| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Jain, Arjun, et al.
(författare)
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Endothelin-1 Induces Endoplasmic Reticulum Stress by Activating the PLC-IP3 Pathway Implications for Placental Pathophysiology in Preeclampsia
- 2012
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Ingår i: American Journal of Pathology. - : Elsevier BV. - 0002-9440 .- 1525-2191. ; 180:6, s. 2309-2320
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Tidskriftsartikel (refereegranskat)abstract
- Recent evidence implicates placental endoplasmic reticulum (ER) stress in the pathophysiological characteristics of preeclampsia. Herein, we investigate whether endothelin (ET)-1, which induces Ca2+ release from the ER, can induce placental ER stress. Loss of ER Ca2+ homeostasis impairs post-translational modification of proteins, triggering ER stress-response pathways. IHC confirmed the presence of both ET-1 and its receptors in the syncytiotrophoblast. Protein levels and immunoreactivity of ET-1 and the endothelin B receptor (ETBR) were increased in preeclamptic samples compared with normotensive controls. JEG-3 and BeWo choriocarcinoma cells treated with ET-1 displayed an increase in ER stress markers. ET-1 induced phospho-activation of the ETBR. Treating cells with BQ788, an ETBR antagonist, or small-interfering RNA knockdown of the receptor inhibited induction of ER stress. ET-1 also stimulated p-phospholipase C (PLC)gamma 1 levels. By using inhibitors of PLC activation, U73122, and the inositol 1,4,5-triphosphate (IP3) receptor, xestospongin-C, we demonstrated that ET-1 induces ER stress via the PLC-IP3 pathway. Furthermore, ET-1 levels increased in the syncytiotrophoblast of explants from normal placentas after hypoxia-reoxygenation in vitro. Conditioned medium from hypoxia-reoxygenation explants also contained higher ET-1 levels, which induced ER stress in JEG-3 cells that was abolished by an ET-1 neutralizing antibody. Collectively, the data show that ET-1 induced ER stress in trophoblasts via the ETBR and initiation of signaling through the PLC-IP3 pathway, with the potential for autocrine stimulation.
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