| 1. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)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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| 2. |
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| 3. |
- 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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| 4. |
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| 5. |
- Campbell, PJ, et al.
(author)
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Pan-cancer analysis of whole genomes
- 2020
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In: Nature. - : Springer Science and Business Media LLC. - 1476-4687 .- 0028-0836. ; 578:7793, s. 82-
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Journal article (peer-reviewed)abstract
- Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale1–3. Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter4; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation5,6; analyses timings and patterns of tumour evolution7; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity8,9; and evaluates a range of more-specialized features of cancer genomes8,10–18.
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| 6. |
- Haim-Vilmovsky, Liora, et al.
(author)
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Mapping Rora expression in resting and activated CD4+ T cells
- 2021
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:5
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Journal article (peer-reviewed)abstract
- The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4 + T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment. Copyright: © 2021 Haim-Vilmovsky et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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| 7. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes
- 2008
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In: Autophagy. - : Landes Bioscience. - 1554-8627 .- 1554-8635. ; 4:2, s. 151-175
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Research review (peer-reviewed)abstract
- Research in autophagy continues to accelerate,1 and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.2,3 There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.
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| 8. |
- Mahajan, Anubha, et al.
(author)
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Multi-ancestry genetic study of type 2 diabetes highlights the power of diverse populations for discovery and translation
- 2022
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In: Nature Genetics. - : Springer Nature. - 1061-4036 .- 1546-1718. ; 54:5, s. 560-572
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Journal article (peer-reviewed)abstract
- We assembled an ancestrally diverse collection of genome-wide association studies (GWAS) of type 2 diabetes (T2D) in 180,834 affected individuals and 1,159,055 controls (48.9% non-European descent) through the Diabetes Meta-Analysis of Trans-Ethnic association studies (DIAMANTE) Consortium. Multi-ancestry GWAS meta-analysis identified 237 loci attaining stringent genome-wide significance (P < 5 x 10(-9)), which were delineated to 338 distinct association signals. Fine-mapping of these signals was enhanced by the increased sample size and expanded population diversity of the multi-ancestry meta-analysis, which localized 54.4% of T2D associations to a single variant with >50% posterior probability. This improved fine-mapping enabled systematic assessment of candidate causal genes and molecular mechanisms through which T2D associations are mediated, laying the foundations for functional investigations. Multi-ancestry genetic risk scores enhanced transferability of T2D prediction across diverse populations. Our study provides a step toward more effective clinical translation of T2D GWAS to improve global health for all, irrespective of genetic background. Genome-wide association and fine-mapping analyses in ancestrally diverse populations implicate candidate causal genes and mechanisms underlying type 2 diabetes. Trans-ancestry genetic risk scores enhance transferability across populations.
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| 9. |
- Qiu, Chunjing, et al.
(author)
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A strong mitigation scenario maintains climate neutrality of northern peatlands
- 2022
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In: One Earth. - : Elsevier BV. - 2590-3330 .- 2590-3322. ; 5:1, s. 86-97
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Journal article (peer-reviewed)abstract
- Northern peatlands store 300–600 Pg C, of which approximately half are underlain by permafrost. Climate warming and, in some regions, soil drying from enhanced evaporation are progressively threatening this large carbon stock. Here, we assess future CO2 and CH4 fluxes from northern peatlands using five land surface models that explicitly include representation of peatland processes. Under Representative Concentration Pathways (RCP) 2.6, northern peatlands are projected to remain a net sink of CO2 and climate neutral for the next three centuries. A shift to a net CO2 source and a substantial increase in CH4 emissions are projected under RCP8.5, which could exacerbate global warming by 0.21°C (range, 0.09–0.49°C) by the year 2300. The true warming impact of peatlands might be higher owing to processes not simulated by the models and direct anthropogenic disturbance. Our study highlights the importance of understanding how future warming might trigger high carbon losses from northern peatlands.
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