scholarly journals Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80

2009 ◽  
Vol 8 (4) ◽  
pp. 530-539 ◽  
Author(s):  
My-Hang Huynh ◽  
Vern B. Carruthers
Keyword(s):  
Toxoplasma Gondii ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Expression Profiles ◽  
Yellow Fluorescent Protein ◽  
Hypothetical Proteins ◽  
Ligation Independent Cloning ◽  
Proteomic Screen ◽  
Pcr Products ◽  
Dna Strand

ABSTRACT As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Δku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Δku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Δku80 strain and the pYFP.LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.

scholarly journals High-Throughput Growth Assay for Toxoplasma gondii Using Yellow Fluorescent Protein

2003 ◽  
Vol 47 (1) ◽  
pp. 309-316 ◽  
Author(s):  
Marc-Jan Gubbels ◽  
Catherine Li ◽  
Boris Striepen
Keyword(s):  
Toxoplasma Gondii ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Host Cells ◽  
Rapid Screening ◽  
Published Data ◽  
Parasite Line ◽  
Wild Type ◽  
Growth Assay

ABSTRACT A high-throughput growth assay for the protozoan parasite Toxoplasma gondii was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the β-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs (cytochalasin D, pyrimethamine, and clindamycin) resulted in values comparable to published data. The delayed parasite death kinetics of clindamycin could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of pyrimethamine was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of mycophenolic acid resistance after transfection of a resistance gene in T. gondii was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.

scholarly journals pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

2015 ◽  
Vol 28 (2) ◽  
pp. 107-121 ◽  
Author(s):  
Xiaoyan Gong ◽  
Oscar Hurtado ◽  
Baohua Wang ◽  
Congqing Wu ◽  
Mihwa Yi ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Yellow Fluorescent Protein ◽  
Effector Proteins ◽  
Fungal Transformation ◽  
In Planta ◽  
Green Fluorescent

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their “directly fused” counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

scholarly journals Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element–specific proteins reveal differentiation of the endomembrane system

2022 ◽  
Vol 119 (1) ◽  
pp. e2112755119
Author(s):  
Yan Liu ◽  
Viktoriya V. Vasina ◽  
Max E. Kraner ◽  
Winfried S. Peters ◽  
Uwe Sonnewald ◽  
...  
Keyword(s):  
Nicotiana Tabacum ◽  
Large Scale ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Proteome Analysis ◽  
Defense Responses ◽  
Sieve Element ◽  
Ferric Reductase ◽  
Endomembrane System ◽  
Sieve Elements

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins (SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

scholarly journals Diethylstilbestrol, a Novel ANO1 Inhibitor, Exerts an Anticancer Effect on Non-Small Cell Lung Cancer via Inhibition of ANO1

2021 ◽  
Vol 22 (13) ◽  
pp. 7100
Author(s):  
Yohan Seo ◽  
Sung Baek Jeong ◽  
Joo Han Woo ◽  
Oh-Bin Kwon ◽  
Sion Lee ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Small Cell ◽  
Channel Activity ◽  
Small Cell Lung ◽  
Protein Levels

Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer-related mortality; thus, therapeutic targets continue to be developed. Anoctamin1 (ANO1), a novel drug target considered for the treatment of NSCLC, is a Ca2+-activated chloride channel (CaCC) overexpressed in various carcinomas. It plays an important role in the development of cancer; however, the role of ANO1 in NSCLC is unclear. In this study, diethylstilbestrol (DES) was identified as a selective ANO1 inhibitor using high-throughput screening. We found that DES inhibited yellow fluorescent protein (YFP) fluorescence reduction caused by ANO1 activation but did not inhibit cystic fibrosis transmembrane conductance regulator channel activity or P2Y activation-related cytosolic Ca2+ levels. Additionally, electrophysiological analyses showed that DES significantly reduced ANO1 channel activity, but it more potently reduced ANO1 protein levels. DES also inhibited the viability and migration of PC9 cells via the reduction in ANO1, phospho-ERK1/2, and phospho-EGFR levels. Moreover, DES induced apoptosis by increasing caspase-3 activity and PARP-1 cleavage in PC9 cells, but it did not affect the viability of hepatocytes. These results suggest that ANO1 is a crucial target in the treatment of NSCLC, and DES may be developed as a potential anti-NSCLC therapeutic agent.

scholarly journals IL-6 contributes to metastatic switch via the differentiation of monocytic-dendritic progenitors into prometastatic immune cells

2021 ◽  
Vol 9 (6) ◽  
pp. e002856
Author(s):  
Ksenia Magidey-Klein ◽  
Tim J Cooper ◽  
Ksenya Kveler ◽  
Rachelly Normand ◽  
Tongwu Zhang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Data Analysis ◽  
Conditioned Medium ◽  
Mononuclear Cells ◽  
Fluorescent Protein ◽  
Metastatic Potential ◽  
Loss Of Function ◽  
Tumor Spread ◽  
Hematopoietic Stem ◽  
Proteomic Screen

BackgroundMetastasis is the major cause of death in patients with cancer. Myeloid skewing of hematopoietic cells is a prominent promoter of metastasis. However, the reservoir of these cells in the bone marrow (BM) compartment and their differentiation pattern from hematopoietic stem and progenitor cells (HSPCs) have not been explored.MethodsWe used a unique model system consisting of tumor cell clones with low metastatic potential or high metastatic potential (met-low and met-high, respectively) to investigate the fate of HSPC differentiation using murine melanoma and breast carcinoma. Single-cell RNA sequencing (scRNA-seq) analysis was performed on HSPC obtained from the BM of met-low and met-high tumors. A proteomic screen of tumor-conditioned medium integrated with the scRNA-seq data analysis was performed to analyze the potential cross talk between cancer cells and HSPCs. Adoptive transfer of tumor-educated HSPC subsets obtained from green fluorescent protein (GFP)+ tagged mice was then carried out to identify the contribution of committed HSPCs to tumor spread. Peripheral mononuclear cells obtained from patients with breast and lung cancer were analyzed for HSPC subsets.ResultsMice bearing met-high tumors exhibited a significant increase in the percentage of HSPCs in the BM in comparison with tumor-free mice or mice bearing met-low tumors. ScRNA-seq analysis of these HSPCs revealed that met-high tumors enriched the monocyte-dendritic progenitors (MDPs) but not granulocyte-monocyte progenitors (GMPs). A proteomic screen of tumor- conditioned medium integrated with the scRNA-seq data analysis revealed that the interleukin 6 (IL-6)–IL-6 receptor axis is highly active in HSPC-derived MDP cells. Consequently, loss of function and gain of function of IL-6 in tumor cells resulted in decreased and increased metastasis and corresponding MDP levels, respectively. Importantly, IL-6-educated MDPs induce metastasis within mice bearing met-low tumors—through further differentiation into immunosuppressive macrophages and not dendritic cells. Consistently, MDP but not GMP levels in peripheral blood of breast and lung cancer patients are correlated with tumor aggressiveness.ConclusionsOur study reveals a new role for tumor-derived IL-6 in hijacking the HSPC differentiation program toward prometastatic MDPs that functionally differentiate into immunosuppressive monocytes to support the metastatic switch.

scholarly journals A High-Content Screening Assay for Small Molecules That Stabilize Mutant Triose Phosphate Isomerase (TPI) as Treatments for TPI Deficiency

2021 ◽  
pp. 247255522110181
Author(s):  
Andreas Vogt ◽  
Samantha L. Eicher ◽  
Tracey D. Myers ◽  
Stacy L. Hrizo ◽  
Laura L. Vollmer ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Large Scale ◽  
Protein Quality ◽  
Fluorescent Protein ◽  
Triose Phosphate Isomerase ◽  
Pharmacological Intervention ◽  
Screening Assay ◽  
Triose Phosphate ◽  
The Common ◽  
Green Fluorescent

Triose phosphate isomerase deficiency (TPI Df) is an untreatable, childhood-onset glycolytic enzymopathy. Patients typically present with frequent infections, anemia, and muscle weakness that quickly progresses with severe neuromusclar dysfunction requiring aided mobility and often respiratory support. Life expectancy after diagnosis is typically ~5 years. There are several described pathogenic mutations that encode functional proteins; however, these proteins, which include the protein resulting from the “common” TPIE105D mutation, are unstable due to active degradation by protein quality control (PQC) pathways. Previous work has shown that elevating mutant TPI levels by genetic or pharmacological intervention can ameliorate symptoms of TPI Df in fruit flies. To identify compounds that increase levels of mutant TPI, we have developed a human embryonic kidney (HEK) stable knock-in model expressing the common TPI Df protein fused with green fluorescent protein (HEK TPIE105D-GFP). To directly address the need for lead TPI Df therapeutics, these cells were developed into an optical drug discovery platform that was implemented for high-throughput screening (HTS) and validated in 3-day variability tests, meeting HTS standards. We initially used this assay to screen the 446-member National Institutes of Health (NIH) Clinical Collection and validated two of the hits in dose–response, by limited structure–activity relationship studies with a small number of analogs, and in an orthogonal, non-optical assay in patient fibroblasts. The data form the basis for a large-scale phenotypic screening effort to discover compounds that stabilize TPI as treatments for this devastating childhood disease.

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