A Three-Tiered Study of Differences in Murine Intrahost Immune Response to Multiple Pneumococcal Strains

We apply a previously developed 4-variable ordinary differential equation model of in-host immune response to pneumococcal pneumonia to study the variability of the immune response of MF1 mice and to explore bacteria-driven differences in disease progression and outcome. In particular, we study the immune response to D39 strain of bacteria missing portions of the pneumolysin protein controlling either the hemolytic activity or complement-activating activity, the response to D39 bacteria deficient in either neuraminidase A or B, and the differences in the response to D39 (serotype 2), 0100993 (serotype 3), and TIGR4 (serotype 4) bacteria. The model accurately reproduces infection kinetics in all cases and provides information about which mechanisms in the immune response have the greatest effect in each case. Results suggest that differences in the ability of bacteria to defeat immune response are primarily due to the ability of the bacteria to elude nonspecific clearance in the lung tissue as well as the ability to create damage to the lung epithelium.     

Monoacylglycerols Activate TRPV1 – A Link between Phospholipase C and TRPV1

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H₁ receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H₁ receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous “entourage” compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.     

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies.     

Detection of Aldehydes and Their Conjugated Hydroperox Ydiene Precursors in Thermally-Stressed Culinary Oils and Fats: Investigations Using High Resolution Proton Nmr Spectroscopy

High field (400 and 600 MHz) proton NMR spectroscopy has been employed to investigate the thermally-induced autoxidation of glycerol-bound polyunsaturated fatty acids present in intact culinary frying oils and fats. Heating of these materials at 180°C for periods of 30, 60 and 90 min. generated a variety of peroxidation products, notably aldehydes (alkanals, trans-2-alkenals and alka-2, 4-dienals) and their conjugated hydroperoxydiene precursors. Since such aldehydes appear to be absorbed into the systemic circulation from the gut in vivo, the toxicological significance of their production during standard frying practices is discussed.     

Solvent cage effects: the influence of radical mass and volume on the recombination dynamics of radical cage pairs generated by photolysis of [CpCH2CH2N(CH3)C(O)(CH2)nCH3Mo(CO)3]2 (n = 3, 8, 13, 18) (Cp = eta 5-C5H4) complexes

The photochemistry of the [(CpR)Mo(CO)(3)](2) molecules, where CpR = eta(5)-C(5)H(4)(CH(2))(2)C(O)NCH(3)(CH(2))(n)CH(3) (n = 3, 8, 13, and 18), was examined using femtosecond pump-probe transient absorption spectroscopy. The goal of this study was to investigate the importance of radical size and mass on the dynamics and efficiency of geminate radical-radical recombination. The femtosecond results demonstrated the lack of any size/mass dependence of the recombination efficiency. This finding contrasts with results from a prior study that did find a size/mass dependence using a steady-state photochemical technique. To explain these conflicting results, it is proposed that the femtosecond pump-probe results are a measurement of the efficiency of primary geminate recombination whereas the steady-state method results are a measurement of the sum of primary and secondary geminate recombination efficiencies. The size/mass dependence is evident in the latter because secondary geminate recombination is a slower diffusive recombination process and therefore depends on the steric properties of the radicals. Although the existence of primary and secondary recombination channels is often taken for granted, experimental differentiation of primary and secondary caging has proven to be difficult because it is not possible for a single experimental technique to span the entire timescale for recombination of a radical cage pair and adequately resolve these recombination pathways.