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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. |
- 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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| 4. |
- Wei, Tao, et al.
(författare)
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Molecular and catalytic characterization of a phi class glutathione transferase from Cathaya argyrophylla
- 2012
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Ingår i: Biochemical Systematics and Ecology. - Oxford : Pergamon Press. - 0305-1978 .- 1873-2925. ; 40, s. 75-85
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Tidskriftsartikel (refereegranskat)abstract
- Plant phi class glutathione transferases (GSTs) play important roles in stress tolerance and detoxification metabolism. This study reports the cloning, expression and biochemical characteristics of a phi GST gene (CaGSTF) from the endemic and endangered conifer Cathaya argyrophylla. The recombinant CaGSTF showed GSH-conjugating activity towards the substrate NED-Cl and CDNB. Kinetic analysis revealed low catalytic efficiency with a k(cat)/K-m(GSH) value of 9.82 mM(-1)S(-1). The CaGSTF proved to be a thermolabile enzyme, at 40 degrees C the enzyme's activity was nearly abolished. Site-directed mutagenesis revealed that Ser12, Lys42, Ile55, Glu67 and Ser68 of CaGSTF are critical components of glutathione-binding sites that contribute to the enzyme's catalytic activity. Compared to other plant phi GSTs and conifer tau GSTs, CaGSTF showed a narrow substrate spectrum, low catalytic efficiency and thermolability. These atypical properties suggest the enzyme may have a limited functional role in the organism's adaptation to environmental stresses in the subtropical regions. (C) 2011 Elsevier Ltd. All rights reserved.
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| 5. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 6. |
- Qu, Chang, et al.
(författare)
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Functional significance of asymmetrical retention of parental alleles in a hybrid pine species complex
- 2024
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Ingår i: Journal of Systematics and Evolution. - : John Wiley & Sons. - 1674-4918 .- 1759-6831. ; 62:1, s. 135-148
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Tidskriftsartikel (refereegranskat)abstract
- Hybrid genomes usually harbor asymmetrical parental contributions. However, it is challenging to infer the functional significance of asymmetrical retention of parental alleles in hybrid populations of conifer trees. Here we investigated the diversity in the glutathione S-transferase (GST) gene family in a hybrid pine Pinus densata and its parents (Pinus tabuliformis and Pinus yunnanensis). Plant GSTs play major roles in protecting plants against biotic and abiotic stresses. In this study, 19 orthologous groups of GST genes were identified and cloned from these three species. We examined their expression in different tissues, and then purified the corresponding proteins to characterize their enzymatic activities and specificities toward different substrates. We found that among the 19 GST orthologous groups, divergence in gene expression and in enzymatic activities toward different substrates was prevalent. P. densata preferentially retained P. yunnanensis-like GSTs for 17 out of the 19 gene loci. We determined the first GST crystal structure from conifer species at a resolution of 2.19 Å. Based on this structure, we performed site-directed mutagenesis to replace amino acid residuals in different wild-types of GSTs to understand their functional impacts. Reciprocal replacement of amino acid residuals in native GSTs of P. densata and P. tabuliformis demonstrated significant changes in enzyme functions and identified key sites controlling GSTs activities. This study illustrates an approach to evaluating the functional significance of sequence variations in conifer genomes. Our study also sheds light on plausible mechanisms for controlling the selective retention of parental alleles in the P. densata genome.
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| 7. |
- Zamioudis, Christos, et al.
(författare)
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Rhizobacterial volatiles and photosynthesis-related signals coordinate MYB72 expression in Arabidopsis roots during onset of induced systemic resistance and iron-deficiency responses
- 2015
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Ingår i: The Plant Journal. - : Wiley. - 0960-7412 .- 1365-313X. ; 84:2, s. 309-322
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Tidskriftsartikel (refereegranskat)abstract
- In Arabidopsis roots, the transcription factor MYB72 plays a dual role in the onset of rhizobacteria-induced systemic resistance (ISR) and plant survival under conditions of limited iron availability. Previously, it was shown that MYB72 coordinates the expression of a gene module that promotes synthesis and excretion of iron-mobilizing phenolic compounds in the rhizosphere, a process that is involved in both iron acquisition and ISR signaling. Here, we show that volatile organic compounds (VOCs) from ISR-inducing Pseudomonas bacteria are important elicitors of MYB72. In response to VOC treatment, MYB72 is co-expressed with the iron uptake-related genes FERRIC REDUCTION OXIDASE2 (FRO2) and IRON-REGULATED TRANSPORTER1 (IRT1) in a manner that is dependent on FER-LIKE IRON DEFICIENCY TRANSCRIPTION FACTOR (FIT), indicating that MYB72 is an intrinsic part of the plant's iron-acquisition response that is typically activated upon iron starvation. However, VOC-induced MYB72 expression is activated independently of iron availability in the root vicinity. Moreover, rhizobacterial VOC-mediated induction of MYB72 requires photosynthesis-related signals, while iron deficiency in the rhizosphere activates MYB72 in the absence of shoot-derived signals. Together, these results show that the ISR- and iron acquisition-related transcription factor MYB72 in Arabidopsis roots is activated by rhizobacterial volatiles and photosynthesis-related signals, and enhances the iron-acquisition capacity of roots independently of the iron availability in the rhizosphere. This work highlights the role of MYB72 in plant processes by which root microbiota simultaneously stimulate systemic immunity and activate the iron-uptake machinery in their host plants. Significance Statement Plant roots intimately interact with plant growth-promoting rhizobacteria that prime the plant immune system and aid in iron uptake two functions facilitated by the root-specific transcription factor MYB72. Here we show how MYB72 and iron uptake responses are systemically activated by photosynthesis-related signals and volatiles produced by plant growth-promoting rhizobacteria, highlighting the important role of beneficial root microbiota in supporting plant growth and health.
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