34 research outputs found
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Multidrug and efflux transporters of the model microbe Dictyostelium discoideum
The evolutionarily ubiquitous multidrug and toxin efflux (MATE) proteins mediate anticancer and antibiotic resistance, while transporting toxins, ions and flavonoids in plants. MATEs of the model amoeba Dictyostelium discoideumhave not been studied although sequences of its pair group with the two Homo sapiens MATEs. Ddmate1 and 2 are both transcribed, Ddmate2 more so, with peaks in vegetative and slug life-cycle stages. Ddmate1 was upregulated in response to a toxin, ethidium bromide, at the lowest concentration tested. Removing MATE function by inhibitor or mutation increased intracellular levels of various compounds, confirming these as efflux transporters. Plasma membrane localisation was revealed using a GFP-MATE1 reporter-line. MATE1 and MATE2 phenotypes indicated roles beyond detoxification: on Klebsiella lawns these mutants produced significantly smaller plaques than WT, and their axenic growth rates were also lower. The transportersâ impact on use of Dictyostelium for novel drug research was tested using flavonoids. LCMS and fluorescence-imaging revealed differential flavonoid uptake. Flavanones such as naringenin did not cross into cells, whereas flavonols localised to mitochondria and cytoplasm. Ddmate1transcription was upregulated, however, in response to naringenin, which is known to reduce levels of kidney-disease protein PKD2 in both Dictyostelium and animal cells. Increased flavonol intracellular concentrations confirmed that efflux not import was impeded in MATE1andMATE2, and kaempferol therefore further reduced MATE1-cellsâ growth. These D. discoideum MATEs may usefully model the HsMATEs, aid understanding of flavonoidsâ effects, and should be considered when using this model eukaryote to screen drugs
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Life cycle-specific secondary metabolites in the model eukaryotic microbe, Dictyostelium
The social amoeba Dictyostelium discoideum is an environmental eukaryote that detects and engulfs bacteria and yeast as a food source. Its life cycle progresses from unicellular to multicellular stages, with cAMP as the chemoattractant and the signal controlling the developmental programme. Chemosensing and signalling, cell-type differentiation, the similar gene content to higher animals, and its phagocytosis of other microbes have all led to Dictyostelium becoming a useful biomedical and cell-biology model organism. Its secondary metabolism is less explored, although interest is increasing. The amoeba encodes genes involved in quite diverse biosynthetic pathways, through to those encoding the proteins carrying out final transport mechanisms. Two multidrug and efflux (MATE) transporters are encoded, orthologues of which are notable for sequestering flavonoids in plant vacuoles, among other functions. Having demonstrated the ability of Dictyostelium MATE proteins to efflux specific polyphenolic substrates, genes were identified in the amoeba that are similar to those for synthesis of plant polyphenolics. These enabled targeted analysis of âplantâ compounds in the amoeba. Life cycle analysis reveals peaks of genetic expression and chemical content at specific stages in development, augmenting the currently limited knowledge of the physical roles of Dictyostelium secondary metabolites
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A day in the life of mitochondria reveals shifting workloads
Mitochondria provide energy for cellular function. We examine daily changing patterns of mitochondrial function and metabolism in Drosophila in vivo in terms of their complex (I-IV) activity, ATP production, glycolysis, and whole fly respiration in the morning, afternoon and night. Complex activity and respiration showed significant and unexpected variation, peaking in the afternoon. However, ATP levels by contrast are >40% greater in the morning and lowest at night when glycolysis peaks. Complex activity modulation was at the protein level with no evidence for differential transcription over the day. Timing differences between increased ATP production and peaks of complex activity may result from more efficient ATP production early in the day leaving complex activity with spare capacity. Optical stimulation of mitochondria is only possible in the mornings when there is such spare capacity.
These results provide first evidence of shifts in cellular energy capacity at the organism level. Understanding their translation may be significant to the chosen timing of energy demanding interventions to improve function and health
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Crop biology under agrivoltaics systemsâ skewed spectrum light
Agrivoltaics refers to the concurrent production of crops with solar-derived electrical generation. It offers a means of generating âgreenâ energy while producing exploitable biomass and enhancing profit by reducing production costs. Semi-transparent, colour-tinted panels, in addition, permit light transmission, produce power for the farm/growth house, and provide tailored lighting wavelengths for different growth processes and sector need. The aim of this project is to evaluate contrasting crop types and the positive and negative effects of growth under tinted systems. Root, leafy and soft fruit crops were compared under the skewed spectrum resulting from orange tinted panels, to understand their growth and biochemistry in such red-enhanced light conditions. Plant growth, morphology and nutrient content were observed to differ in all crop types. Rocket and tomato biomass under tinted conditions showed a smaller reduction, when compared with neutral density shaded plants, than that of radish varieties. An increased protein content previously reported was consistently observed. Further aspects of biochemistry and fruit characteristics suggest other high value features may compensate for deleterious biomass effects
Building a Field: The Future of Astronomy with Gravitational Waves
Harnessing the sheer discovery potential of GW Astronomy will require bold, deliberate,and sustained efforts to train and develop the requisite workforce. The next decaderequires a strategic plan to build - from the ground up - a robust, open, andwell-connected GW Astronomy community with deep participation from traditionalastronomers, physicists, data scientists, and instrumentalists. This basic infrastructure issorely needed as an enabling foundation for research. We outline a set ofrecommendations for funding agencies, universities, and professional societies to helpbuild a thriving, diverse, and inclusive new field
Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes
This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application
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Production, titration, neutralisation and storage of SARS-CoV-2 lentiviral pseudotypes
This protocol details a rapid and reliable method for the production and titration of high-titer viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G variant) on a lentiviral vector core, and use for neutralization assays in target cells expressing ACE2 and TMPRSS2. It additionally provides detailed instruction on substituting in new spike variants via gene cloning, and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titers to SARS-CoV and MERS-CoV pseudotypes, that they can be neutralized by human convalescent plasma and monoclonal antibodies and that they can be stored at a range of laboratory temperatures and lyophilized for distribution
TRY plant trait database â enhanced coverage and open access
Plant traits - the morphological, anatomical, physiological, biochemical and phenological characteristics of plants - determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of traitâbased plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits - almost complete coverage for âplant growth formâ. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and traitâenvironmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives