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At least 1,495 records · Page 83

Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

Non-NASA Center↗

Coalescence of DNA Double Strand Breaks Induced by Galactic Cosmic Radiation is Modulated by Genetics in 15 Inbred Strains of Mice

In this manuscript we address the challenges associated with the ability to predict radiation sensitivity associated with exposure to either cosmic radiation or X-rays in a population study, by monitoring DNA damage sensing protein 53BP1 forming small nuclear radiation-induced foci (RIF) as a surrogate biomarker of DNA double strand breaks (DSB). 76 primary skin fibroblasts were isolated from 10 collaborative cross strains and five reference inbred mice (C57Bl/6, BALB/CByJ, B6C3, C3H and CBA/CaJ) and exposed to three different charged nuclei of increasing LET (350 MeV/n Si, 350 MeV/n Ar and 600 MeV/n Fe) and X-ray. Our data brings strong evidence against the classic "contact-first" model where DSBs are assumed to be immobile and repaired at the lesion site. In contrast, our model suggests nearby DSBs move into single repair unit characterized by large RIF before the repair machinery kicks in. Such model has the advantage of being much more efficient molecularly but is poorly suited to deal with cosmic radiation, where energy is concentrated along the particle trajectory, inducing a large density of DSBs along each particle track. In accordance with this model, RIF quantification after X-ray exposition showed a saturated dose response for early time points post-irradiation for all strains. Similarly, the high-LET response showed that RIF number matched the number of track per cell, not the number of expected DSB per cell (1). At the temporal level, we noted that the percentage of unrepaired high-LET tracks over a 48 hour time-course increased with LET, confirming that the DNA repair process becomes more difficult as more DSB coalesce into single RIF. There was also good agreement between persistent RIF levels measured in-vitro in the primary skin cultures and survival levels of T-cells and B-cells collected in blood samples from 10 CC strains 24 hours after 0.1 Gy whole-body dose of X-ray. This suggests that persistent RIF 24 hour post-IR is a good surrogate in-vitro biomarker for in-vivo radiation toxicity. Finally, at the genomic level, large differences in repair rates between strains for high-LET allowed us to identify suggestive genetic loci associated with radiation sensitivity. Interestingly, the two highest LETs provided the most strain variation with a common locus on Chromosome 10 highly enriched for DNA repair associated genes we discussed in detail.

Radiation-Induced Foci↗

Insights into mixing of non-isothermal multi-polymer melts for complex plastics recycling

Catalytic recycling or upcycling of plastics is often limited not by catalyst performance, but by transport, arising from highly viscous, non-Newtonian polymer melts. In this work, we develop a reactor-scale framework that integrates rheological measurements, constitutive modeling, computational fluid dynamics (CFD), and experiments to quantify mixing, heat transfer, and dispersion in surrogate hydrocarbon melts representing mixed plastics systems. Temperature- and shear rate-dependent viscosity of low-density polyethylene (LDPE) and high-density polyethylene (HDPE) is measured to create two surrogate polymers (PLD and PHD) that capture the dominant shear-thinning flow behavior while neglecting strong elastic effects, enabling tractable simulation of non-isothermal, polymer-melt mixing using a Carreau-Arrhenius generalized Newtonian framework. Three-dimensional CFD simulations are employed to evaluate impeller performance in PLD using mixing time, cavern volume, thermal uniformity, and interfacial area for regimes in which viscoelastic effects are not dominant. We show that magnetic stir bars commonly used in lab-scale studies produce large thermal gradients (~60 °C) and poor mixing, even under idealized power delivery and polymer flow conditions. In contrast, close-clearance anchor impellers achieve near-isothermal operation, reduce mixing times by up to 5×, and provide >90% active circulation volume. We further demonstrate that, at low pseudo-Deborah number (De*), motor power requirements can be predicted directly from shear rate-dependent rheology using the Carreau-Arrhenius framework, enabling rational selection of operating conditions. Extension to surrogate immiscible multi-polymer systems based on PLD and PHD shows that interfacial area is highly sensitive to operating conditions and impeller design, with coaxial anchor-turbine configurations enhancing dispersion by up to 4 × .

Close-clearance impellers↗

Improved nuclear-structure corrections to the hyperfine splitting of electronic and muonic deuterium

We calculate the nuclear-structure correction to the hyperfine splitting in both electronic and muonic deuterium using interactions from chiral effective field theory. We explore the sensitivity to different parameterizations of the nucleon-nucleon force, study the convergence pattern in the order-by-order chiral expansion, and estimate remaining uncertainties. Our results are consistent with earlier calculations from pionless effective field theory, offering new insights for a robust uncertainty quantification. Thanks to the order-of-magnitude reduction in uncertainty achieved with chiral effective field theory, the two-photon exchange contribution in electronic deuterium agrees with experimental extractions within 0.5σ, in contrast to the 2.6σ discrepancy observed in muonic deuterium. This study lays the groundwork for extending TPE calculations to HFS in heavier atomic systems.

Chiral effective field theory↗

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed, respiration, mitonuclear protein imbalance or down-stream signaling may be altered. - Highlights: • Chloramphenicol inhibits mitochondrial translation and respiration in HeLa and PC12. • Respiration is normal in chloramphenicol treated INS-1E cells and human islets. • Chloramphenicol inhibits mitochondrial translation in insulin secreting INS-1E cells. • Antibiotics induce a mitochondrial unfolded protein response in INS-1E and PC12. • Chloramphenicol slightly promotes metabolism-secretion coupling in INS-1E cells.

60 APPLIED LIFE SCIENCES↗

Ratcheted electrophoresis of Brownian particles

The realization of nanoscale machines requires efficient methods by which to rectify unbiased perturbations to perform useful functions in the presence of significant thermal noise. The performance of such Brownian motors often depends sensitively on their operating conditions—in particular, on the relative rates of diffusive and deterministic motions. In this letter, we present a type of Brownian motor that uses contact charge electrophoresis of a colloidal particle within a ratcheted channel to achieve directed transport or perform useful work against an applied load. We analyze the stochastic dynamics of this model ratchet to show that it functions under any operating condition—even in the limit of strong thermal noise and in contrast to existing ratchets. The theoretical results presented here suggest that ratcheted electrophoresis could provide a basis for electrochemically powered, nanoscale machines capable of transport and actuation of nanoscale components.

71 CLASSICAL AND QUANTUM MECHANICS, GENERAL PHYSIC↗

Modified sensing element of a fibre-optic current sensor based on a low-eigenellipticity spun fibre

We have proposed and investigated a modified sensing element of a spun fibre current sensor for the case when the beat length of the built-in linear birefringence of the fibre is equal to or less than the spin pitch of its helical structure. The proposed configuration makes it possible to restore the interferometer contrast reduced because of the decrease in the ellipticity of the wavelength-averaged polarisation state of radiation propagating in such spun fibre. The modified sensing element contains two polarisation state converters: one, located at the spun fibre input, produces polarisation with ellipticity equal to the eigenellipticity of the fibre, and the other ensures conversion of the elliptical polarisation to an orthogonal one through mirror reflection at the fibre output. We have also demonstrated that the magneto-optical sensitivity decreases slightly for the analysed spectrum-averaged parameters of the polarisation state of radiation in the spun fibre. Experimental data lend support to the theoretical predictions. (fibre-optic sensors)

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

AWARE: The Atmospheric Radiation Measurement (ARM) West Antarctic Radiation Experiment

The U.S. Department of Energy Atmospheric Radiation Measurement (ARM) West Antarctic Radiation Experiment (AWARE) performed comprehensive meteorological and aerosol measurements and ground-based atmospheric remote sensing at two Antarctic stations using the most advanced instrumentation available. A suite of cloud research radars, lidars, spectral and broadband radiometers, aerosol chemical and microphysical sampling equipment, and meteorological instrumentation was deployed at McMurdo Station on Ross Island from December 2015 through December 2016. A smaller suite of radiometers and meteorological equipment, including radiosondes optimized for surface energy budget measurement, was deployed on the West Antarctic Ice Sheet between 4 December 2015 and 17 January 2016. AWARE provided Antarctic atmospheric data comparable to several well-instrumented high Arctic sites that have operated for many years and that reveal numerous contrasts with the Arctic in aerosol and cloud microphysical properties. These include persistent differences in liquid cloud occurrence, cloud height, and cloud thickness. Antarctic aerosol properties are also quite different from the Arctic in both seasonal cycle and composition, due to the continent’s isolation from lower latitudes by Southern Ocean storm tracks. Antarctic aerosol number and mass concentrations are not only non-negligible but perhaps play a more important role than previously recognized because of the higher sensitivities of clouds at the very low concentrations caused by the large-scale dynamical isolation. Antarctic aerosol chemical composition, particularly organic components, has implications for local cloud microphysics. The AWARE dataset, fully available online in the ARM Program data archive, offers numerous case studies for unique and rigorous evaluation of mixed-phase cloud parameterization in climate models.

54 ENVIRONMENTAL SCIENCES↗

Insulin binding and glucose uptake of adipocytes in rats adapted to hypergravitational force

Rats were exposed to 4.15 g for 1 yr and weight and age matched, and lean noncentrifuged rats were used as control groups. Rats exposed to chronic hypergravity (hypergravic rats) were found to show lower ambient insulin levels, greater food intake with smaller body weight gain, and decreased size of isolated adipocytes. The ability of adipocytes from the hypergravic rats to bind insulin was increased. With Scatchard analysis, both number and affinity of receptors were increased. In contrast to the increased binding, glucose transport was found to be decreased in adipocytes from these animals. However, when the data were expressed as a percentage of maximal effect, the half maximal insulin effect for both the hypergravic and lean control groups was produced at an insulin concentration of 0.23 + or - 0.02 ng/ml, which was lower than the insulin concentration of 0.31 + or - 0.02 ng/ml for the weight-matched control group (P less than 0.05). This increased insulin sensitivity in the hypergravic group was accounted for by an increased number of receptors.

Kobayashi, M.↗

Microwave Remote Sensing of Soil Moisture

Because of the large contrast between the dielectric constant of liquid water and that of dry soil at microwave wavelength, there is a strong dependence of the thermal emission and radar backscatter from the soil on its moisture content. This dependence provides a means for the remote sensing of the moisture content in a surface layer approximately 5 cm thick. The feasibility of these techniques is demonstrated from field, aircraft and spacecraft platforms. The soil texture, surface roughness, and vegetative cover affect the sensitivity of the microwave response to moisture variations with vegetation being the most important. It serves as an attenuating layer which can totally obscure the surface. Research indicates that it is possible to obtain five or more levels of moisture discrimination and that a mature corn crop is the limiting vegetation situation.

Schmugge, T. J.↗

Microwave remote sensing of soil moisture

Because of the large contrast between the dielectric constant of liquid water and that of dry soil at microwave wavelength, there is a strong dependence of the thermal emission and radar backscatter from the soil on its moisture content. This dependence provides a means for the remote sensing of the moisture content in a surface layer approximately 5 cm thick. The feasibility of these techniques is demonstrated from field, aircraft and spacecraft platforms. The soil texture, surface roughness, and vegetative cover affect the sensitivity of the microwave response to moisture variations with vegetation being the most important. It serves as an attenuating layer which can totally obscure the surface. Research indicates that it is possible to obtain five or more levels of moisture discrimination and that a mature corn crop is the limiting vegetation situation.

Schmugge, T. J.↗

AWARE: The Atmospheric Radiation Measurement (ARM) West Antarctic Radiation Experiment

The US Department of Energy Atmospheric Radiation Measurement (ARM) West Antarctic Radiation Experiment (AWARE) performed comprehensive meteorological and aerosol measurements, and ground-based atmospheric remote sensing at two Antarctic stations using the most advanced instrumentation available. A suite of cloud research radars, lidars, spectral and broadband radiometers, aerosol chemical and microphysical sampling equipment, and meteorological instrumentation was deployed at McMurdo Station on Ross Island from December 2015 through December 2016. A smaller suite of radiometers and meteorological equipment including radiosondes, optimized for surface energy budget measurement, was deployed on the West Antarctic Ice Sheet between 4 December 2015 and 17 January 2016. AWARE provided Antarctic atmospheric data comparable to several well-instrumented high Arctic sites that have operated for many years and that reveal numerous contrasts with the Arctic in aerosol and cloud microphysical properties. These include persistent differences in liquid cloud occurrence, cloud height and cloud thickness. Antarctic aerosol properties are also quite different from the Arctic in both seasonal cycle and composition, due to the continent’s isolation from lower latitudes by Southern Ocean storm tracks. Antarctic aerosol number and mass concentrations are not only non-negligible but perhaps play a more important role than previously recognized because of the higher sensitivities of clouds at the very low concentrations caused by the large-scale dynamical isolation. Antarctic aerosol chemical composition, particularly organic components, has implications for local cloud microphysics. The AWARE data set, fully available online in the ARM Program data archive, offers numerous case studies for unique and rigorous evaluation of mixed-phase cloud parameterization in climate models.

Atmospheric Radiation Measurement (ARM)↗

Reduction of structural impacts and distinction of photosynthetic pathways in a global estimation of GPP from space-borne solar-induced chlorophyll fluorescence

Quantifying global photosynthesis remains a challenge due to a lack of accurate remote sensing proxies. Solar-induced chlorophyll fluorescence (SIF) has been shown to be a good indicator of photosynthetic activity across various spatial scales. However, a global and spatially challenging estimate of terrestrial gross primary production (GPP) based on satellite SIF remains unresolved due to the confounding effects of species-specific physical and physiological traits and external factors, such as canopy structure or photosynthetic pathway (C3orC4). Here we analyze an ensemble of far-red SIF data from OCO-2 satellite and ground observations at multiple sites, using the spectral invariant theory to reduce the effects of canopy structure and to retrieve a structure-corrected total canopy SIF emission (SIF total). We find that the relationships between observed canopy-leavingSIF and ecosystem GPP vary significantly among biomes. In contrast, the relationships between SIF total and GPP converge around two unique models, one for C3and one for C4plants. We show that the two single empirical models can be used to globally scale satellite SIF observations to terrestrial GPP. We obtain an independent estimate of global terrestrial GPP of 129.56 ± 6.54 PgC/year for the 2015–2017 period, which is consistent with the state-of-the-art data- and process-oriented models. The new GPP product shows improved sensitivity to previously undetected ‘hotspots’ of productivity, being able to resolve the double-peak in GPP due to rotational cropping systems. We suggest that the direct scheme to estimate GPP presented here, which is based on satelliteSIF, may open up new possibilities to resolve the dynamics of global terrestrial GPP across space and time.

scale satellite SIF observations to terrestrial GP↗

Heating, moisture, and water budgets of tropical and midlatitude squall lines - Comparisons and sensitivity to longwave radiation

A 2D time-dependent and nonhydrostatic numerical cloud model is presently used to estimate the heating, moisture, and water budgets in the convective and stratiform regions for both a tropical and a midlatitude squall line. The model encompasses a parameterized, three-class ice phase microphysical scheme and longwave radiative transfer process. It is noted that the convective region plays an important role in the generation of stratiform rainfall for both cases. While a midlevel minimum in the moisture profile for the tropical case is due to vertical eddy transport in the convective region, the contribution to the heating budget by the cloud-scale fluxes is minor; by contrast, the vertical eddy heat-flux is relatively important for the midlatitude case due to the stronger vertical velocities present in the convective cells.

Tao, W.-K.↗

Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

60 APPLIED LIFE SCIENCES↗

Bunker probe: A plasma potential probe almost insensitive to its orientation with the magnetic field

Due to their ability to suppress a large part of the electron current and thus measuring directly the plasma potential, ion sensitive probes have begun to be widely tested and used in fusion devices. For these probes to work, almost perfect alignment with the total magnetic field is necessary. This condition cannot always be fulfilled due to the curvature of magnetic fields, complex magnetic structure, or magnetic field reconnection. In this perspective, we have developed a plasma potential probe (named Bunker probe) based on the principle of the ion sensitive probe but almost insensitive to its orientation with the total magnetic field. Therefore it can be used to measure the plasma potential inside fusion devices, especially in regions with complex magnetic field topology. Experimental results are presented and compared with Ball-Pen probe measurements taken under identical conditions. We have observed that the floating potential of the Bunker probe is indeed little affected by its orientation with the magnetic field for angles ranging from 90° to 30°, in contrast to the Ball-Pen probe whose floating potential decreases towards that of a Langmuir probe if not properly aligned with the magnetic field.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

A Method Based on Radiative Cooling for Detecting Structural Changes in Undercooled Metallic Liquids

We introduce a structure-sensitive parameter for undercooled melts which can be measured in containerless processing experiments. We have established that the ratio, R(T), of hemispherical total emissivity epsilon(sub T)(T) to constant-pressure specific heat c(sub p)(T) can serve as an indicator which is sensitive to any changes in short range atomic order in undercooled metallic melts. R(T) (triple bonds) epsilon(sub T)(T)/c(sub p)(T) values for nickel, zirconium, and silicon have been obtained using the high temperature electrostatic levitator while the levitated melts were undergoing purely radiative cooling into the deeply undercooled region. R(T) plots for undercooled liquid nickel and zirconium indicate no significant change in short-range structure from their melting temperatures to 15% undercooling. In contrast, liquid silicon shows marked short-range structural changes beginning above its melting temperature and extending throughout the undercooled region. The short-range structure of liquid silicon is related to the highly-directional covalent bonding which characterizes its solid form. The nickel and zirconium data show that epsilon(sub T) varies linearly with T, in support of metal emissivity theories.

Rulison, Aaron J.↗

Potassium uptake supporting plant growth in the absence of AKT1 channel activity: Inhibition by ammonium and stimulation by sodium

A transferred-DNA insertion mutant of Arabidopsis that lacks AKT1 inward-rectifying K+ channel activity in root cells was obtained previously by a reverse-genetic strategy, enabling a dissection of the K+-uptake apparatus of the root into AKT1 and non-AKT1 components. Membrane potential measurements in root cells demonstrated that the AKT1 component of the wild-type K+ permeability was between 55 and 63% when external [K+] was between 10 and 1,000 microM, and NH4+ was absent. NH4+ specifically inhibited the non-AKT1 component, apparently by competing for K+ binding sites on the transporter(s). This inhibition by NH4+ had significant consequences for akt1 plants: K+ permeability, 86Rb+ fluxes into roots, seed germination, and seedling growth rate of the mutant were each similarly inhibited by NH4+. Wild-type plants were much more resistant to NH4+. Thus, AKT1 channels conduct the K+ influx necessary for the growth of Arabidopsis embryos and seedlings in conditions that block the non-AKT1 mechanism. In contrast to the effects of NH4+, Na+ and H+ significantly stimulated the non-AKT1 portion of the K+ permeability. Stimulation of akt1 growth rate by Na+, a predicted consequence of the previous result, was observed when external [K+] was 10 microM. Collectively, these results indicate that the AKT1 channel is an important component of the K+ uptake apparatus supporting growth, even in the "high-affinity" range of K+ concentrations. In the absence of AKT1 channel activity, an NH4+-sensitive, Na+/H+-stimulated mechanism can suffice.

Non-NASA Center↗