scholarly journals Agrosuppression: A Bioassay for the Hypersensitive Response Suited to High-Throughput Screening

2003 ◽  
Vol 16 (1) ◽  
pp. 7-13 ◽  
Author(s):  
Sophien Kamoun ◽  
Walid Hamada ◽  
Edgar Huitema
Keyword(s):  
High Throughput ◽  
Hypersensitive Response ◽  
High Throughput Screening ◽  
Tumor Formation ◽  
Functional Screening ◽  
Mechanical Wounding ◽  
Intact Plants ◽  
Oomycete Pathogen ◽  
Inf1 Elicitin ◽  
Functional Analyses

We describe a novel method, agrosuppression, that addresses the need for an assay of the hypersensitive response (HR) in intact plants that is rapid and adapted to high-throughput functional screening of plant and pathogen genes. The agrosuppression assay is based on inoculation of intact plants with a mixture of Agrobacterium tumefaciens strains carrying (i) a binary plasmid with one or more candidate HR-inducing genes and (ii) a tumor-inducing (oncogenic) T-DNA. In the absence of HR induction, tumor formation is initiated, resulting in a typical crown gall phenotype. However, upon induction of the HR, tumor formation by the oncogenic T-DNA is suppressed, resulting in a phenotype that can be readily scored. We tested and optimized agrosuppression in Nicotiana benthamiana using the inf1 elicitin gene from the oomycete pathogen Phytophthora infestans, which specifically induces the HR in Nicotiana spp., and the gene-for-gene pair Avr9/Cf-9 from the fungal pathogen Cladosporium fulvum and Lycopersicon pimpinellifolium (currant tomato), respectively. Agrosuppression protocols that can be rapidly performed using simple mechanical wounding of petioles of intact N. benthamiana plants were developed and appeared particularly adapted to intensive high-throughput screening. This assay promises to greatly facilitate the cloning of novel plant R genes and pathogen Avr genes and to accelerate functional analyses and structure-function studies of these genes.

scholarly journals Identification of Angiogenesis Inhibitors Using a Co-culture Cell Model in a High-Content and High-Throughput Screening Platform

2017 ◽  
Vol 23 (3) ◽  
pp. 217-225 ◽  
Author(s):  
Shuaizhang Li ◽  
Chia-Wen Hsu ◽  
Srilatha Sakamuru ◽  
Chaozhong Zou ◽  
Ruili Huang ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cell Model ◽  
Hypoxia Inducible Factor ◽  
Angiogenesis Inhibitors ◽  
Culture Cell ◽  
Tube Formation ◽  
Tumor Formation ◽  
Human Telomerase Reverse Transcriptase

Angiogenesis is an important hallmark of cancer, contributing to tumor formation and metastasis. In vitro angiogenesis models for analyzing tube formation serve as useful tools to study these processes. However, current in vitro co-culture models using primary cells have limitations in usefulness and consistency. Therefore, in the present study, an in vitro co-culture assay system was optimized in a 1536-well format for high-throughput screening using human telomerase reverse transcriptase (hTERT)–immortalized mesenchymal stem cells and aortic endothelial cells. The National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (NPC) library containing 2816 drugs was evaluated using the in vitro co-culture assay. From the screen, 35 potent inhibitors (IC50 ≤1 µM) were identified, followed by 15 weaker inhibitors (IC50 1–50 µM). Moreover, many known angiogenesis inhibitors were identified, such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study, including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia-inducible factor-1α (HIF-1α) and the nuclear factor-kappa B (NF-κB) pathways. The co-culture model developed by using hTERT-immortalized cell lines described in this report provides a consistent and robust in vitro system for antiangiogenic drug screening.

scholarly journals A high throughput bispecific antibody discovery pipeline

2021 ◽  
Author(s):  
Aude I. Segaliny ◽  
Jayapriya Jayaraman ◽  
Xiaoming Chen ◽  
Jonathan Chong ◽  
Ryan Luxon ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Cytotoxicity ◽  
Functional Screening ◽  
Sequence Composition ◽  
Single Cell Pcr ◽  
Screening Efficiency ◽  
And Function

AbstractBispecific antibodies (BsAbs) represent an emerging class of immunotherapy but inefficiency in the current BsAb discovery paradigm has limited their broad clinical availability. Here we report a high throughput, agnostic, single-cell-based BsAb functional screening pipeline, comprising molecular and cell engineering for efficient generation of BsAb library cells, followed by functional interrogation at the single-cell level to identify and sort positive clones and downstream sequence identification with single-cell PCR and sequencing and functionality characterization. Using a CD19xCD3 bispecific T cell engager (BiTE) as a model system, we demonstrate that our single cell platform possesses a high throughput screening efficiency of up to one and half million variant library cells per run and can isolate rare functional clones at low abundance of 0.008%. Using a complex CD19xCD3 BiTE-expressing cell library with approximately 22,300 unique variants comprising combinatorially varied scFvs, connecting linkers and VL/VH orientations, we have identified 98 unique clones including extremely rare ones (∼ 0.001% abundance). We also discovered BiTEs that exhibit novel properties contradictory to conventional wisdom, including harboring rigid scFv connecting peptide linkers yet with in vitro cytotoxicity comparable to that of clinically approved Blinatumomab. Through sequencing analyses on sorted BiTE clones, we discovered multiple design variable preferences for functionality including the CD19VL-VH– CD3VH-VL and CD19VH-VL–CD3VH-VL arrangements being the most favored orientation. Sequence analysis further interrogated the sequence composition of the CDRH3 domain in scFvs and identified amino acid residues conserved for function. We expect our single cell platform to not only significantly increase the development speed of high quality of new BsAb therapeutics for cancer and other disorders, but also enable identifying generalizable design principles for new BsAbs and other immunotherapeutics based on an in-depth understanding of the inter-relationships between sequence, structure, and function.

scholarly journals Identification of Small Molecules That Inhibit the Interaction of TEM8 with Anthrax Protective Antigen Using a FRET Assay

2013 ◽  
Vol 18 (6) ◽  
pp. 714-725 ◽  
Author(s):  
Lorna M. Cryan ◽  
Kaiane A. Habeshian ◽  
Thomas P. Caldwell ◽  
Meredith T. Morris ◽  
P. Christine Ackroyd ◽  
...  
Keyword(s):  
Blood Vessels ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protective Antigen ◽  
Resonance Energy ◽  
Cysteine Residue ◽  
Tumor Formation ◽  
Screening Assay ◽  
High Throughput Screening Assay

Tumor marker endothelial 8 (TEM8) is a receptor for the protective antigen (PA) component of anthrax toxin. TEM8 is upregulated on endothelial cells lining the blood vessels within tumors, compared with normal blood vessels. A number of studies have demonstrated a pivotal role for TEM8 in developmental and tumor angiogenesis. We have also shown that targeting the anthrax receptors with a mutated form of PA inhibits angiogenesis and tumor formation in vivo. Here we describe the development and testing of a high-throughput fluorescence resonance energy transfer assay to identify molecules that strongly inhibit the interaction of PA and TEM8. The assay we describe is sensitive and robust, with a Z’ value of 0.8. A preliminary screen of 2310 known bioactive library compounds identified ebselen and thimerosal as inhibitors of the TEM8-PA interaction. These molecules each contain a cysteine-reactive transition metal, and complementary studies indicate that their inhibition of interaction is due to modification of a cysteine residue in the TEM8 extracellular domain. This is the first demonstration of a high-throughput screening assay that identifies inhibitors of TEM8, with potential application for antianthrax and antiangiogenic diseases.

scholarly journals Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

2020 ◽  
pp. 1-12
Author(s):  
Sikander Ali ◽  
Syed Shahid Hussain
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Immense Potential

Concerned with the construction and design of novel biocatalysts, the enzyme engineering served to overcome the limitations of native enzymes, in order to create biocatalysts with tailored functions, to facilitate industrial applications. The enzymes, being recognized by screening and discovery workflows and further tailored by engineering platforms, are of immense potential as improved biocatalysts. Functional metagenomics is a powerful tool to identify novel enzymes followed by the construction of metagenome-based enzyme libraries. And the subsequent screening of these enzyme libraries is in turn facilitated by ultra-high-throughput-based, for example FACS or microfluidics, enzyme engineering technologies. Relies on the compartmentalization of reaction components, in order to detect and measure assay signal within the reaction compartments, the enzyme engineering platforms are designed which include cell-as-compartment platforms, droplet-based platforms and micro-chamber-based platforms. The metagenomics approach and high-throughput screening by these three prime enzyme engineer platforms are the focus of this review.

scholarly journals High-throughput screening and validation of antibodies against synaptic proteins to explore opioid signaling dynamics

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Mariana Lemos Duarte ◽  
Nikita A. Trimbake ◽  
Achla Gupta ◽  
Christine Tumanut ◽  
Xiaomin Fan ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Temporal Dynamics ◽  
Single Cells ◽  
Synaptic Proteins ◽  
Functional Screening ◽  
Signaling Dynamics ◽  
Antibody Libraries ◽  
Multiple State ◽  
Opioid Signaling

AbstractAntibodies represent powerful tools to examine signal transduction pathways. Here, we present a strategy integrating multiple state-of-the-art methods to produce, validate, and utilize antibodies. Focusing on understudied synaptic proteins, we generated 137 recombinant antibodies. We used yeast display antibody libraries from the B cells of immunized rabbits, followed by FACS sorting under stringent conditions to identify high affinity antibodies. The antibodies were validated by high-throughput functional screening, and genome editing. Next, we explored the temporal dynamics of signaling in single cells. A subset of antibodies targeting opioid receptors were used to examine the effect of treatment with opiates that have played central roles in the worsening of the ‘opioid epidemic.’ We show that morphine and fentanyl exhibit differential temporal dynamics of receptor phosphorylation. In summary, high-throughput approaches can lead to the identification of antibody-based tools required for an in-depth understanding of the temporal dynamics of opioid signaling.

scholarly journals High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe

2015 ◽  
Vol 21 (1) ◽  
pp. 12-23 ◽  
Author(s):  
Thilo Riedl ◽  
Egon van Boxtel ◽  
Martijn Bosch ◽  
Paul W. H. I. Parren ◽  
Arnout F. Gerritsen
Keyword(s):  
Fluorescence Imaging ◽  
High Throughput ◽  
Tyrosine Kinases ◽  
High Throughput Screening ◽  
Receptor Internalization ◽  
Target Cells ◽  
Functional Screening ◽  
Surface Binding ◽  
Surface Receptors ◽  
Antigen Interaction

Antibody-drug conjugates (ADCs) represent a rapidly growing class of biotherapeutics that deliver drugs specifically to target cells by binding of the antibody component to surface receptors. The majority of ADCs require receptor internalization depending on intrinsic features of the specific ADC-antigen interaction. The development of potent ADCs would greatly benefit from the identification of efficiently internalizing antibodies at early stages of discovery. We developed a highly sensitive and rapid antibody internalization assay using an indirect Cypher5E label. The pH-activated CypHer5E label becomes fluorescent upon internalization into the acidic environment of endocytic organelles, whereas background fluorescence of noninternalized CypHer5E is minimal. The pH-dependency of the CypHer5E signal enables robust discrimination of antibody internalization from surface binding. The favorable signal-over-background ratio allows a homogeneous assay design with high-throughput fluorescence imaging in 384- and 1536-well formats. The biophysical readout of the primary internalization event substantially shortens incubation times compared to killing assays using toxin internalization. The assay was validated with tumor-relevant targets, including receptor tyrosine kinases (EGFR and HER2) and a class II cytokine receptor (TF) expressed by A431, AU565, and SKOV-3 cells and transient expression systems (CHO-S). Our method enables functional screening of large antibody libraries to identify therapeutic antibody candidates with internalization characteristics favorable for the development of ADCs.

scholarly journals A 1536-Well Quantitative High-Throughput Screen to Identify Compounds Targeting Cancer Stem Cells

2012 ◽  
Vol 17 (9) ◽  
pp. 1231-1242 ◽  
Author(s):  
Lesley A. Mathews ◽  
Jonathan M. Keller ◽  
Bonnie L. Goodwin ◽  
Rajarshi Guha ◽  
Paul Shinn ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Growth ◽  
Cancer Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Selection Process ◽  
Tumor Formation ◽  
New Drugs ◽  
Growth Media ◽  
Tumor Initiating Cells

Tumor cell subpopulations called cancer stem cells (CSCs) or tumor-initiating cells (TICs) have self-renewal potential and are thought to drive metastasis and tumor formation. Data suggest that these cells are resistant to current chemotherapy and radiation therapy treatments, leading to cancer recurrence. Therefore, finding new drugs and/or drug combinations that cause death of both the differentiated tumor cells as well as CSC populations is a critical unmet medical need. Here, we describe how cancer-derived CSCs are generated from cancer cell lines using stem cell growth media and nonadherent conditions in quantities that enable high-throughput screening (HTS). A cell growth assay in a 1536-well microplate format was developed with these CSCs and used to screen a focused collection of oncology drugs and clinical candidates to find compounds that are cytotoxic against these highly aggressive cells. A hit selection process that included potency and efficacy measurements during the primary screen allowed us to efficiently identify compounds with potent cytotoxic effects against spheroid-derived CSCs. Overall, this research demonstrates one of the first miniaturized HTS assays using CSCs. The procedures described here should enable further testing of the effect of compounds on CSCs and help determine which pathways need to be targeted to kill them.

scholarly journals High-Throughput Screening for Norepinephrine Transporter Inhibitors Using the FLIPRTetra

2007 ◽  
Vol 12 (3) ◽  
pp. 436-441 ◽  
Author(s):  
Ryan Wagstaff ◽  
Michael Hedrick ◽  
Jun Fan ◽  
Paul D. Crowe ◽  
Daniel DiSepio
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Radioligand Binding ◽  
Norepinephrine Transporter ◽  
Functional Screening ◽  
Monoamine Transporters ◽  
Binding Assays ◽  
Screening Assay ◽  
Fluorescent Substrate

Monoamine transporters regulate the concentration of neurotransmitters in the synapse following neurotransmission and are very important drug targets in the pharmaceutical industry. Because of the labor-intensive nature of functional uptake assays using radioactive substrates, high-throughput screening for monoamine transporter inhibitors has been limited to radioligand binding assays. In this article, the authors describe the development of a 384-well, high-throughput functional screening assay for norepinephrine transporter inhibitors using the FLIPRTetra and a recently identified fluorescent substrate, 4-(4-dimethylaminostyryl)- N-methyl-pyridinium (ASP+). ( Journal of Biomolecular Screening 2007:436-441)

High-throughput screening program for botanical extracts

Planta Medica ◽  
2012 ◽  
Vol 78 (11) ◽  
Author(s):  
L Hingorani ◽  
NP Seeram ◽  
B Ebersole
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Screening Program ◽  
Botanical Extracts
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