scholarly journals Porcine OCT4 reporter system as a tool for monitoring pluripotency states

2021 ◽  
Vol 36 (4) ◽  
pp. 175-182
Author(s):  
Seung-Hun Kim ◽  
Chang-Kyu Lee
Keyword(s):  
Reporter System

Identification of N-Substituted Triazolo-azetidines as Novel Antibacterials using pDualrep2 HTS Platform

2019 ◽  
Vol 22 (5) ◽  
pp. 346-354
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Large Scale ◽  
Underlying Mechanism ◽  
Luciferase Assay ◽  
Reporter System ◽  
E Coli ◽  
Starting Point ◽  
High Concentrations ◽  
Mic Value

Aim and Objective: Antibiotic resistance is a serious constraint to the development of new effective antibacterials. Therefore, the discovery of the new antibacterials remains one of the main challenges in modern medicinal chemistry. This study was undertaken to identify novel molecules with antibacterial activity. Materials and Methods: Using our unique double-reporter system, in-house large-scale HTS campaign was conducted for the identification of antibacterial potency of small-molecule compounds. The construction allows us to visually assess the underlying mechanism of action. After the initial HTS and rescreen procedure, luciferase assay, C14-test, determination of MIC value and PrestoBlue test were carried out. Results: HTS rounds and rescreen campaign have revealed the antibacterial activity of a series of Nsubstituted triazolo-azetidines and their isosteric derivatives that has not been reported previously. Primary hit-molecule demonstrated a MIC value of 12.5 µg/mL against E. coli Δ tolC with signs of translation blockage and no SOS-response. Translation inhibition (26%, luciferase assay) was achieved at high concentrations up to 160 µg/mL, while no activity was found using C14-test. The compound did not demonstrate cytotoxicity in the PrestoBlue assay against a panel of eukaryotic cells. Within a series of direct structural analogues bearing the same or bioisosteric scaffold, compound 2 was found to have an improved antibacterial potency (MIC=6.25 µg/mL) close to Erythromycin (MIC=2.5-5 µg/mL) against the same strain. In contrast to the parent hit, this compound was more active and selective, and provided a robust IP position. Conclusion: N-substituted triazolo-azetidine scaffold may be used as a versatile starting point for the development of novel active and selective antibacterial compounds.

scholarly journals Engineering subtilisin proteases that specifically degrade active RAS

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yingwei Chen ◽  
Eric A. Toth ◽  
Biao Ruan ◽  
Eun Jung Choi ◽  
Richard Simmerman ◽  
...  
Keyword(s):  
Active Form ◽  
High Specificity ◽  
Kinetic Properties ◽  
Target Sequence ◽  
Reporter System ◽  
Human Cell Culture ◽  
Conserved Sequence ◽  
Cofactor Binding ◽  
Selective Proteolysis

AbstractWe describe the design, kinetic properties, and structures of engineered subtilisin proteases that degrade the active form of RAS by cleaving a conserved sequence in switch 2. RAS is a signaling protein that, when mutated, drives a third of human cancers. To generate high specificity for the RAS target sequence, the active site was modified to be dependent on a cofactor (imidazole or nitrite) and protease sub-sites were engineered to create a linkage between substrate and cofactor binding. Selective proteolysis of active RAS arises from a 2-step process wherein sub-site interactions promote productive binding of the cofactor, enabling cleavage. Proteases engineered in this way specifically cleave active RAS in vitro, deplete the level of RAS in a bacterial reporter system, and also degrade RAS in human cell culture. Although these proteases target active RAS, the underlying design principles are fundamental and will be adaptable to many target proteins.

scholarly journals Structure-guided engineering of adenine base editor with minimized RNA off-targeting activity

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jianan Li ◽  
Wenxia Yu ◽  
Shisheng Huang ◽  
Susu Wu ◽  
Liping Li ◽  
...  
Keyword(s):  
Rna Editing ◽  
Potential Interaction ◽  
Reporter System ◽  
Independent Manner ◽  
Robust Detection ◽  
Guide Rna ◽  
Highly Efficient ◽  
Adenine Base

AbstractBoth adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.

scholarly journals Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol

2021 ◽  
Vol 9 (2) ◽  
pp. 295
Author(s):  
Yvann Bourigault ◽  
Andrea Chane ◽  
Corinne Barbey ◽  
Sylwia Jafra ◽  
Robert Czajkowski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Infected Plant ◽  
Soft Rot ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Cell Physiology ◽  
In Planta ◽  
Reporter System ◽  
Cell Wall Lysis

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

scholarly journals The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Shintaro Sasuga ◽  
Toshiya Osada
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Biological Activities ◽  
Inducible Promoter ◽  
Upstream Region ◽  
Reporter Plasmid ◽  
Reporter System ◽  
High Background ◽  
Downstream Region ◽  
Green Fluorescent

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, themam2upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.

scholarly journals Novel activities by ebolavirus and marburgvirus interferon antagonists revealed using a standardized in vitro reporter system

Virology ◽  
2017 ◽  
Vol 501 ◽  
pp. 147-165 ◽  
Author(s):  
Jonathan C. Guito ◽  
César G. Albariño ◽  
Ayan K. Chakrabarti ◽  
Jonathan S. Towner
Keyword(s):  
Reporter System ◽  
Interferon Antagonists
Sign in / Sign up

Export Citation Format

Share Document