scholarly journals Found in Translation: High-Throughput Chemical Screening in Arabidopsis thaliana Identifies Small Molecules That Reduce Fusarium Head Blight Disease in Wheat

2011 ◽  
Vol 24 (6) ◽  
pp. 640-648 ◽  
Author(s):  
Karl J. Schreiber ◽  
Charles G. Nasmith ◽  
Ghislaine Allard ◽  
Jasbir Singh ◽  
Rajagopal Subramaniam ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Small Molecules ◽  
High Throughput ◽  
Crop Protection ◽  
Indole Alkaloid ◽  
Small Scale ◽  
Head Blight ◽  
Biologically Relevant ◽  
Chemical Screening ◽  
Model Plant Arabidopsis Thaliana

Despite the tremendous economic impact of cereal crop pathogens such as the fungus Fusarium graminearum, the development of strategies for enhanced crop protection is hampered by complex host genetics and difficulties in performing high-throughput analyses. To bypass these challenges, we have developed an assay in which the interaction between F. graminearum and the model plant Arabidopsis thaliana is monitored in liquid media in 96-well plates. In this assay, fungal infection is associated with the development of dark lesion-like spots on the cotyledons of Arabidopsis seedlings by 4 days postinoculation. These symptoms can be alleviated by the application of known defense-activating small molecules and in previously described resistant host genetic backgrounds. Based on this infection phenotype, we conducted a small-scale chemical screen to identify small molecules that protect Arabidopsis seedlings from infection by F. graminearum. We identified sulfamethoxazole and the indole alkaloid gramine as compounds with strong protective activity in the liquid assay. Remarkably, these two chemicals also significantly reduced the severity of F. graminearum infection in wheat. As such, the Arabidopsis-based liquid assay represents a biologically relevant surrogate system for high-throughput studies of agriculturally important plant–pathogen interactions.

Differential Induction of Glutathione Transferases and Glucosyltransferases in Wheat, Maize and Arabidopsis thaliana by Herbicide Safeners

2005 ◽  
Vol 60 (3-4) ◽  
pp. 307-316 ◽  
Author(s):  
Robert Edwards ◽  
Daniele Del Buono ◽  
Michael Fordham ◽  
Mark Skipsey ◽  
Melissa Brazier ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Crop Protection ◽  
Detoxification Enzymes ◽  
Glutathione Transferases ◽  
Wide Range ◽  
Breeding Programmes ◽  
Model Plant Arabidopsis Thaliana ◽  
Herbicide Safeners ◽  
Species Specific ◽  
The Right

Abstract By learning lessons from weed science we have adopted three approaches to make plants more effective in phytoremediation: 1. The application of functional genomics to identify key components involved in the detoxification of, or tolerance to, xenobiotics for use in subsequent genetic engineering/breeding programmes. 2. The rational metabolic engineering of plants through the use of forced evolution of protective enzymes, or alternatively transgenesis of detoxification pathways. 3. The use of chemical treatments which protect plants from herbicide injury. In this paper we examine the regulation of the xenome by herbicide safeners, which are chemicals widely used in crop protection due to their ability to enhance herbicide selectivity in cereals. We demonstrate that these chemicals act to enhance two major groups of phase 2 detoxification enzymes, notably the glutathione transferases and glucosyltransferases, in both cereals and the model plant Arabidopsis thaliana, with the safeners acting in a chemical- and species-specific manner. Our results demonstrate that by choosing the right combination of safener and plant it should be possible to enhance the tolerance of diverse plants to a wide range of xenobiotics including pollutants.

Validation of the high-throughput marker technology DArT using the model plant Arabidopsis thaliana

2005 ◽  
Vol 274 (1) ◽  
pp. 30-39 ◽  
Author(s):  
Alexander H. J. Wittenberg ◽  
Theo van der Lee ◽  
Cyril Cayla ◽  
Andrzej Kilian ◽  
Richard G. F. Visser ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
High Throughput ◽  
Model Plant ◽  
Model Plant Arabidopsis Thaliana ◽  
Plant Arabidopsis Thaliana

scholarly journals A High-Throughput Cell-Based Gaussia Luciferase Reporter Assay for Identifying Modulators of Fibulin-3 Secretion

2012 ◽  
Vol 18 (6) ◽  
pp. 647-658 ◽  
Author(s):  
John D. Hulleman ◽  
Steven J. Brown ◽  
Hugh Rosen ◽  
Jeffery W. Kelly
Keyword(s):  
Small Molecules ◽  
Cell Lines ◽  
High Throughput ◽  
Age Related Macular Degeneration ◽  
Luciferase Reporter ◽  
Macular Dystrophy ◽  
Gaussia Luciferase ◽  
Chemical Screening ◽  
Age Related ◽  
Pharmacologically Active

An R345W mutation in fibulin-3 causes its inefficient secretion, increased intracellular steady-state levels, and the macular dystrophy, Malattia Leventinese (ML), a disease similar to age-related macular degeneration. It is unknown whether R345W causes ML through increased intracellular levels, by the secretion of a potentially aggregation-prone protein, or both. To identify small molecules that alter the secretion of fibulin-3, we developed ARPE19 retinal cell lines that inducibly express wild-type (WT) or R345W fibulin-3 fused to an enhanced Gaussia luciferase (eGLuc2). Screening of the Library of Pharmacologically Active Compounds demonstrated that these cell lines and the GLuc assay are suitable for high-throughput chemical screening. Two estrogen-related compounds enhanced fibulin-3 secretion, whereas a diverse series of small molecules reduced fibulin-3 secretion. A counterscreen identified compounds that did not substantially alter the secretion of unfused eGLuc2, demonstrating at least partial selectivity for fibulin-3. A secondary assay using untagged fibulin-3 confirmed that the top three inhibitory compounds reduced R345W fibulin-3 secretion. Interestingly, in untagged fibulin-3 studies, one compound, phorbol 12-myristate 13-acetate, reduced R345W fibulin-3 secretion while minimally enhancing WT fibulin-3 secretion, the desired activity and selectivity we sought for ML. The identified compounds could serve as tools for probing the etiology of fibulin-3–related diseases.

scholarly journals Development of a Dimethylarginine Dimethylaminohydrolase (DDAH) Assay for High-Throughput Chemical Screening

2012 ◽  
Vol 17 (5) ◽  
pp. 651-661 ◽  
Author(s):  
Yohannes T. Ghebremariam ◽  
Daniel A. Erlanson ◽  
Keisuke Yamada ◽  
John P. Cooke
Keyword(s):  
Nitric Oxide ◽  
Small Molecules ◽  
High Throughput ◽  
Asymmetric Dimethylarginine ◽  
Vascular Diseases ◽  
Dimethylarginine Dimethylaminohydrolase ◽  
Physiological Concentration ◽  
Chemical Screening ◽  
Migraine Headaches ◽  
Oxide Synthase

Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.

scholarly journals 4241 Identification of small molecules that facilitate the efficient differentiation of stem cell derived β-cells

2020 ◽  
Vol 4 (s1) ◽  
pp. 8-8
Author(s):  
Yuhao Min ◽  
Chris Clifford ◽  
Quinn P. Peterson
Keyword(s):  
Stem Cells ◽  
Small Molecules ◽  
High Throughput ◽  
Single Cells ◽  
Embryonic Stem ◽  
Immunocytochemical Staining ◽  
Β Cells ◽  
Cell Stage ◽  
Chemical Screening ◽  
Screening Platform

OBJECTIVES/GOALS: In this study, we established a high-throughput chemical screening platform to identify small molecules that facilitates efficient differentiation of stem cells derived β (SC-β) cells. Using this platform, we identified several compounds that potentially increase the differentiation efficiency. METHODS/STUDY POPULATION: Differentiation of human embryonic stem cells (HUES8) into SC-β was carried out using previously published protocols in a 3D cell suspension. Single cells were replated in Matrigel-coated well plates at the start of different stages depending on experiments. Differentiation medium supplemented with small molecules at a final concentration of 2 M and 0.2 M was used throughout the stage. All the cells were then fixed and permeabilized. Immunocytochemical staining was performed. Images of each well were taken and analyzed. Numbers of the total cell, insulin-positive cell, NKX6.1-positive cell, and co-positive cell were recorded. Candidate compounds were validated using flow cytometry or ICC. RESULTS/ANTICIPATED RESULTS: We identified several hit compounds that significantly increase the NKX6.1 positive cell percentage compared to the DMSO-treated controls when treated at the PP1 cell stage. Follow up assays demonstrated that at least one of these putative hits reproducibly increased NKX6.1 expression. In addition, we identified other compounds that significantly increase the insulin and NKX6.1 copositive SC-β cell population when treated at the later PP2 cell stage during the differentiation. We expect a dosage-dependent response when the candidate hits are validated using more accurate assays. DISCUSSION/SIGNIFICANCE OF IMPACT: We established a high-throughput screening platform to identify small molecules that increase the efficiency of SC-β direct differentiation. Successful generation of SC-β allows cell replacement therapy in diabetes patients, and a better understanding of pancreatic biology and development.

scholarly journals Mapping the osmotic component of salinity tolerance in Arabidopsis thaliana

2015 ◽  
Author(s):  
Mariam Awlia ◽  
Mark Tester
Keyword(s):  
Arabidopsis Thaliana ◽  
Salt Stress ◽  
Salinity Tolerance ◽  
High Throughput ◽  
Imaging Techniques ◽  
Early Response ◽  
Photochemical Quenching ◽  
Tolerance Index ◽  
Contributing Factors ◽  
Model Plant Arabidopsis Thaliana

Salt stress significantly reduces plant growth, as well as crop yield, and is becoming an increasing problem with the growing presence of salt-affected and arid land. To enhance and better understand the mechanisms of salinity tolerance, we propose to dissect the early responses to salinity. This early response phase is termed the osmotic component of salinity tolerance (sensu Munns & Tester, 2008), which has been further described as the shoot ion independent tolerance (Roy et al., 2014). High throughput and non-destructive imaging techniques have been utilised to discover the genes affecting the osmotic component of salinity tolerance in the model plant Arabidopsis thaliana. Non-invasive phenotyping can capture multiple growth measurements and other phenotypic parameters upon imposition of salt stress, enabling quantification of “osmotic tolerance”. Doing this for members of association mapping populations enables the identification of quantitative trait loci (QTL), and ultimately genes, underlying the shoot ion independent tolerance, thus, providing insights into the controlling processes. By testing the high-throughput phenotyping system at Photon Systems Instruments (PSI), we have been able to optimise the GWAS experimental design and select the most promising parameters that best illustrate the salt effect in the early shoot ion independent phase. The acquired data from these experiments will be presented, including general observations on the shoot ion independent tolerance index and other contributing factors such as changes in leaf colour, rosette temperature and non-photochemical quenching. The analysis of these datasets has facilitated for us to design a GWAS experiment using 350 Arabidopsis accessions that will be evaluated in terms of their shoot ion independent response.

scholarly journals High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

2014 ◽  
Vol 13 (3) ◽  
pp. 412-426 ◽  
Author(s):  
Paula MacGregor ◽  
Alasdair Ivens ◽  
Steven Shave ◽  
Iain Collie ◽  
David Gray ◽  
...  
Keyword(s):  
Small Molecules ◽  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Morphological Changes ◽  
Specific Gene ◽  
African Trypanosomes ◽  
Chemical Screening

ABSTRACT In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei , exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo . Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.

scholarly journals Druggable Transient Pockets in Protein Kinases

Molecules ◽  
2021 ◽  
Vol 26 (3) ◽  
pp. 651
Author(s):  
Koji Umezawa ◽  
Isao Kii
Keyword(s):  
Drug Discovery ◽  
Small Molecules ◽  
Protein Kinases ◽  
Binding Sites ◽  
Kinase Inhibitors ◽  
Screening Methods ◽  
Research Attention ◽  
Folding Process ◽  
Chemical Screening ◽  
Inhibitory Mechanisms

Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of “cryptic inhibitor-binding sites.” These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.

Sign in / Sign up

Export Citation Format

Share Document