| 1. |
- Beal, Jacob, et al.
(author)
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Robust estimation of bacterial cell count from optical density
- 2020
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Journal article (peer-reviewed)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. |
- Gunja, Sethu Madhava Rao, 1986-, et al.
(author)
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PARN acts as a maternal factor required for normal embryogenesis and telomere length maintenance in zebrafish
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Other publication (other academic/artistic)abstract
- Poly(A)-specific ribonuclease (PARN) is a 3’-5’ exoribonuclease that removes poly(A) tails of mRNAs and ncRNAs in eukaryotes. Mutations in the human PARN gene are associated with developmental delay and/or telomere biology disorders (TBDs). Here we have established a parn loss-of-function zebrafish model that recapitulates TBD symptoms and phenotypes displayed in human patients. Homozygous parn deficient zebrafish exhibited aberrant snoRNA profile, telomerase RNA maturation and shortening of telomere length over generations. In addition, we found that zygotic parn mutant embryos (Zparn) generated by crossing homozygous male and heterozygous females developed a spectrum of growth/developmental defects from embryonic stage to adult stage. The mutant embryos showed developmental defects with lethality during blastula and gastrulation where the maternal mRNAs need to be destabilized with the activation of zygotic transcription; larvae that surpassed the embryonic stage developed severe developmental defects like bent tail and cardiac edema; the fish that survived to adulthood have severe growth defects. Overall, the array of disease phenotypes observed in PARN mutant fish explains the importance of PARN at different stages of life and could provide a link to the mechanism of TBD penetrance in humans.
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| 3. |
- Visnes, Torkild, et al.
(author)
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Targeting OGG1 arrests cancer cell proliferation by inducing replication stress
- 2020
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In: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 48:21, s. 12234-12251
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Journal article (peer-reviewed)abstract
- Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.
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