scholarly journals High throughput screening of Toxoplasma gondii clones by PCR after limitation cloning

2013 ◽  
Author(s):  
Ting-Kai Liu
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
High Throughput Screening ◽  
Early Stage ◽  
Gene Knockout ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Serological Studies ◽  
Gene Defects ◽  
Global Population

Toxoplasma gondii is a kind of obligate intracellular parasites that are capable of infecting virtually all warm-blooded animals. It is one of the most common parasites in human. Serological studies estimated that up to a third of the global population has been chronically infected with the parsites. Toxoplasma gondii is also used as a model orgnisim of Apicomplexans that includes Plasmodium – the parasites that cause malaria. Gene knockout is a very important way to study gene function in all organisms. When it comes to Toxoplasma, it’s very difficult as this haploid parasite has strong adaptability to circumvent the condition of gene defects by gene duplication. High throughput screening at very early stage of transfection is very important for generating a true knockout of this parasite. For some genes that are required forToxoplasma gondii, the high throughput screening is necessary. Different labs use different protocols. After reading and practicing protocols from different papers or different labs, here I established an efficient pipeline for this purpose.

scholarly journals High throughput screening of Toxoplasma gondii clones by PCR after limitation cloning

2013 ◽  
Author(s):  
Ting-Kai Liu
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
High Throughput Screening ◽  
Early Stage ◽  
Gene Knockout ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Serological Studies ◽  
Gene Defects ◽  
Global Population

Toxoplasma gondii is a kind of obligate intracellular parasites that are capable of infecting virtually all warm-blooded animals. It is one of the most common parasites in human. Serological studies estimated that up to a third of the global population has been chronically infected with the parsites. Toxoplasma gondii is also used as a model orgnisim of Apicomplexans that includes Plasmodium – the parasites that cause malaria. Gene knockout is a very important way to study gene function in all organisms. When it comes to Toxoplasma, it’s very difficult as this haploid parasite has strong adaptability to circumvent the condition of gene defects by gene duplication. High throughput screening at very early stage of transfection is very important for generating a true knockout of this parasite. For some genes that are required forToxoplasma gondii, the high throughput screening is necessary. Different labs use different protocols. After reading and practicing protocols from different papers or different labs, here I established an efficient pipeline for this purpose.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2019 ◽  
Vol 25 (1) ◽  
pp. 9-20 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

Cell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes, and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed the cell-based cytotoxicity of nearly 10,000 compounds in the National Institutes of Health, National Center for Advancing Translational Sciences annotated libraries and more than 100,000 compounds in a diversity library against four normal cell lines (HEK 293, NIH 3T3, CRL-7250, and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity, and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitute a valuable resource for the scientific community and provide insight into the extent of cytotoxic compounds in screening libraries, allowing for the identification and avoidance of compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals High-throughput screening for developability during early-stage antibody discovery using self-interaction nanoparticle spectroscopy

mAbs ◽  
2013 ◽  
Vol 6 (2) ◽  
pp. 483-492 ◽  
Author(s):  
Yuqi Liu ◽  
Isabelle Caffry ◽  
Jiemin Wu ◽  
Steven B Geng ◽  
Tushar Jain ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Early Stage ◽  
Antibody Discovery

scholarly journals Cytotoxic Profiling of Annotated and Diverse Chemical Libraries Using Quantitative High-Throughput Screening

2018 ◽  
Author(s):  
Olivia W. Lee ◽  
Shelley Austin ◽  
Madison Gamma ◽  
Dorian M. Cheff ◽  
Tobie D. Lee ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Large Scale ◽  
Early Stage ◽  
Cancer Cell Line ◽  
Hek 293 ◽  
Cytotoxic Compounds

AbstractCell-based phenotypic screening is a commonly used approach to discover biological pathways, novel drug targets, chemical probes and high-quality hit-to-lead molecules. Many hits identified from high-throughput screening campaigns are ruled out through a series of follow-up potency, selectivity/specificity, and cytotoxicity assays. Prioritization of molecules with little or no cytotoxicity for downstream evaluation can influence the future direction of projects, so cytotoxicity profiling of screening libraries at an early stage is essential for increasing the likelihood of candidate success. In this study, we assessed cell-based cytotoxicity of nearly 10,000 compounds in NCATS annotated libraries, and over 100,000 compounds in a diversity library, against four ‘normal’ cell lines (HEK 293, NIH 3T3, CRL-7250 and HaCat) and one cancer cell line (KB 3-1, a HeLa subline). This large-scale library profiling was analyzed for overall screening outcomes, hit rates, pan-activity and selectivity. For the annotated library, we also examined the primary targets and mechanistic pathways regularly associated with cell death. To our knowledge, this is the first study to use high-throughput screening to profile a large screening collection (>100,000 compounds) for cytotoxicity in both normal and cancer cell lines. The results generated here constitutes a valuable resource for the scientific community and provides insight on the extent of cytotoxic compounds in screening libraries, identifying and avoiding compounds with cytotoxicity during high-throughput screening campaigns.

scholarly journals Involvement of multiple influx and efflux transporters in the accumulation of cationic fluorescent dyes byEscherichia coli

2019 ◽  
Author(s):  
Srijan Jindal ◽  
Lei Yang ◽  
Philip J. Day ◽  
Douglas B. Kell
Keyword(s):  
Steady State ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Dyes ◽  
Gene Knockout ◽  
Sybr Green ◽  
Efflux Transporters ◽  
General Strategy ◽  
Individual Gene

AbstractWe used high-throughput flow cytometry to assess the ability of individual gene knockout strains ofE colito take up two membrane-permeable, cationic fluorescent dyes, viz the carbocyanine diS-C3(5) and the DNA dye SYBR Green. Individual strains showed a large range of distributions of uptake. The range of modal steady-state uptakes for the carbocyanine between the different strains was 36-fold. Knockouts of the ATP synthase α- and β-subunits greatly inhibited uptake, implying that most uptake was ATP-driven rather than being driven by say a membrane potential. Dozens of transporters changed the steady-state uptake of the dye by more than 50% with respect to that of the wild type, in both directions (increased or decreased); knockouts in known influx and efflux transporters behaved as expected, giving confidence in the general strategy. Many of the knockouts with the most reduced uptake were transporter genes of unknown function (‘y-genes’). Similarly, several overexpression variants in the ‘ASKA’ collection had the anticipated, opposite effects. Similar findings were made with SYBR Green (the range being some 69-fold), though despite it too containing a benzimidazole motif there was negligible correlation between its uptake and that of the carbocyanine when compared across the various strains. Overall, we conclude that the uptake of these dyes may be catalysed by a great many transporters of possibly broad and presently unknown specificity. This casts serious doubt upon the use of such dyes as quantitative stains for representing either bioenergetic parameters or the amount of cellular DNA in unfixed cells (in vivo). By contrast, it opens up their potential use as transporter assay substrates in high-throughput screening.

scholarly journals Establishment of Novel High-Standard Chemiluminescent Assay for NTPase in Two Protozoans and Its High-Throughput Screening

Marine Drugs ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 161
Author(s):  
Masamitsu Harada ◽  
Jun Nagai ◽  
Riho Kurata ◽  
Kenji Shimizu ◽  
Xiaofeng Cui ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
Marine Bacteria ◽  
High Throughput Screening ◽  
Neospora Caninum ◽  
Organ Transplant ◽  
High Standard ◽  
Nucleoside Triphosphate ◽  
Synthetic Compound ◽  
Compound Libraries

Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.

scholarly journals Vitamin and cofactor acquisition in apicomplexans: Synthesis versus salvage

2019 ◽  
Vol 295 (3) ◽  
pp. 701-714 ◽  
Author(s):  
Aarti Krishnan ◽  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Dominique Soldati-Favre
Keyword(s):  
Toxoplasma Gondii ◽  
Nutrient Availability ◽  
Metabolic Reconstruction ◽  
Mammalian Host ◽  
Free Living ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Functional Studies ◽  
Intracellular Parasites ◽  
Large Repertoire

The Apicomplexa phylum comprises diverse parasitic organisms that have evolved from a free-living ancestor. These obligate intracellular parasites exhibit versatile metabolic capabilities reflecting their capacity to survive and grow in different hosts and varying niches. Determined by nutrient availability, they either use their biosynthesis machineries or largely depend on their host for metabolite acquisition. Because vitamins cannot be synthesized by the mammalian host, the enzymes required for their synthesis in apicomplexan parasites represent a large repertoire of potential therapeutic targets. Here, we review recent advances in metabolic reconstruction and functional studies coupled to metabolomics that unravel the interplay between biosynthesis and salvage of vitamins and cofactors in apicomplexans. A particular emphasis is placed on Toxoplasma gondii, during both its acute and latent stages of infection.

scholarly journals A Perspective on the Future of High-Throughput RNAi Screening

2015 ◽  
Vol 20 (8) ◽  
pp. 1040-1051 ◽  
Author(s):  
Jessica Taylor ◽  
Simon Woodcock
Keyword(s):  
Functional Genomics ◽  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Gene Knockout ◽  
Strand Break ◽  
Loss Of Function ◽  
Rnai Screening ◽  
Guide Rna ◽  
Cas A

For more than a decade, RNA interference (RNAi) has brought about an entirely new approach to functional genomics screening. Enabling high-throughput loss-of-function (LOF) screens against the human genome, identifying new drug targets, and significantly advancing experimental biology, RNAi is a fast, flexible technology that is compatible with existing high-throughput systems and processes; however, the recent advent of clustered regularly interspaced palindromic repeats (CRISPR)-Cas, a powerful new precise genome-editing (PGE) technology, has opened up vast possibilities for functional genomics. CRISPR-Cas is novel in its simplicity: one piece of easily engineered guide RNA (gRNA) is used to target a gene sequence, and Cas9 expression is required in the cells. The targeted double-strand break introduced by the gRNA–Cas9 complex is highly effective at removing gene expression compared to RNAi. Together with the reduced cost and complexity of CRISPR-Cas, there is the realistic opportunity to use PGE to screen for phenotypic effects in a total gene knockout background. This review summarizes the exciting development of CRISPR-Cas as a high-throughput screening tool, comparing its future potential to that of well-established RNAi screening techniques, and highlighting future challenges and opportunities within these disciplines. We conclude that the two technologies actually complement rather than compete with each other, enabling greater understanding of the genome in relation to drug discovery.

scholarly journals Development of a Fluorescence-Based, Ultra High-Throughput Screening Platform for Nanoliter-Scale Cytochrome P450 Microarrays

2009 ◽  
Vol 14 (6) ◽  
pp. 668-678 ◽  
Author(s):  
Sumitra M. Sukumaran ◽  
Benjamin Potsaid ◽  
Moo-Yeal Lee ◽  
Douglas S. Clark ◽  
Jonathan S. Dordick
Keyword(s):  
Enzyme Activity ◽  
Cytochrome P450 ◽  
High Throughput ◽  
High Throughput Screening ◽  
Imaging System ◽  
Early Stage ◽  
Wide Field ◽  
Cytochrome P450 Enzyme ◽  
Assay Sensitivity ◽  
Screening Platform

Cytochrome P450 enzyme (CYP450s) assays are critical enzymes in early-stage lead discovery and optimization in drug development. Currently available fluorescence-based reaction assays provide a rapid and reliable method for monitoring CYP450 enzyme activity but are confined to medium-throughput well-plate systems. The authors present a high-throughput, integrated screening platform for CYP450 assays combining enzyme encapsulation techniques, microarraying methods, and wide-field imaging. Alginate-containing microarrays consisting of up to 1134 CYP450 reaction elements were fabricated on functionalized glass slides (reaction volumes 20 to 80 nL, total enzyme content in pg) and imaged to yield endpoint activity, stability, and kinetic data. A charge-coupled device imager acquired quantitative, high-resolution images of a 20 × 20 mm area/snapshot using custom-built wide-field optics with telecentric lenses and easily interchangeable filter sets. The imaging system offered a broad dynamic intensity range (linear over 3 orders of magnitude) and sensitivity down to fluorochrome quantities of <5 fmols, with read accuracy similar to a laser scanner or a fluorescence plate reader but with higher throughput. Rapid image acquisition enabled analysis of CYP450 kinetics. Fluorogenic assays with CYP3A4, CYP2C9, and CYP2D6 on the alginate microarrays exhibited Z′ factors ranging from 0.75 to 0.85, sensitive detection of inhibitory compounds, and reactivity comparable to that in solution, thereby demonstrating the reliability and accuracy of the microarray platform. This system enables for the first time a significant miniaturization of CYP enzyme assays with significant conservation of assay reagents, greatly increased throughput, and no apparent loss of enzyme activity or assay sensitivity. ( Journal of Biomolecular Screening 2009:668-678)

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