Macrophage pro-inflammatory cytokine secretion is enhanced following interaction with autologous platelets

<p>Abstract</p> <p>Background</p> <p>Macrophages are the dominant phagocyte at sites of wound healing and inflammation, and the cellular and acellular debris encountered by macrophages can have profound effects on their inflammatory profile. Following interaction with apoptotic cells, macrophages are known to switch to an anti-inflammatory phenotype. Activated platelets, however, are also a major component of inflammatory lesions and have been proposed to be pro-inflammatory mediators. In the present study, we tested the hypothesis that macrophage interaction with activated platelets results in an inflammatory response that differs from the response following phagocytosis of apoptotic cells.</p> <p>Methods</p> <p>Human monocyte-derived macrophages (hMDMs) were co-incubated with autologous activated platelets (AAPs) and the platelet-macrophage interaction was examined by electron microscopy and flow cytometry. The cytokines TNF-α, IL-6, and IL-23 were also measured during LPS-activated hMDM co-incubation with AAPs, which was compared to co-incubation with apoptotic lymphocytes. Cytokine secretion was also compared to platelets pre-treated with the gluococorticoid dexamethasone.</p> <p>Results</p> <p>Macrophages trapped and phagocytized AAPs utilizing a mechanism that was significantly inhibited by the scavenger receptor ligand fucoidan. LPS-induced macrophage secretion of TNF-α, IL-6, and IL-23 was inhibited by co-incubation with apoptotic cells, but enhanced by co-incubation with AAPs. The platelet-dependent enhancement of LPS-induced cytokines could be reversed by pre-loading the platelets with the glucocorticoid dexamethasone.</p> <p>Conclusions</p> <p>The interaction of human macrophages with autologous platelets results in scavenger-receptor-mediated platelet uptake and enhancement of LPS-induced cytokines. Therefore, the presence of activated platelets at sites of inflammation may exacerbate pro-inflammatory macrophage activation. The possibility of reversing macrophage activation with dexamethasone-loaded platelets is a promising therapeutic approach to treating unresolved inflammation.</p

Ensemble Methods for Monitoring Enzyme Translocation along Single Stranded Nucleic Acids

We review transient kinetic methods developed to study the mechanism of translocation of nucleic acid motor proteins. One useful stopped-flow fluorescence method monitors arrival of the translocase at the end of a fluorescently labeled nucleic acid. When conducted under single-round conditions the time courses can be analyzed quantitatively using n-step sequential models to determine the kinetic parameters for translocation (rate, kinetic step size and processivity). The assay and analysis discussed here can be used to study enzyme translocation along a linear lattice such as ssDNA or ssRNA. We outline the methods for experimental design and two approaches, along with their limitations, that can be used to analyze the time courses. Analysis of the full time courses using n-step sequential models always yields an accurate estimate of the translocation rate. An alternative semi-quantitative “time to peak” analysis yields accurate estimates of translocation rates only if the enzyme initiates translocation from a unique site on the nucleic acid. However, if initiation occurs at random sites along the nucleic acid, then the “time to peak” analysis can yield inaccurate estimates of even the rates of translocation depending on the values of other kinetic parameters, especially the rate of dissociation of the translocase. Thus, in those cases analysis of the full time course is needed to obtain accurate estimates of translocation rates

Single stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis

E. coli UvrD is an SF1A helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded (ss) DNA translocase, but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM) we proposed a non-uniform stepping mechanism in which a UvrD monomer translocates with biased (3′ to 5′) directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step-size of 4–5 nucleotides/step, suggesting a pause occurs every 4–5 nucleotides translocated. To further test this mechanism we examined UvrD translocation over a range of lower ATP concentrations (10–500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ~1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM) indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4–5 nucleotides/step down to 50 μM ATP, but increases to ~7 nucleotides/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP, but with little futile ATP hydrolysis

Characterisation by EIS of ternary Mg alloys synthesised by mechanical alloying

Electrochemical impedance spectroscopy has been used to study the electrochemical behaviour of ternary Mg60Ti10Si30 and Mg88Ti5Si7 alloy samples fabricated by mechanical alloying of the elemental powders in an argon atmosphere. The influence of different milling times up to 25 h and heat treatment on the electrochemical behaviour of the samples, after compacting under pressure into disks, has been investigated in 0.1 M Na2SO4 and 0.01 M NaCl electrolyte solutions. Complementary measurements of open circuit potential, polarisation curves, and surface and microstructural analysis have been carried out. The experimental results revealed that corrosion is greater for Mg88Ti5Si7 which contains free magnesium; however, in sulphate solution a protective oxide layer formed can reduce the corrosion rate. In Mg60Ti10Si30, heat treatment increases corrosion, which is explained through a greater tendency for pitting corrosion. Comparison is made between the electrochemical impedance data and the nanophase structure as well as with the electrochemical behaviour of other magnesium alloys.http://www.sciencedirect.com/science/article/B6TG0-4H21KGF-3/1/564bda28bb49702cb8769e10e8e350a

Temperature stability of the sky quality meter

The stability of radiance measurements taken by the Sky Quality Meter (SQM)was tested under rapidly changing temperature conditions during exposure to a stable light field in the laboratory. The reported radiance was found to be negatively correlated with temperature, but remained within 7% of the initial reported radiance over a temperature range of -15 °C to 35 °C, and during temperature changes of -33 °C/h and +70 °C/h.This is smaller than the manufacturer’s quoted unit-to-unit systematic uncertainty of 10%,indicating that the temperature compensation of the SQM is adequate under expected outdoor operating conditions

Macrophage Pro-Inflammatory Cytokine Secretion is Enhanced Following Interaction with Autologous Platelets

Abstract Background Macrophages are the dominant phagocyte at sites of wound healing and inflammation, and the cellular and acellular debris encountered by macrophages can have profound effects on their inflammatory profile. Following interaction with apoptotic cells, macrophages are known to switch to an anti-inflammatory phenotype. Activated platelets, however, are also a major component of inflammatory lesions and have been proposed to be pro-inflammatory mediators. In the present study, we tested the hypothesis that macrophage interaction with activated platelets results in an inflammatory response that differs from the response following phagocytosis of apoptotic cells. Methods Human monocyte-derived macrophages (hMDMs) were co-incubated with autologous activated platelets (AAPs) and the platelet-macrophage interaction was examined by electron microscopy and flow cytometry. The cytokines TNF-α, IL-6, and IL-23 were also measured during LPS-activated hMDM co-incubation with AAPs, which was compared to co-incubation with apoptotic lymphocytes. Cytokine secretion was also compared to platelets pre-treated with the gluococorticoid dexamethasone. Results Macrophages trapped and phagocytized AAPs utilizing a mechanism that was significantly inhibited by the scavenger receptor ligand fucoidan. LPS-induced macrophage secretion of TNF-α, IL-6, and IL-23 was inhibited by co-incubation with apoptotic cells, but enhanced by co-incubation with AAPs. The platelet-dependent enhancement of LPS-induced cytokines could be reversed by pre-loading the platelets with the glucocorticoid dexamethasone. Conclusions The interaction of human macrophages with autologous platelets results in scavenger-receptor-mediated platelet uptake and enhancement of LPS-induced cytokines. Therefore, the presence of activated platelets at sites of inflammation may exacerbate pro-inflammatory macrophage activation. The possibility of reversing macrophage activation with dexamethasone-loaded platelets is a promising therapeutic approach to treating unresolved inflammation

Citizen Science Provides Valuable Data for Monitoring Global Night Sky Luminance

The skyglow produced by artificial lights at night is one of the most dramatic anthropogenic modifications of Earth’s biosphere. The GLOBE at Night citizen science project allows individual observers to quantify skyglow using star maps showing different levels of light pollution. We show that aggregated GLOBE at Night data depend strongly on artificial skyglow, and could be used to track lighting changes worldwide. Naked eye time series can be expected to be very stable, due to the slow pace of human eye evolution. The standard deviation of an individual GLOBE at Night observation is found to be 1.2 stellar magnitudes. Zenith skyglow estimates from the ‘‘First World Atlas of Artificial Night Sky Brightness’’ are tested using a subset of the GLOBE at Night data. Although we find the World Atlas overestimates sky brightness in the very center of large cities, its predictions for Milky Way visibility are accurate

Effects of nucleosome stability on remodeler-catalyzed repositioning

Chromatin remodelers are molecular motors that play essential roles in the regulation of nucleosome positioning and chromatin accessibility. These machines couple the energy obtained from the binding and hydrolysis of ATP to the mechanical work of manipulating chromatin structure through processes that are not completely understood. Here we present a quantitative analysis of nucleosome repositioning by the imitation switch (ISWI) chromatin remodeler and demonstrate that nucleosome stability significantly impacts the observed activity. We show how DNA damage induced changes in the affinity of DNA wrapping within the nucleosome can affect ISWI repositioning activity and demonstrate how assay-dependent limitations can bias studies of nucleosome repositioning. Together, these results also suggest that some of the diversity seen in chromatin remodeler activity can be attributed to the variations in the thermodynamics of interactions between the remodeler, the histones, and the DNA, rather than reflect inherent properties of the remodeler itself