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| 2. |
- 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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| 3. |
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- 2019
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Tidskriftsartikel (refereegranskat)
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| 4. |
- 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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| 5. |
- Chang, Christine Chi-Chen, 1974-
(författare)
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Mechanisms and genes controlling the signalling network for biotic and abiotic stress defences in Arabidopsis thaliana (L.) Heyhn : Functional cross-talk between photo-produced reactive oxygen species, photosynthesis and plant disease defence responses
- 2005
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Excess excitation energy, mechanical injury and defence against pathogens, each trigger rapid production of reactive oxygen species (ROS) in Arabidopsis thaliana leaves. ROS, such as hydrogen peroxide (H2O2), are required for the induction of systemic acquired acclimation and may lead to redox changes in photosynthetic electron transport (PET). On one hand, enhanced ROS production during stress can destroy cells, and on the other, ROS can also act as signals for the activation of stress responsive and defensive pathways.In this work, physiological and molecular analyses of Arabidopsis mutants and transgenic lines were applied to investigate the signalling network controlling biotic and abiotic stress responses. A key enzyme of the antioxidant network is encoded by ASCORBATE PEROXIDASE 2 (APX2). Wounded leaves showed low induction of APX2 expression and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid, chitosan and abscisic acid were not involved in the wound-induced expression of APX2, but PET was required, and APX2 induction was preceded by a depressed rate of CO2 fixation.Analysis of lsd1 (LESION SIMULATING DISEASE 1) strongly suggests that light acclimatory processes and pathogen defences are genetically and functionally linked. It is important to know that LSD1 type of mutants have mainly been studied with regard to pathogenesis. From this work, it reveals that association of LSD1 with hypersensitive response may only be supplementary.GLUTATHIONE PEROXIDASES (GPXs) are another major family of ROS scavenging enzymes. Analysis of the Arabidopsis genome database revealed a new open-reading frame, thus increasing the total number of AtGPX gene family to eight (AtGPX1-AtGPX8). Arabidopsis thaliana transgenic lines with reduced expression of both putative chloroplastic isoforms (AtGPX1 and AtGPX7) and AtGPX7 knock-out mutant (ko-GPX7) were more sensitive to photo-oxidative stress but had a reduced bacterial growth rate when inoculated with virulent strains Pseudomonas syringae pv. tomato DC3000 and P.s.t. maculicola strain ES4326, indicating increased resistance to pathogenesis. This, to our knowledge, is the first functional and genetic analysis of chloroplastic GPXs in plants, and confirms that light and chloroplastic ROS metabolism is important for basal resistance against virulent pathogens.The above results confirm that light sensing, light acclimatory processes and photo-produced ROS also govern pathogen defence pathways. This has a great ecological relevance for Darwinian fitness of plants growing in the natural environment, where simultaneous pathogen attack and fluctuations in light, temperature and other environmental factors make rapid acclimation a constant necessity. Molecular, biochemical and physiological analysis of pathogen responses in mutants impaired in light sensing, EEE-dissipatory mechanisms, and similar analysis of light acclimatory processes in mutants impaired in pathogen defences may prove to be seminal.
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| 6. |
- Mateo, Alfonso, et al.
(författare)
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LESION SIMULATING DISEASE 1 is required for acclimation to conditions that promote excess excitation energy
- 2004
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 136:1, s. 2818-2830
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Tidskriftsartikel (refereegranskat)abstract
- The lsd1 mutant of Arabidopsis fails to limit the boundaries of hypersensitive cell death response during avirulent pathogen infection and initiates unchecked lesions in long day photoperiod. giving rise to the runaway cell death (rcd) phenotype. We link here the initiation and propagation of rcd to the activity of photosystem II, stomatal conductance and ultimately to photorespiratory H2O2. A cross of lsd1 with the chlorophyll a/b binding harvesting-organelle specific (designated cao) mutant, which has a reduced photosystem II antenna, led to reduced lesion formation in the lsd1/cao double mutant. This lsd1 mutant also had reduced stomatal conductance and catalase activity in short-day permissive conditions and induced H2O2 accumulation followed by rcd when stomatal gas exchange was further impeded. All of these traits depended on the defense regulators EDS1 and PAD4. Furthermore, nonphotorespiratory conditions retarded propagation of lesions in lsd1. These data suggest that lsd1 failed to acclimate to light conditions that promote excess excitation energy (EEE) and that LSD1 function was required for optimal catalase activity. Through this regulation LSD1 can influence the effectiveness of photorespiration in dissipating EEE and consequently may be a key determinant of acclimatory processes. Salicylic acid, which induces stomatal closure, inhibits catalase activity and triggers the rcd phenotype in lsd1, also impaired acclimation of wild-type plants to conditions that promote EEE. We propose that the roles of LSD1 in light acclimation and in restricting pathogen-induced cell death are functionally linked.
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