scholarly journals Sorting out antibiotics' mechanisms of action: a double fluorescent protein reporter for high throughput screening of ribosome and DNA biosynthesis inhibitors

2016 ◽  
pp. AAC.02117-16 ◽  
Author(s):  
Ilya A. Osterman ◽  
Ekaterina S. Komarova ◽  
Dmitry I. Shiryaev ◽  
Ilya A. Korniltsev ◽  
Irina M. Khven ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Cost Effective ◽  
High Signal ◽  
Red Fluorescent Protein ◽  
Reporter System ◽  
Ribosome Stalling

In order to accelerate drug discovery, a simple, reliable and cost-effective system for high-throughput identification of a potential antibiotic mechanism of action is required. To facilitate such screening of new antibiotics, we created a double reporter system for not only antimicrobial activity detection, but also for simultaneous sorting of potential antimicrobials into those that cause ribosome stalling, and others that induce SOS response due to DNA damage. In this reporter system the red fluorescent protein generfpwas placed under the control of the SOS-induciblesulApromoter. The far-red fluorescent protein genekatushka2Swas inserted downstream the tryptophan attenuator where two tryptophan codons were replaced by alanine codons, with simultaneous replacement of the complementary part of the attenuator, to preserve the ability to form secondary structures that influence transcription termination. This genetically modified attenuator makes possible Katushka2S expression only upon exposure to any ribosome stalling compounds. The application of red and far-red fluorescent proteins provides a high signal-to-background ratio without any need in enzymatic substrates for detection of the reporter activity. This reporter was shown to be efficient in high-throughput screening of both synthetic and natural chemicals.

scholarly journals A Reporter System for Reverse Transfection Cell Arrays

2003 ◽  
Vol 8 (6) ◽  
pp. 620-623 ◽  
Author(s):  
Brian L. Webb ◽  
Begoña Díaz ◽  
G. Steven Martin ◽  
Fang Lai
Keyword(s):  
Map Kinase ◽  
High Throughput ◽  
Gene Function ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Signaling Proteins ◽  
Kinase Signaling ◽  
Reporter System ◽  
Map Kinase Signaling ◽  
Reverse Transfection

The incredible speed of gene cloning and sequencing brought about by the genomic revolution has begun to outpace conven tional gene discovery approaches in the pharmaceutical industry. High-throughput approaches for studying gene function in vivo are greatly needed. One potential answer to this challenge is reverse transfection, a high-throughput gene expression method for examining the function of hundreds to thousands of genes in parallel. One limitation of reverse transfection tech nology is the need for posttransfection processing of the arrays to analyze the activity of the expressed proteins. The authors have investigated the use of a reporter construct cotransfected with other genes of interest to monitor and screen gene function on reverse transfection microarrays. They developed a serum response element (SRE) reporter linked to the green fluorescent protein (GFP) that is cotransfected with target genes on reverse transfection arrays for monitoring mitogen-activated protein (MAP) kinase signaling by multiple targets in parallel. The authors show that this reporter system is able to detect inhibition of upstream MAP kinase signaling proteins by the MEK inhibitor U0126. The ability to monitor the activity of multiple signaling proteins in a multiwell format suggests the utility of reverse transfection reporter arrays for high-throughput screening applications.

scholarly journals Reassembling green fluorescent protein for in vitro evaluation of trans-translation

2020 ◽  
Vol 48 (4) ◽  
pp. e22-e22
Author(s):  
Charlotte Guyomar ◽  
Marion Thépaut ◽  
Sylvie Nonin-Lecomte ◽  
Agnès Méreau ◽  
Renan Goude ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
High Throughput ◽  
Messenger Rna ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Chemical Compounds ◽  
Reporter System ◽  
New Antibiotics ◽  
Green Fluorescent

Abstract In order to discover new antibiotics with improved activity and selectivity, we created a reliable in vitro reporter system to detect trans-translation activity, the main mechanism for recycling ribosomes stalled on problematic messenger RNA (mRNA) in bacteria. This system is based on an engineered tmRNA variant that reassembles the green fluorescent protein (GFP) when trans-translation is active. Our system is adapted for high-throughput screening of chemical compounds by fluorescence.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

scholarly journals Novel Regeneration Approach for Creating Reusable FO-SPR Probes with NTA Surface Chemistry

Nanomaterials ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 186
Author(s):  
Jia-Huan Qu ◽  
Karen Leirs ◽  
Remei Escudero ◽  
Žiga Strmšek ◽  
Roman Jerala ◽  
...  
Keyword(s):  
Surface Chemistry ◽  
Fluorescent Protein ◽  
Cost Effective ◽  
Nitrilotriacetic Acid ◽  
Antibody Fragments ◽  
Rapid Screening ◽  
Red Fluorescent Protein ◽  
Effective Manner ◽  
Surface Regeneration ◽  
Sensitivity Specificity

To date, surface plasmon resonance (SPR) biosensors have been exploited in numerous different contexts while continuously pushing boundaries in terms of improved sensitivity, specificity, portability and reusability. The latter has attracted attention as a viable alternative to disposable biosensors, also offering prospects for rapid screening of biomolecules or biomolecular interactions. In this context here, we developed an approach to successfully regenerate a fiber-optic (FO)-SPR surface when utilizing cobalt (II)-nitrilotriacetic acid (NTA) surface chemistry. To achieve this, we tested multiple regeneration conditions that can disrupt the NTA chelate on a surface fully saturated with His6-tagged antibody fragments (scFv-33H1F7) over ten regeneration cycles. The best surface regeneration was obtained when combining 100 mM EDTA, 500 mM imidazole and 0.5% SDS at pH 8.0 for 1 min with shaking at 150 rpm followed by washing with 0.5 M NaOH for 3 min. The true versatility of the established approach was proven by regenerating the NTA surface for ten cycles with three other model system bioreceptors, different in their size and structure: His6-tagged SARS-CoV-2 spike fragment (receptor binding domain, RBD), a red fluorescent protein (RFP) and protein origami carrying 4 RFPs (Tet12SN-RRRR). Enabling the removal of His6-tagged bioreceptors from NTA surfaces in a fast and cost-effective manner can have broad applications, spanning from the development of biosensors and various biopharmaceutical analyses to the synthesis of novel biomaterials.

scholarly journals Non-invasive and high-throughput interrogation of exon-specific isoform expression

2021 ◽  
Author(s):  
Dong-Jiunn Jeffery Truong ◽  
Teeradon Phlairaharn ◽  
Bianca Eßwein ◽  
Christoph Gruber ◽  
Deniz Tümen ◽  
...  
Keyword(s):  
Stem Cells ◽  
High Throughput ◽  
High Throughput Screening ◽  
Embryonic Stem ◽  
High Sensitivity ◽  
Therapeutic Interventions ◽  
Dominant Factor ◽  
Reporter System ◽  
Isoform Expression ◽  
Induced Pluripotent

AbstractExpression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.

scholarly journals Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

2009 ◽  
Vol 53 (11) ◽  
pp. 4825-4834 ◽  
Author(s):  
Kao-Lu Pan ◽  
Jin-Ching Lee ◽  
Hsing-Wen Sung ◽  
Teng-Yuang Chang ◽  
John T.-A. Hsu
Keyword(s):  
Cell Culture ◽  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Infection ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter System ◽  
Viral Protease ◽  
Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

scholarly journals A Novel Reporter System for Molecular Imaging and High-Throughput Screening of Anticancer Drugs

ChemBioChem ◽  
2013 ◽  
Vol 14 (12) ◽  
pp. 1494-1503 ◽  
Author(s):  
Jingping Xie ◽  
Chunxia Wang ◽  
John Virostko ◽  
H. Charles Manning ◽  
Wellington Pham ◽  
...  
Keyword(s):  
Molecular Imaging ◽  
Anticancer Drugs ◽  
High Throughput ◽  
High Throughput Screening ◽  
Reporter System

scholarly journals Beeporter: a high-throughput tool for non-invasive analysis of honey bee virus infection

2021 ◽  
Author(s):  
Jay D. Evans ◽  
Olubukola Banmeke ◽  
Evan C. Palmer-Young ◽  
Yanping Chen ◽  
Eugene V. Ryabov
Keyword(s):  
Virus Infection ◽  
Honey Bee ◽  
High Throughput ◽  
Honey Bees ◽  
High Throughput Screening ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Uv Light ◽  
Light Sources ◽  
Deformed Wing Virus

ABSTRACTHoney bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Development of antiviral therapeutics and virus-resistant honey bee lines to control DWV in honey bees is slowed by the lack of a cost-effective high-throughput screening of DWV infection. Currently, analysis of virus infection and screening for antiviral treatments in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we utilize a cDNA clone of DWV tagged with green fluorescent protein (GFP) to develop the Beeporter assay, a method for detection and quantification of DWV infection in live honey bees. The assay involves infection of honey bee pupae by injecting a standardized DWV-GFP inoculum, followed by incubation for up to 44 hours. GFP fluorescence is recorded at intervals via commonly available long-wave UV light sources and a smartphone camera or a standard ultraviolet transilluminator gel imaging system. Nonlethal DWV monitoring allows high-throughput screening of antiviral candidates and a direct breeding tool for identifying honey bee parents with increased antivirus resistance. For even more rapid drug screening, we also describe a method for screening bees using 96-well trays and a spectrophotometer.

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