may play a role in the pathogenesis of glioma by promoting cellular apoptosis and differentiation through the RA signaling pathway. © 2015 UICC.

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.

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.

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.

Cellular Telephones Measure Activity and Lifespace in Community-Dwelling Adults: Proof of Principle

PubMed Central

Schenk, Ana Katrin; Witbrodt, Bradley C.; Hoarty, Carrie A.; Carlson, Richard H.; Goulding, Evan H.; Potter, Jane F.; Bonasera, Stephen J.

2011-01-01

OBJECTIVES To describe a system that uses off-the-shelf sensor and telecommunication technologies to continuously measure individual lifespace and activity levels in a novel way. DESIGN Proof of concept involving three field trials of 30, 30, and 21 days. SETTING Omaha, Nebraska, metropolitan and surrounding rural region. PARTICIPANTS Three participants (48-year-old man, 33-year-old woman, and 27-year-old male), none with any functional limitations. MEASUREMENTS Cellular telephones were used to detect in-home position and in-community location and to measure physical activity. Within the home, cellular telephones and Bluetooth transmitters (beacons) were used to locate participants at room-level resolution. Outside the home, the same cellular telephones and global positioning system (GPS) technology were used to locate participants at a community-level resolution. Physical activity was simultaneously measured using the cellular telephone accelerometer. RESULTS This approach had face validity to measure activity and lifespace. More importantly, this system could measure the spatial and temporal organization of these metrics. For example, an individual’s lifespace was automatically calculated across multiple time intervals. Behavioral time budgets showing how people allocate time to specific regions within the home were also automatically generated. CONCLUSION Mobile monitoring shows much promise as an easily deployed system to quantify activity and lifespace, important indicators of function, in community-dwelling adults. PMID:21288235

Quantitative imaging with fluorescent biosensors.

PubMed

Okumoto, Sakiko; Jones, Alexander; Frommer, Wolf B

2012-01-01

Molecular activities are highly dynamic and can occur locally in subcellular domains or compartments. Neighboring cells in the same tissue can exist in different states. Therefore, quantitative information on the cellular and subcellular dynamics of ions, signaling molecules, and metabolites is critical for functional understanding of organisms. Mass spectrometry is generally used for monitoring ions and metabolites; however, its temporal and spatial resolution are limited. Fluorescent proteins have revolutionized many areas of biology-e.g., fluorescent proteins can report on gene expression or protein localization in real time-yet promoter-based reporters are often slow to report physiologically relevant changes such as calcium oscillations. Therefore, novel tools are required that can be deployed in specific cells and targeted to subcellular compartments in order to quantify target molecule dynamics directly. We require tools that can measure enzyme activities, protein dynamics, and biophysical processes (e.g., membrane potential or molecular tension) with subcellular resolution. Today, we have an extensive suite of tools at our disposal to address these challenges, including translocation sensors, fluorescence-intensity sensors, and Förster resonance energy transfer sensors. This review summarizes sensor design principles, provides a database of sensors for more than 70 different analytes/processes, and gives examples of applications in quantitative live cell imaging.

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

Highly sensitive and quantitative detection of rare pathogens through agarose droplet microfluidic emulsion PCR at the single-cell level.

PubMed

Zhu, Zhi; Zhang, Wenhua; Leng, Xuefei; Zhang, Mingxia; Guan, Zhichao; Lu, Jiangquan; Yang, Chaoyong James

2012-10-21

Genetic alternations can serve as highly specific biomarkers to distinguish fatal bacteria or cancer cells from their normal counterparts. However, these mutations normally exist in very rare amount in the presence of a large excess of non-mutated analogs. Taking the notorious pathogen E. coli O157:H7 as the target analyte, we have developed an agarose droplet-based microfluidic ePCR method for highly sensitive, specific and quantitative detection of rare pathogens in the high background of normal bacteria. Massively parallel singleplex and multiplex PCR at the single-cell level in agarose droplets have been successfully established. Moreover, we challenged the system with rare pathogen detection and realized the sensitive and quantitative analysis of a single E. coli O157:H7 cell in the high background of 100,000 excess normal K12 cells. For the first time, we demonstrated rare pathogen detection through agarose droplet microfluidic ePCR. Such a multiplex single-cell agarose droplet amplification method enables ultra-high throughput and multi-parameter genetic analysis of large population of cells at the single-cell level to uncover the stochastic variations in biological systems.

Levels of reconstruction as complementarity in mixed methods research: a social theory-based conceptual framework for integrating qualitative and quantitative research.

PubMed

Carroll, Linda J; Rothe, J Peter

2010-09-01

Like other areas of health research, there has been increasing use of qualitative methods to study public health problems such as injuries and injury prevention. Likewise, the integration of qualitative and quantitative research (mixed-methods) is beginning to assume a more prominent role in public health studies. Likewise, using mixed-methods has great potential for gaining a broad and comprehensive understanding of injuries and their prevention. However, qualitative and quantitative research methods are based on two inherently different paradigms, and their integration requires a conceptual framework that permits the unity of these two methods. We present a theory-driven framework for viewing qualitative and quantitative research, which enables us to integrate them in a conceptually sound and useful manner. This framework has its foundation within the philosophical concept of complementarity, as espoused in the physical and social sciences, and draws on Bergson's metaphysical work on the 'ways of knowing'. Through understanding how data are constructed and reconstructed, and the different levels of meaning that can be ascribed to qualitative and quantitative findings, we can use a mixed-methods approach to gain a conceptually sound, holistic knowledge about injury phenomena that will enhance our development of relevant and successful interventions.

AN APPROACH TO THE DEVELOPMENT OF MODELS TO QUANTITATIVELY ASSESS THE EFFECTS OF EXPOSURE TO ENVIRONMENTALLY RELEVANT LEVELS OF ENDOCRINE DISRUPTORS

EPA Science Inventory

An approach to the development of quantitative models to assess the effects of exposure to environmentally relevant levels of endocrine disruptors on homeostasis in adults.

Ben-Jonathan N, Cooper RL, Foster P, Hughes CL, Hoyer PB, Klotz D, Kohn M, Lamb DJ, Stancel GM.
<...

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

NASA Astrophysics Data System (ADS)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress

PubMed Central

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response. PMID:26442059

Expression of Arabidopsis FCS-Like Zinc finger genes is differentially regulated by sugars, cellular energy level, and abiotic stress.

PubMed

Jamsheer K, Muhammed; Laxmi, Ashverya

2015-01-01

Cellular energy status is an important regulator of plant growth, development, and stress mitigation. Environmental stresses ultimately lead to energy deficit in the cell which activates the SNF1-RELATED KINASE 1 (SnRK1) signaling cascade which eventually triggering a massive reprogramming of transcription to enable the plant to survive under low-energy conditions. The role of Arabidopsis thaliana FCS-Like Zinc finger (FLZ) gene family in energy and stress signaling is recently come to highlight after their interaction with kinase subunits of SnRK1 were identified. In a detailed expression analysis in different sugars, energy starvation, and replenishment series, we identified that the expression of most of the FLZ genes is differentially modulated by cellular energy level. It was found that FLZ gene family contains genes which are both positively and negatively regulated by energy deficit as well as energy-rich conditions. Genetic and pharmacological studies identified the role of HEXOKINASE 1- dependent and energy signaling pathways in the sugar-induced expression of FLZ genes. Further, these genes were also found to be highly responsive to different stresses as well as abscisic acid. In over-expression of kinase subunit of SnRK1, FLZ genes were found to be differentially regulated in accordance with their response toward energy fluctuation suggesting that these genes may work downstream to the established SnRK1 signaling under low-energy stress. Taken together, the present study provides a conceptual framework for further studies related to SnRK1-FLZ interaction in relation to sugar and energy signaling and stress response.

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

Integrated cellular network of transcription regulations and protein-protein interactions.

PubMed

Wang, Yu-Chao; Chen, Bor-Sen

2010-03-08

With the accumulation of increasing omics data, a key goal of systems biology is to construct networks at different cellular levels to investigate cellular machinery of the cell. However, there is currently no satisfactory method to construct an integrated cellular network that combines the gene regulatory network and the signaling regulatory pathway. In this study, we integrated different kinds of omics data and developed a systematic method to construct the integrated cellular network based on coupling dynamic models and statistical assessments. The proposed method was applied to S. cerevisiae stress responses, elucidating the stress response mechanism of the yeast. From the resulting integrated cellular network under hyperosmotic stress, the highly connected hubs which are functionally relevant to the stress response were identified. Beyond hyperosmotic stress, the integrated network under heat shock and oxidative stress were also constructed and the crosstalks of these networks were analyzed, specifying the significance of some transcription factors to serve as the decision-making devices at the center of the bow-tie structure and the crucial role for rapid adaptation scheme to respond to stress. In addition, the predictive power of the proposed method was also demonstrated. We successfully construct the integrated cellular network which is validated by literature evidences. The integration of transcription regulations and protein-protein interactions gives more insight into the actual biological network and is more predictive than those without integration. The method is shown to be powerful and flexible and can be used under different conditions and for different species. The coupling dynamic models of the whole integrated cellular network are very useful for theoretical analyses and for further experiments in the fields of network biology and synthetic biology.

Integrated Smartphone-App-Chip System for On-Site Parts-Per-Billion-Level Colorimetric Quantitation of Aflatoxins.

PubMed

Li, Xiaochun; Yang, Fan; Wong, Jessica X H; Yu, Hua-Zhong

2017-09-05

We demonstrate herein an integrated, smartphone-app-chip (SPAC) system for on-site quantitation of food toxins, as demonstrated with aflatoxin B1 (AFB1), at parts-per-billion (ppb) level in food products. The detection is based on an indirect competitive immunoassay fabricated on a transparent plastic chip with the assistance of a microfluidic channel plate. A 3D-printed optical accessory attached to a smartphone is adapted to align the assay chip and to provide uniform illumination for imaging, with which high-quality images of the assay chip are captured by the smartphone camera and directly processed using a custom-developed Android app. The performance of this smartphone-based detection system was tested using both spiked and moldy corn samples; consistent results with conventional enzyme-linked immunosorbent assay (ELISA) kits were obtained. The achieved detection limit (3 ± 1 ppb, equivalent to μg/kg) and dynamic response range (0.5-250 ppb) meet the requested testing standards set by authorities in China and North America. We envision that the integrated SPAC system promises to be a simple and accurate method of food toxin quantitation, bringing much benefit for rapid on-site screening.

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

PubMed Central

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

2013-01-01

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

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

Definitions and validation criteria for biomarkers and surrogate endpoints: development and testing of a quantitative hierarchical levels of evidence schema.

PubMed

Lassere, Marissa N; Johnson, Kent R; Boers, Maarten; Tugwell, Peter; Brooks, Peter; Simon, Lee; Strand, Vibeke; Conaghan, Philip G; Ostergaard, Mikkel; Maksymowych, Walter P; Landewe, Robert; Bresnihan, Barry; Tak, Paul-Peter; Wakefield, Richard; Mease, Philip; Bingham, Clifton O; Hughes, Michael; Altman, Doug; Buyse, Marc; Galbraith, Sally; Wells, George

2007-03-01

There are clear advantages to using biomarkers and surrogate endpoints, but concerns about clinical and statistical validity and systematic methods to evaluate these aspects hinder their efficient application. Our objective was to review the literature on biomarkers and surrogates to develop a hierarchical schema that systematically evaluates and ranks the surrogacy status of biomarkers and surrogates; and to obtain feedback from stakeholders. After a systematic search of Medline and Embase on biomarkers, surrogate (outcomes, endpoints, markers, indicators), intermediate endpoints, and leading indicators, a quantitative surrogate validation schema was developed and subsequently evaluated at a stakeholder workshop. The search identified several classification schema and definitions. Components of these were incorporated into a new quantitative surrogate validation level of evidence schema that evaluates biomarkers along 4 domains: Target, Study Design, Statistical Strength, and Penalties. Scores derived from 3 domains the Target that the marker is being substituted for, the Design of the (best) evidence, and the Statistical strength are additive. Penalties are then applied if there is serious counterevidence. A total score (0 to 15) determines the level of evidence, with Level 1 the strongest and Level 5 the weakest. It was proposed that the term "surrogate" be restricted to markers attaining Levels 1 or 2 only. Most stakeholders agreed that this operationalization of the National Institutes of Health definitions of biomarker, surrogate endpoint, and clinical endpoint was useful. Further development and application of this schema provides incentives and guidance for effective biomarker and surrogate endpoint research, and more efficient drug discovery, development, and approval.

Cellular fibronectin response to supervised moderate aerobic training in patients with type 2 diabetes

PubMed Central

Alghadir, Ahmad H.; Gabr, Sami A.; Al-Eisa, Einas

2016-01-01

[Purpose] Physical activity is one of the most pivotal targets for the prevention and management of vascular complications, especially endothelial dysfunctions. Cellular fibronectin is an endothelium-derived protein involved in subendothelial matrix assembly. Its plasma levels reflect matrix alterations and vessel wall destruction in patients with type II diabetes. This study investigated the influence of 12 weeks of supervised aerobic training on cellular fibronectin and its relationship with insulin resistance and body weight in type II diabetic subjects. [Subjects and Methods] This study included 50 men with type II diabetes who had a mean age of 48.8 ± 14.6 years and were randomly divided into two groups: an aerobic exercise group (12 weeks, three 50 minutes sessions per week) and control group. To examine changes in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile, 5 ml of blood was taken from the brachial vein of patients before and 48 hours after completion of the exercise period and after 12 hours of fasting at rest. Data analysis was performed using the SPSS-16 software with the independent and paired t-tests. [Results] A significant decrease was observed in body mass index and body fat percentage in the experimental group. Compared with the control group, the aerobic exercise group showed a significant decrease in cellular fibronectin, glycosylated hemoglobin, insulin resistance, fasting insulin, fasting blood sugar, and lipid profile after 12 weeks of aerobic exercise. The change in cellular fibronectin showed positive significant correlation with body mass index, diabetic biomarkers, and physical activity level. [Conclusion] The results showed that supervised aerobic exercise as a stimulus can change the levels of cellular fibronectin as matrix metalloproteinase protein a long with improvement of insulin sensitivity and glycosylated hemoglobin in order to prevent

Cellular internalization, transcellular transport, and cellular effects of silver nanoparticles in polarized Caco-2 cells following apical or basolateral exposure

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

Imai, Shunji; Morishita, Yuki; Hata, Tomoyuki

When considering the safety of ingested nanomaterials, it is important to quantitate their transfer across intestinal cells; however, little information exists about the effects of nanomaterial size or exposure side (apical versus basolateral epithelial surface) on nanomaterial transfer. Here, we examined cellular internalization and transcellular transport, and the effects of nanomaterials on Caco-2 monolayers after apical or basolateral exposure to Ag or Au nanoparticles with various sizes. After apical treatment, both internalization and transfer to the basolateral side of the monolayers were greater for smaller Ag nanoparticles than for larger Ag nanoparticles. In contrast, after basolateral treatment, larger Ag nanoparticlesmore » were more internalized than smaller Ag nanoparticles, but the transfer to the apical side was greater for smaller Ag nanoparticles. Au nanoparticles showed different rules of internalization and transcellular transport compared with Ag nanoparticles. Furthermore, the paracellular permeability of the Caco-2 monolayers was temporarily increased by Ag nanoparticles (5 μg/mL; diameters, ≤10 nm) following basolateral but not apical exposure. We conclude that the internalization, transfer, and effects of nanomaterials in epithelial cell monolayers depend on the size and composition of nanomaterials, and the exposure side. - Highlights: • Ag and Au nanoparticles can transfer across Caco-2 monolayers. • Cellular uptake of nanoparticles change between apical and basolateral exposure. • Basolateral Ag nanoparticle exposure increases the permeability of Caco-2 monolayers.« less

Cellular volume regulation and substrate stiffness modulate the detachment dynamics of adherent cells

NASA Astrophysics Data System (ADS)

Yang, Yuehua; Jiang, Hongyuan

2018-03-01

Quantitative characterizations of cell detachment are vital for understanding the fundamental mechanisms of cell adhesion. Experiments have found that cell detachment shows strong rate dependence, which is mostly attributed to the binding-unbinding kinetics of receptor-ligand bond. However, our recent study showed that the cellular volume regulation can significantly regulate the dynamics of adherent cell and cell detachment. How this cellular volume regulation contributes to the rate dependence of cell detachment remains elusive. Here, we systematically study the role of cellular volume regulation in the rate dependence of cell detachment by investigating the cell detachments of nonspecific adhesion and specific adhesion. We find that the cellular volume regulation and the bond kinetics dominate the rate dependence of cell detachment at different time scales. We further test the validity of the traditional Johnson-Kendall-Roberts (JKR) contact model and the detachment model developed by Wyart and Gennes et al (W-G model). When the cell volume is changeable, the JKR model is not appropriate for both the detachments of convex cells and concave cells. The W-G model is valid for the detachment of convex cells but is no longer applicable for the detachment of concave cells. Finally, we show that the rupture force of adherent cells is also highly sensitive to substrate stiffness, since an increase in substrate stiffness will lead to more associated bonds. These findings can provide insight into the critical role of cell volume in cell detachment and might have profound implications for other adhesion-related physiological processes.

Mitochondrial peptides modulate mitochondrial function during cellular senescence.

PubMed

Kim, Su-Jeong; Mehta, Hemal H; Wan, Junxiang; Kuehnemann, Chisaka; Chen, Jingcheng; Hu, Ji-Fan; Hoffman, Andrew R; Cohen, Pinchas

2018-06-10

Cellular senescence is a complex cell fate response that is thought to underlie several age-related pathologies. Despite a loss of proliferative potential, senescent cells are metabolically active and produce energy-consuming effectors, including senescence-associated secretory phenotypes (SASPs). Mitochondria play crucial roles in energy production and cellular signaling, but the key features of mitochondrial physiology and particularly of mitochondria-derived peptides (MDPs), remain underexplored in senescence responses. Here, we used primary human fibroblasts made senescent by replicative exhaustion, doxorubicin or hydrogen peroxide treatment, and examined the number of mitochondria and the levels of mitochondrial respiration, mitochondrial DNA methylation and the mitochondria-encoded peptides humanin, MOTS-c, SHLP2 and SHLP6. Senescent cells showed increased numbers of mitochondria and higher levels of mitochondrial respiration, variable changes in mitochondrial DNA methylation, and elevated levels of humanin and MOTS-c. Humanin and MOTS-c administration modestly increased mitochondrial respiration and selected components of the SASP in doxorubicin-induced senescent cells partially via JAK pathway. Targeting metabolism in senescence cells is an important strategy to reduce SASP production for eliminating the deleterious effects of senescence. These results provide insight into the role of MDPs in mitochondrial energetics and the production of SASP components by senescent cells.

NAD(H) and NADP(H) Redox Couples and Cellular Energy Metabolism.

PubMed

Xiao, Wusheng; Wang, Rui-Sheng; Handy, Diane E; Loscalzo, Joseph

2018-01-20

The nicotinamide adenine dinucleotide (NAD + )/reduced NAD + (NADH) and NADP + /reduced NADP + (NADPH) redox couples are essential for maintaining cellular redox homeostasis and for modulating numerous biological events, including cellular metabolism. Deficiency or imbalance of these two redox couples has been associated with many pathological disorders. Recent Advances: Newly identified biosynthetic enzymes and newly developed genetically encoded biosensors enable us to understand better how cells maintain compartmentalized NAD(H) and NADP(H) pools. The concept of redox stress (oxidative and reductive stress) reflected by changes in NAD(H)/NADP(H) has increasingly gained attention. The emerging roles of NAD + -consuming proteins in regulating cellular redox and metabolic homeostasis are active research topics. The biosynthesis and distribution of cellular NAD(H) and NADP(H) are highly compartmentalized. It is critical to understand how cells maintain the steady levels of these redox couple pools to ensure their normal functions and simultaneously avoid inducing redox stress. In addition, it is essential to understand how NAD(H)- and NADP(H)-utilizing enzymes interact with other signaling pathways, such as those regulated by hypoxia-inducible factor, to maintain cellular redox homeostasis and energy metabolism. Additional studies are needed to investigate the inter-relationships among compartmentalized NAD(H)/NADP(H) pools and how these two dinucleotide redox couples collaboratively regulate cellular redox states and cellular metabolism under normal and pathological conditions. Furthermore, recent studies suggest the utility of using pharmacological interventions or nutrient-based bioactive NAD + precursors as therapeutic interventions for metabolic diseases. Thus, a better understanding of the cellular functions of NAD(H) and NADP(H) may facilitate efforts to address a host of pathological disorders effectively. Antioxid. Redox Signal. 28, 251-272.

Cellular genetic therapy.

PubMed

Del Vecchio, F; Filareto, A; Spitalieri, P; Sangiuolo, F; Novelli, G

2005-01-01

Cellular genetic therapy is the ultimate frontier for those pathologies that are consequent to a specific nonfunctional cellular type. A viable cure for there kinds of diseases is the replacement of sick cells with healthy ones, which can be obtained from the same patient or a different donor. In fact, structures can be corrected and strengthened with the introduction of undifferentiated cells within specific target tissues, where they will specialize into the desired cellular types. Furthermore, consequent to the recent results obtained with the transdifferentiation experiments, a process that allows the in vitro differentiation of embryonic and adult stem cells, it has also became clear that many advantages may be obtained from the use of stem cells to produce drugs, vaccines, and therapeutic molecules. Since stem cells can sustain lineage potentials, the capacity for differentiation, and better tolerance for the introduction of exogenous genes, they are also considered as feasible therapeutic vehicles for gene therapy. In fact, it is strongly believed that the combination of cellular genetic and gene therapy approaches will definitely allow the development of new therapeutic strategies as well as the production of totipotent cell lines to be used as experimental models for the cure of genetic disorders.

Cellular telephone interference with medical equipment.

PubMed

Tri, Jeffrey L; Severson, Rodney P; Firl, Allen R; Hayes, David L; Abenstein, John P

2005-10-01

To assess the potential electromagnetic interference (EMI) effects that new or current-generation cellular telephones have on medical devices. For this study, performed at the Mayo Clinic in Rochester, Minn, between March 9, 2004, and April 24, 2004, we tested 16 different medical devices with 6 cellular telephones to assess the potential for EMI. Two of the medical devices were tested with both new and old interface modules. The 6 cellular telephones chosen represent the different cellular technology protocols in use: Code Division Multiple Access (2 models), Global System for Mobile communications, Integrated Digital Enhanced Network, Time Division Multiple Access, and analog. The cellular telephones were tested when operating at or near their maximum power output. The medical devices, connected to clinical simulators during testing, were monitored by observing the device displays and alarms. Of 510 tests performed, the incidence of clinically important interference was 1.2%; EMI was Induced in 108 tests (21.2%). Interference occurred in 7 (44%) of the 16 devices tested. Cellular telephones can interfere with medical equipment. Technology changes in both cellular telephones and medical equipment may continue to mitigate or may worsen clinically relevant interference. Compared with cellular telephones tested in previous studies, those currently in use must be closer to medical devices before any interference is noticed. However, periodic testing of cellular telephones to determine their effects on medical equipment will be required.

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.

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.

Mathematical Modeling of Cellular Metabolism.

PubMed

Berndt, Nikolaus; Holzhütter, Hermann-Georg

Cellular metabolism basically consists of the conversion of chemical compounds taken up from the extracellular environment into energy (conserved in energy-rich bonds of organic phosphates) and a wide array of organic molecules serving as catalysts (enzymes), information carriers (nucleic acids), and building blocks for cellular structures such as membranes or ribosomes. Metabolic modeling aims at the construction of mathematical representations of the cellular metabolism that can be used to calculate the concentration of cellular molecules and the rates of their mutual chemical interconversion in response to varying external conditions as, for example, hormonal stimuli or supply of essential nutrients. Based on such calculations, it is possible to quantify complex cellular functions as cellular growth, detoxification of drugs and xenobiotic compounds or synthesis of exported molecules. Depending on the specific questions to metabolism addressed, the methodological expertise of the researcher, and available experimental information, different conceptual frameworks have been established, allowing the usage of computational methods to condense experimental information from various layers of organization into (self-) consistent models. Here, we briefly outline the main conceptual frameworks that are currently exploited in metabolism research.

Molecular Dissection of a Major Gene Effect on a Quantitative Trait: The Level of Alcohol Dehydrogenase Expression in Drosophila Melanogaster

PubMed Central

Stam, L. F.; Laurie, C. C.

1996-01-01

A molecular mapping experiment shows that a major gene effect on a quantitative trait, the level of alcohol dehydrogenase expression in Drosophila melanogaster, is due to multiple polymorphisms within the Adh gene. These polymorphisms are located in an intron, the coding sequence, and the 3' untranslated region. Because of nonrandom associations among polymorphisms at different sites, the individual effects combine (in some cases epistatically) to produce ``superalleles'' with large effect. These results have implications for the interpretation of major gene effects detected by quantitative trait locus mapping methods. They show that large effects due to a single locus may be due to multiple associated polymorphisms (or sequential fixations in isolated populations) rather than individual mutations of large effect. PMID:8978044

Quantitative in vivo assessment of lung microstructure at the alveolar level with hyperpolarized 3He diffusion MRI

NASA Astrophysics Data System (ADS)

Yablonskiy, Dmitriy A.; Sukstanskii, Alexander L.; Leawoods, Jason C.; Gierada, David S.; Bretthorst, G. Larry; Lefrak, Stephen S.; Cooper, Joel D.; Conradi, Mark S.

2002-03-01

The study of lung emphysema dates back to the beginning of the 17th century. Nevertheless, a number of important questions remain unanswered because a quantitative localized characterization of emphysema requires knowledge of lung structure at the alveolar level in the intact living lung. This information is not available from traditional imaging modalities and pulmonary function tests. Herein, we report the first in vivo measurements of lung geometrical parameters at the alveolar level obtained with 3He diffusion MRI in healthy human subjects and patients with severe emphysema. We also provide the first experimental data demonstrating that 3He gas diffusivity in the acinus of human lung is highly anisotropic. A theory of anisotropic diffusion is presented. Our results clearly demonstrate substantial differences between healthy and emphysematous lung at the acinar level and may provide new insights into emphysema progression. The technique offers promise as a clinical tool for early diagnosis of emphysema.

Is central dogma a global property of cellular information flow?

PubMed

Piras, Vincent; Tomita, Masaru; Selvarajoo, Kumar

2012-01-01

The central dogma of molecular biology has come under scrutiny in recent years. Here, we reviewed high-throughput mRNA and protein expression data of Escherichia coli, Saccharomyces cerevisiae, and several mammalian cells. At both single cell and population scales, the statistical comparisons between the entire transcriptomes and proteomes show clear correlation structures. In contrast, the pair-wise correlations of single transcripts to proteins show nullity. These data suggest that the organizing structure guiding cellular processes is observed at omics-wide scale, and not at single molecule level. The central dogma, thus, globally emerges as an average integrated flow of cellular information.

Curcumin enhances recovery of pancreatic islets from cellular stress induced inflammation and apoptosis in diabetic rats

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

Rashid, Kahkashan; Sil, Parames C., E-mail: parames@jcbose.ac.in

The phytochemical, curcumin, has been reported to play many beneficial roles. However, under diabetic conditions, the detail mechanism of its beneficial action in the glucose homeostasis regulatory organ, pancreas, is poorly understood. The present study has been designed and carried out to explore the role of curcumin in the pancreatic tissue of STZ induced and cellular stress mediated diabetes in eight weeks old male Wistar rats. Diabetes was induced with a single intraperitoneal dose of STZ (65 mg/kg body weight). Post to diabetes induction, animals were treated with curcumin at a dose of 100 mg/kg body weight for eight weeks.more » Underlying molecular and cellular mechanism was determined using various biochemical assays, DNA fragmentation, FACS, histology, immunoblotting and ELISA. Treatment with curcumin reduced blood glucose level, increased plasma insulin and mitigated oxidative stress related markers. In vivo and in vitro experimental results revealed increased levels of proinflammatory cytokines (TNF-α, IL1-β and IFN-γ), reduced level of cellular defense proteins (Nrf-2 and HO-1) and glucose transporter (GLUT-2) along with enhanced levels of signaling molecules of ER stress dependent and independent apoptosis (cleaved Caspase-12/9/8/3) in STZ administered group. Treatment with curcumin ameliorated all the adverse changes and helps the organ back to its normal physiology. Results suggest that curcumin protects pancreatic beta-cells by attenuating inflammatory responses, and inhibiting ER/mitochondrial dependent and independent pathways of apoptosis and crosstalk between them. This uniqueness and absence of any detectable adverse effect proposes the possibility of using this molecule as an effective protector in the cellular stress mediated diabetes mellitus. - Highlights: • STZ induced cellular stress plays a vital role in pancreatic dysfunction. • Cellular stress causes inflammation, pancreatic islet cell death and diabetes. • Deregulation of

Effect of liniment levamisole on cellular immune functions of patients with chronic hepatitis B.

PubMed

Wang, Ke-Xia; Zhang, Li-Hua; Peng, Jiang-Long; Liang, Yong; Wang, Xue-Feng; Zhi, Hui; Wang, Xiang-Xia; Geng, Huan-Xiong

2005-12-07

To explore the effects of liniment levamisole on cellular immune functions of patients with chronic hepatitis B. The levels of T lymphocyte subsets and mIL-2R in peripheral blood mononuclear cells (PBMCs) were measured by biotin-streptavidin (BSA) technique in patients with chronic hepatitis B before and after the treatment with liniment levamisole. After one course of treatment with liniment levamisole, the levels of CD3(+), CD4(+), and the ratio of CD4(+)/CD8(+) increased as compared to those before the treatment but the level of CD8(+) decreased. The total expression level of mIL-2R in PBMCs increased before and after the treatment with liniment levamisole. Liniment levamisole may reinforce cellular immune functions of patients with chronic hepatitis B.

Magnetic fingerprints of rolling cells for quantitative flow cytometry in whole blood

NASA Astrophysics Data System (ADS)

Reisbeck, Mathias; Helou, Michael Johannes; Richter, Lukas; Kappes, Barbara; Friedrich, Oliver; Hayden, Oliver

2016-09-01

Over the past 50 years, flow cytometry has had a profound impact on preclinical and clinical applications requiring single cell function information for counting, sub-typing and quantification of epitope expression. At the same time, the workflow complexity and high costs of such optical systems still limit flow cytometry applications to specialized laboratories. Here, we present a quantitative magnetic flow cytometer that incorporates in situ magnetophoretic cell focusing for highly accurate and reproducible rolling of the cellular targets over giant magnetoresistance sensing elements. Time-of-flight analysis is used to unveil quantitative single cell information contained in its magnetic fingerprint. Furthermore, we used erythrocytes as a biological model to validate our methodology with respect to precise analysis of the hydrodynamic cell diameter, quantification of binding capacity of immunomagnetic labels, and discrimination of cell morphology. The extracted time-of-flight information should enable point-of-care quantitative flow cytometry in whole blood for clinical applications, such as immunology and primary hemostasis.

Quantitative Analysis of Trace Element Impurity Levels in Some Gem-Quality Diamonds

NASA Astrophysics Data System (ADS)

McNeill, J. C.; Klein-Bendavid, O.; Pearson, D. G.; Nowell, G. M.; Ottley, C. J.; Chinn, I.; Malarkey, J.

2009-05-01

Perhaps the most important information required to understand the origin of diamonds is the nature of the fluid that they crystallise from. Constraining the identity of the diamond-forming fluid for high purity gem diamonds is hampered by analytical challenges because of the very low analyte levels involved. Here we use a new ultra- low blank 'off-line' laser ablation method coupled to sector-field ICPMS for the quantitative analysis of fluid-poor gem diamonds. Ten diamonds comprised of both E- and P-type parageneses, from the Premier Mine, South Africa, were analysed for trace element abundances. We assume that the elemental signatures arise from low densities of sub-microscopic fluid inclusions that are analogous to the much higher densities of fluid inclusions commonly found within fluid-rich diamonds exhibiting fibrous growth. Repeatability of multiple (>20) blanks yielded consistently low values so that using the current procedure our limits of quantitation (10-ã blank) are <1pg for most trace elements, except for Sr, Zr, Ba, from 2-9pg and Pb ~30pg. Trace element patterns of the Premier diamond suite show enrichment of LREE over HREE. Abundances broadly decrease with increasing elemental compatibility. As a suite the chondrite normalised diamond patterns show negative Sr, Zr, Ti and Y anomalies and positive U, and Pb anomalies. All sample abundances are very depleted relative to chondrites (0.1 to 0.001X ch). HREE range from 0.1 to 1ppb as do Y, Nb, Cs. Other lighter elements vary from 2-30ppb. Pb reaches several ppb and Ti ranges from ppb values up to 2ppm. No significant difference were observed between the trace element systematics of the eclogitic and peridotitic diamonds. Overall, these initial data have inter-element fractionation patterns similar to those evident from fluid-rich fibrous diamonds and can be sued to infer that both types of diamond-forming fluids share a common origin.

Derivation of large-scale cellular regulatory networks from biological time series data.

PubMed

de Bivort, Benjamin L

2010-01-01

Pharmacological agents and other perturbants of cellular homeostasis appear to nearly universally affect the activity of many genes, proteins, and signaling pathways. While this is due in part to nonspecificity of action of the drug or cellular stress, the large-scale self-regulatory behavior of the cell may also be responsible, as this typically means that when a cell switches states, dozens or hundreds of genes will respond in concert. If many genes act collectively in the cell during state transitions, rather than every gene acting independently, models of the cell can be created that are comprehensive of the action of all genes, using existing data, provided that the functional units in the model are collections of genes. Techniques to develop these large-scale cellular-level models are provided in detail, along with methods of analyzing them, and a brief summary of major conclusions about large-scale cellular networks to date.

Design and evaluation of cellular power converter architectures

NASA Astrophysics Data System (ADS)

Perreault, David John

. This cellular system implements entirely distributed control, and achieves performance levels unattainable with an equivalent single converter. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)

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.