| 1. |
- McCrae, Cristopher, et al.
(author)
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Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells
- 2018
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In: American Journal of Respiratory Cell and Molecular Biology. - 1044-1549. ; 59:6, s. 713-722
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Journal article (peer-reviewed)abstract
- Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-beta. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.
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| 2. |
- Dzgoev, A., et al.
(author)
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Optimization of a charge coupled device imaging enzyme linked immuno sorbent assay and supports for the simultaneous determination of multiple 2,4-D samples
- 1997
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In: Analytica Chimica Acta. - 0003-2670. ; 347:1-2, s. 87-93
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Journal article (peer-reviewed)abstract
- A chemiluminescent microformat enzyme linked immune sorbent assay (ELISA) has been optimized for the simultaneous determination of multiple 2,4-dichlorophenoxyacetic acid (2,4-D) samples. The competitive immunoassay employed a 2,4-D-BSA conjugate, anti-2,4-D monoclonal antibodies and alkaline phosphatase (AP) labelled anti-mouse IgG. The bound AP conjugate was determined by quantitating the chemiluminescence emission from the enzymatic decomposition of the luminogenic substrate, CSPD, by AP using a cooled charge coupled device (CCD) camera. The detection limit for the simultaneous determination of multiple samples was 4.3x10-10 M corresponding to 96 pg ml-1 or 192 fg well with a coefficient of variation (CV, %) of 12.5%. The linear range of the assay was 4.5 x 10-7-4.5 x 10-10 M. The ability of gold coated silicon wafers and glass capillaries to serve as solid phase supports in the imaging ELISA was investigated. The highly reflective gold surfaces improved both the linear range and the sensitivity of the assay, as compared to thick-film patterned surfaces. The capillary supports, on the other hand, lead to a reduction in the linear range and the sensitivity of the assay, as compared to the thick-film patterned surfaces. Initial studies indicate that the capillaries guide the light and may provide a built-in mechanism for collecting the emitted light. Strategies for further development of support materials for imaging-based detectors will be discussed.
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