| 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. |
- 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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| 3. |
- Wei, Ting, et al.
(författare)
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Developed and developing world responsibilities for historical climate change and CO2 mitigation
- 2012
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 109:32, s. 12911-12915
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Tidskriftsartikel (refereegranskat)abstract
- At the United Nations Framework Convention on Climate Change Conference in Cancun, in November 2010, the Heads of State reached an agreement on the aim of limiting the global temperature rise to 2 degrees C relative to preindustrial levels. They recognized that long-term future warming is primarily constrained by cumulative anthropogenic greenhouse gas emissions, that deep cuts in global emissions are required, and that action based on equity must be taken to meet this objective. However, negotiations on emission reduction among countries are increasingly fraught with difficulty, partly because of arguments about the responsibility for the ongoing temperature rise. Simulations with two earth-system models (NCAR/CESM and BNU-ESM) demonstrate that developed countries had contributed about 60-80%, developing countries about 20-40%, to the global temperature rise, upper ocean warming, and sea-ice reduction by 2005. Enacting pledges made at Cancun with continuation to 2100 leads to a reduction in global temperature rise relative to business as usual with a 1/3-2/3 (CESM 33-67%, BNU-ESM 35-65%) contribution from developed and developing countries, respectively. To prevent a temperature rise by 2 degrees C or more in 2100, it is necessary to fill the gap with more ambitious mitigation efforts.
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| 4. |
- Ren, Luyao, et al.
(författare)
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Quartet DNA reference materials and datasets for comprehensively evaluating germline variant calling performance
- 2023
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Ingår i: Genome Biology. - : BioMed Central (BMC). - 1465-6906 .- 1474-760X. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Genomic DNA reference materials are widely recognized as essential for ensuring data quality in omics research. However, relying solely on reference datasets to evaluate the accuracy of variant calling results is incomplete, as they are limited to benchmark regions. Therefore, it is important to develop DNA reference materials that enable the assessment of variant detection performance across the entire genome.RESULTS: We established a DNA reference material suite from four immortalized cell lines derived from a family of parents and monozygotic twins. Comprehensive reference datasets of 4.2 million small variants and 15,000 structural variants were integrated and certified for evaluating the reliability of germline variant calls inside the benchmark regions. Importantly, the genetic built-in-truth of the Quartet family design enables estimation of the precision of variant calls outside the benchmark regions. Using the Quartet reference materials along with study samples, batch effects are objectively monitored and alleviated by training a machine learning model with the Quartet reference datasets to remove potential artifact calls. Moreover, the matched RNA and protein reference materials and datasets from the Quartet project enables cross-omics validation of variant calls from multiomics data.CONCLUSIONS: The Quartet DNA reference materials and reference datasets provide a unique resource for objectively assessing the quality of germline variant calls throughout the whole-genome regions and improving the reliability of large-scale genomic profiling.
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