| 1. |
- 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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| 2. |
- Fresard, Laure, et al.
(författare)
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Identification of rare-disease genes using blood transcriptome sequencing and large control cohorts
- 2019
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Ingår i: Nature Medicine. - : NATURE PUBLISHING GROUP. - 1078-8956 .- 1546-170X. ; 25:6, s. 911-919
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Tidskriftsartikel (refereegranskat)abstract
- It is estimated that 350 million individuals worldwide suffer from rare diseases, which are predominantly caused by mutation in a single gene(1). The current molecular diagnostic rate is estimated at 50%, with whole-exome sequencing (WES) among the most successful approaches(2-5). For patients in whom WES is uninformative, RNA sequencing (RNA-seq) has shown diagnostic utility in specific tissues and diseases(6-8). This includes muscle biopsies from patients with undiagnosed rare muscle disorders(6,9), and cultured fibroblasts from patients with mitochondrial disorders(7). However, for many individuals, biopsies are not performed for clinical care, and tissues are difficult to access. We sought to assess the utility of RNA-seq from blood as a diagnostic tool for rare diseases of different pathophysiologies. We generated whole-blood RNA-seq from 94 individuals with undiagnosed rare diseases spanning 16 diverse disease categories. We developed a robust approach to compare data from these individuals with large sets of RNA-seq data for controls (n = 1,594 unrelated controls and n = 49 family members) and demonstrated the impacts of expression, splicing, gene and variant filtering strategies on disease gene identification. Across our cohort, we observed that RNA-seq yields a 7.5% diagnostic rate, and an additional 16.7% with improved candidate gene resolution.
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| 3. |
- Clegg, Simon L., et al.
(författare)
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Chemical speciation models based upon the Pitzer activity coefficient equations, including the propagation of uncertainties. II. Tris buffers in artificial seawater at 25 °C, and an assessment of the seawater ‘Total’ pH scale
- 2022
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Ingår i: Marine Chemistry. - : Elsevier BV. - 0304-4203. ; 244
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Tidskriftsartikel (refereegranskat)abstract
- The substance Tris (or THAM, 2-amino-2-hydroxymethyl-1,3-propanediol, CAS 77–86-1), and its protonated form TrisH+, is used in the preparation of pH buffer solutions for applications in seawater chemistry. The development of an acid-base chemical speciation model of buffer solutions containing Tris, TrisH+, and the major ions of seawater is desirable so that: (i) the effects of changes in the composition of the medium on pH can be calculated; (ii) pH on the free (a measure of [H+]) and total (a measure of ([H+] + [HSO4−])) scales can be interconverted; (iii) approximations inherent in the definition of the total pH scale can be quantified; (iv) electrode pairs such as H+/Cl− and H+/Na+ can more easily be calibrated for the measurement of pH. As a first step towards these goals we have extended the Pitzer-based speciation model of Waters and Millero (Mar. Chem. 149, 8–22, 2013) for artificial seawater to include Tris and TrisH+, at 25 °C. Estimates of the variances and covariances of the additional interaction parameters were obtained by Monte Carlo simulation. This enables the total uncertainty of any model-calculated quantity (e.g., pH, speciation) to be estimated, as well as the individual contributions of all interaction parameters and equilibrium constants. This is important for model development, because it allows the key interactions to be identified. The model was tested against measured EMFs of cells containing Tris buffer in artificial seawater at 25 °C, and the mean deviation was found to be 0.13 ± 0.070 mV for salinities 20 to 40. Total variances for calculated electromotive forces of the buffer solutions are dominated by contributions from just a few interaction parameters, making it likely that the model can readily be improved. The model was used to quantify the difference between various definitions of total pH and –log10([H+] + [HSO4−]) in Tris buffer solutions at 25 °C, for the first time (item (iii) above). The results suggest that the total pH scale can readily be extended to low salinities using the established approach for substituting TrisH+ for Na+ in the buffer solutions, especially if the speciation model is used to quantify the effect on pH of the substitution. The relationships between electromotive force (EMF), and pH on the total scale, with buffer molality in artificial seawater at constant salinity are shown to be linear above about 0.01 to 0.02 mol kg−1 buffer molality. The pH of Tris buffers containing ratios of TrisH+ to Tris that vary from unity can be calculated very simply. Technical aspects of the total pH scale, such as the extrapolation of pH to zero buffer (at constant salinity), are clarified. Recommendations are made for further work to extend the model to the temperature range 0–45 °C, and improve accuracy, so that requirements (i) to (iv) above can be fully met.
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| 4. |
- Humphreys, Matthew P., et al.
(författare)
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Chemical speciation models based upon the Pitzer activity coefficient equations, including the propagation of uncertainties: Artificial seawater from 0 to 45 °C
- 2022
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Ingår i: Marine Chemistry. - : Elsevier BV. - 0304-4203. ; 244
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Tidskriftsartikel (refereegranskat)abstract
- Accurate chemical speciation models of solutions containing the ions of seawater have applications in the calculation of carbonate system equilibria and trace metal speciation in natural waters, and the determination of pH. Existing models, based on the Pitzer formalism for the calculation of activity coefficients, do not yet agree with key experimental data (potentiometric determinations of H+ and Cl− activity products in acidified artificial seawaters) and, critically, do not include uncertainty estimates. This hampers applications of the models, and also their further development (for which the uncertainty contributions of individual ion interactions and equilibrium constants need to be known). We have therefore implemented the models of Waters and Millero (Mar. Chem. 149, 8-22, 2013) and Clegg and Whitfield (Geochim. et Cosmochim. Acta 59, 2403-2421, 1995) for artificial seawater, within a generalised treatment of uncertainties, as a first step towards a more complete model of standard seawater and pH buffers. This addition to the models enables both the total uncertainty of any model-calculated quantity (e.g., pH, speciation) to be estimated, and also the contributions of all interaction parameters and equilibrium constants. Both models have been fully documented (and some corrections made). Estimates of the variances and covariances of the interaction parameters were obtained by Monte Carlo simulation, with simplifying assumptions. The models were tested against measured electromotive forces (EMFs) of cells containing acidified artificial seawaters. The mean offsets (measured – calculated) at 25 °C for the model of Waters and Millero are: 0.046 ± 0.11 mV (artificial seawater without sulphate, 0.280 mol kg−1 to 0.879 mol kg−1 ionic strength); and −0.199 ± 0.070 mV (artificial seawater, salinities 5 to 45). Results are similar at other temperatures. These differences compare with an overall uncertainty in the measured EMFs of about 0.04 mV. Total uncertainties for calculated EMFs of the solutions were dominated by just a few contributions: mainly H+-Cl−, Na+-Cl−, and H+-Na+-Cl− ionic interactions, and the thermodynamic dissociation constant of HSO4−. This makes it likely that the accuracy of the models can readily be improved, and recommendations for further work are made. It is shown that standard EMFs used in the calibration of the marine ‘total’ pH scale can be accurately predicted with only slight modification to the original models, suggesting that they can contribute to the extension of the scale to lower salinities.
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| 5. |
- Kamal, Nadia, et al.
(författare)
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The mosaic oat genome gives insights into a uniquely healthy cereal crop
- 2022
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 606:7912, s. 113-119
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Tidskriftsartikel (refereegranskat)abstract
- Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.
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