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- 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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- Klionsky, Daniel J., et al.
(författare)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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Ingår i: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Forskningsöversikt (refereegranskat)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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- 2019
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Tidskriftsartikel (refereegranskat)
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- Lu, Yingchang, et al.
(författare)
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Identification of Novel Loci and New Risk Variant in Known Loci for Colorectal Cancer Risk in East Asians
- 2020
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Ingår i: Cancer Epidemiology, Biomarkers and Prevention. - : American Association for Cancer Research. - 1055-9965 .- 1538-7755. ; 29:2, s. 477-486
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Tidskriftsartikel (refereegranskat)abstract
- Background: Risk variants identified so far for colorectal cancer explain only a small proportion of milial risk of this cancer, particularly in Asians.Methods: We performed a genome-wide association study (GWAS) of colorectal cancer in East Asians, cluding 23,572 colorectal cancer cases and 48,700 controls. To identify novel risk loci, we selected 60 omising risk variants for replication using data from 58,131 colorectal cancer cases and 67,347 controls European descent. To identify additional risk variants in known colorectal cancer loci, we performed nditional analyses in East Asians.Results: An indel variant, rs67052019 at 1p13.3, was found to be associated with colorectal cancer risk P = 3.9 x 10(-8) in Asians (OR per allele deletion = 1.13, 95% confidence interval = 1.08-1.18). This sociation was replicated in European descendants using a variant (rs2938616) in complete linkage sequilibrium with rs67052019 (P = 7.7 x 10(-3)). Of the remaining 59 variants, 12 showed an association P < 0.05 in the European-ancestry study, including rs11108175 and rs9634162 at P < 5 x 10(-8) and o variants with an association near the genome-wide significance level (rs60911071, P = 5.8 x 10(-8); 62558833, P = 7.5 x 10(-8)) in the combined analyses of Asian- and European-ancestry data. In addition, ing data from East Asians, we identified 13 new risk variants at 11 loci reported from previous GWAS.Conclusions: In this large GWAS, we identified three novel risk loci and two highly suggestive loci for lorectal cancer risk and provided evidence for potential roles of multiple genes and pathways in the iology of colorectal cancer. In addition, we showed that additional risk variants exist in many colorectal ncer risk loci identified previously.Impact: Our study provides novel data to improve the understanding of the genetic basis for colorectal ncer risk.
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