A high-throughput yellow fluorescent protein (YFP) cell-based screen identifies autophagy modulators to increase the effectiveness of radioiodine therapy

2019 ◽  
Author(s):  
Martin Read ◽  
Katie Baker ◽  
Alice Fletcher ◽  
Caitlin Thornton ◽  
Mohammed Alshahrani ◽  
...  
Keyword(s):  
High Throughput ◽  
Fluorescent Protein ◽  
Radioiodine Therapy ◽  
Yellow Fluorescent Protein

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 Versatile phenotype-activated cell sorting

2020 ◽  
Vol 6 (43) ◽  
pp. eabb7438
Author(s):  
Jihwan Lee ◽  
Zhuohe Liu ◽  
Peter H. Suzuki ◽  
John F. Ahrens ◽  
Shujuan Lai ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Human Cells ◽  
Target Cells ◽  
Temporal Properties ◽  
Single Target ◽  
New Variant ◽  
Improved Methods

Unraveling the genetic and epigenetic determinants of phenotypes is critical for understanding and re-engineering biology and would benefit from improved methods to separate cells based on phenotypes. Here, we report SPOTlight, a versatile high-throughput technique to isolate individual yeast or human cells with unique spatiotemporal profiles from heterogeneous populations. SPOTlight relies on imaging visual phenotypes by microscopy, precise optical tagging of single target cells, and retrieval of tagged cells by fluorescence-activated cell sorting. To illustrate SPOTlight’s ability to screen cells based on temporal properties, we chose to develop a photostable yellow fluorescent protein for extended imaging experiments. We screened 3 million cells expressing mutagenesis libraries and identified a bright new variant, mGold, that is the most photostable yellow fluorescent protein reported to date. We anticipate that the versatility of SPOTlight will facilitate its deployment to decipher the rules of life, understand diseases, and engineer new molecules and cells.

scholarly journals Development and utilization of human decidualization reporter cell line uncovers new modulators of female fertility

2019 ◽  
Vol 116 (39) ◽  
pp. 19541-19551
Author(s):  
Meade Haller ◽  
Yan Yin ◽  
Liang Ma
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Patient Specific ◽  
Reporter Cell Line ◽  
Reporter Cell ◽  
A Genome ◽  
Decidual Cells

Failure of embryo implantation accounts for a significant percentage of female infertility. Exquisitely coordinated molecular programs govern the interaction between the competent blastocyst and the receptive uterus. Decidualization, the rapid proliferation and differentiation of endometrial stromal cells into decidual cells, is required for implantation. Decidualization defects can cause poor placentation, intrauterine growth restriction, and early parturition leading to preterm birth. Decidualization has not yet been systematically studied at the genetic level due to the lack of a suitable high-throughput screening tool. Herein we describe the generation of an immortalized human endometrial stromal cell line that uses yellow fluorescent protein under the control of the prolactin promoter as a quantifiable visual readout of the decidualization response (hESC-PRLY cells). Using this cell line, we performed a genome-wide siRNA library screen, as well as a screen of 910 small molecules, to identify more than 4,000 previously unrecognized genetic and chemical modulators of decidualization. Ontology analysis revealed several groups of decidualization modulators, including many previously unappreciated transcription factors, sensory receptors, growth factors, and kinases. Expression studies of hits revealed that the majority of decidualization modulators are acutely sensitive to ovarian hormone exposure. Gradient treatment of exogenous factors was used to identify EC50 values of small-molecule hits, as well as verify several growth factor hits identified by the siRNA screen. The high-throughput decidualization reporter cell line and the findings described herein will aid in the development of patient-specific treatments for decidualization-based recurrent pregnancy loss, subfertility, and infertility.

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 Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

2013 ◽  
Vol 20 (4) ◽  
pp. 540-550 ◽  
Author(s):  
Thomas J. Gardner ◽  
Cynthia Bolovan-Fritts ◽  
Melissa W. Teng ◽  
Veronika Redmann ◽  
Thomas A. Kraus ◽  
...  
Keyword(s):  
Virus Infection ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Screening Tools ◽  
Surface Glycoprotein ◽  
Immediate Early ◽  
Viral Gene ◽  
Cmv Infection ◽  
High Throughput Assay

ABSTRACTInfection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infectionin vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFPcell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection.

High-Throughput Flow Cytometry–Based Assay to Identify Apoptosis-Inducing Proteins

2007 ◽  
Vol 12 (4) ◽  
pp. 510-520 ◽  
Author(s):  
Mamatha Sauermann ◽  
Florian Hahne ◽  
Christian Schmidt ◽  
Meher Majety ◽  
Heiko Rosenfelder ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
Specific Antibody ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Nuclear Fragmentation ◽  
Protein Fluorescence ◽  
Transfected Cells ◽  
High Throughput Assay

After sequencing the human genome, the challenge ahead is to systematically analyze the functions and disease relation of the proteins encoded. Here the authors describe the application of a flow cytometry—based high-throughput assay to screen for apoptosis-activating proteins in transiently transfected cells. The assay is based on the detection of activated caspase-3 with a specific antibody, in cells overexpressing proteins tagged C- or N-terminally with yellow fluorescent protein. Fluorescence intensities are measured using a flow cytometer integrated with a high-throughput autosampler. The applicability of this screen has been tested in a pilot screen with 200 proteins. The candidate proteins were all verified in an independent microscopy-based nuclear fragmentation assay, finally resulting in the identification of 6 apoptosis inducers. ( Journal of Biomolecular Screening 2007:510-520)

scholarly journals Optimizing the Expression of Recombinant αβγ GABAA Receptors in HEK293 Cells for High-Throughput Screening

2008 ◽  
Vol 14 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Daniel Gilbert ◽  
Abolghasem Esmaeili ◽  
Joseph W. Lynch
Keyword(s):  
High Throughput ◽  
Gabaa Receptors ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Effective Means ◽  
Hek293 Cells ◽  
Gaba A ◽  
Protein Assay ◽  
Lead Compounds

Despite being important clinical targets, it is not straightforward to reliably express recombinant trimeric αβγ GABA-A receptors (GABAARs) for high-throughput screening. This study therefore sought to devise a simple and reliable means of transiently expressing α1β1γ1 and α1β1γ2 GABAARs in HEK293 cells. Expression efficiencies resulting from 5 different transfection strategies were assessed by flow cytometry and pharmacological analysis using an anion-sensitive yellow fluorescent protein-based assay. PolyFect™ and Effectene™, employed according to the manufacturers' instructions, conferred the strongest and most reliable expression of trimeric αβγ GABAARs. Functional analysis via the yellow fluorescent protein assay revealed dramatic differences in the pharmacological properties of γ1- and γ2-containing receptors, consistent with previous electrophysiological characterizations. The authors conclude that this method of expressing and screening recombinant GABAARs provides an effective means of discovering novel GABAAR modulators for use as therapeutic lead compounds and pharmacological probes. ( Journal of Biomolecular Screening 2009:86-91)

A yellow fluorescent protein-based assay for high-throughput screening of glycine and GABAA receptor chloride channels

2005 ◽  
Vol 380 (3) ◽  
pp. 340-345 ◽  
Author(s):  
Wade Kruger ◽  
Daniel Gilbert ◽  
Rebecca Hawthorne ◽  
Deanne H. Hryciw ◽  
Stephan Frings ◽  
...  
Keyword(s):  
Gabaa Receptor ◽  
High Throughput ◽  
High Throughput Screening ◽  
Chloride Channels ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein

scholarly journals New mechanisms of radioiodide uptake revealed via a novel high throughput drug screening approach in thyroid cancer

2020 ◽  
Author(s):  
Martin L. Read ◽  
Katie Brookes ◽  
Caitlin E.M. Thornton ◽  
Alice Fletcher ◽  
Mohammed Alshahrani ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Cancer Genes ◽  
Iodide Uptake ◽  
Approved Drugs ◽  
The Impact ◽  
Fda Approved Drugs

ABSTRACTNew combinatorial drug strategies are urgently needed to improve radioiodide (RAI) uptake and efficiently ablate thyroid cancer cells, thereby addressing recurrent and metastatic disease. Cellular iodide uptake is accomplished solely by the sodium iodide symporter (NIS), but the complexity of NIS functional regulation and a lack of amenable high-throughput screening assays has impeded progress. We utilised mutated yellow fluorescent protein (YFP) as a surrogate biosensor of intracellular iodide for ∼1200 FDA-approved drugs, allowing us to appraise the impact of 73 leading compounds at 10 doses on 125I uptake in thyroid cancer cell lines. Subsequent mechanistic analysis suggests three predominant modes of drug action: Firstly, a number of drugs inhibited specific regulation of NIS function by the protein VCP. Secondly, some drugs enhanced transcriptional or post-transcriptional regulation of NIS expression. Thirdly, several drugs strongly implicated proteasomal degradation and the unfolded protein response in the cellular processing of NIS. Exploiting these mechanistic insights, multiple compounds gave striking increases in radioiodide uptake when combined with the drug SAHA. Importantly, our new drug combination strategies were also effective in human primary thyrocytes, suggesting they target endogenous NIS physiology. In patients with papillary thyroid cancer, genes involved in proteostasis were remarkably altered and predicted significantly worse outcome, but only in those patients who received RAI therapy. Collectively, we therefore propose a new model of intracellular NIS processing, and identify key nodes which may now be druggable in patients with aggressive thyroid cancer.SUMMARYOur data identify FDA-approved drugs that enhance radioiodide uptake outside of the canonical pathways of NIS processing, leading to a new mechanistic understanding of endogenous NIS function which is subverted in cancer.

scholarly journals A High-throughput Screening Method to Identify Proteins Involved in Unfolded Protein Response Signaling in Plants

2019 ◽  
Author(s):  
André Alcântara ◽  
Denise Seitner ◽  
Fernando Navarrete ◽  
Armin Djamei
Keyword(s):  
Unfolded Protein Response ◽  
High Throughput ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Screening Method ◽  
Screening Assay ◽  
Unfolded Protein ◽  
High Throughput Screening Assay ◽  
Protein Response

AbstractBackgroundThe unfolded protein response (UPR) is a highly conserved process in eukaryotic organisms that plays a crucial role in adaptation and development. While the most ubiquitous components of this pathway have been characterized, current efforts are focused on identifying and characterizing other UPR factors that play a role in specific conditions, such as developmental changes, abiotic cues, and biotic interactions. Considering the central role of protein secretion in plant pathogen interactions, there has also been a recent focus on understanding how pathogens manipulate their host’s UPR to facilitate infection.ResultsWe developed a high-throughput screening assay to identify proteins that interfere with UPR signalingin planta. A set of 35 genes from a library of secreted proteins from the maize pathogenUstilago maydiswere transiently co-expressed with a reporter construct that upregulates enhanced yellow fluorescent protein (eYFP) expression upon UPR stress inNicotiana benthamianaplants. After UPR stress induction, leaf discs were placed in 96 well plates and eYFP expression was measured. This allowed us to identify a previously undescribed fungal protein that inhibits plant UPR signaling, which was then confirmed using the classical but more laborious qRT-PCR method.ConclusionsWe have established a rapid and reliable fluorescence-based method to identify heterologously expressed proteins involved in UPR stress in plants. This system can be used for initial screens with libraries of proteins and potentially other molecules to identify candidates for further validation and characterization.

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