scholarly journals A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis

2018 ◽  
Author(s):  
Juliane Ollinger ◽  
Anuradha Kumar ◽  
David M. Roberts ◽  
Mai A. Bailey ◽  
Allen Casey ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
High Throughput ◽  
Whole Cell ◽  
Fluorescent Reporters ◽  
Recombinant Strains ◽  
Phenotypic Screen ◽  
Novel Drugs

AbstractTuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed ≥ 90% growth inhibition. We ranked hits and identified three chemical classes of interest – the phenoxyalkylbenzamidazoles, the benzothiophene 1–1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.

Growth Inhibition of Nasopharyngeal Carcinoma Cells Mediated by p53 Gene-Containing Nanolipid Composites

2020 ◽  
Vol 20 (10) ◽  
pp. 6026-6032
Author(s):  
Yongshan Cheng ◽  
Shanying Wu ◽  
Xinting Tie ◽  
Xiaodong Huang ◽  
Lihua Cui
Keyword(s):  
Cell Cycle ◽  
Flow Cytometry ◽  
Nasopharyngeal Carcinoma ◽  
Growth Inhibition ◽  
Transfection Efficiency ◽  
P53 Gene ◽  
Theoretical Support ◽  
Novel Drugs ◽  
Cellular Apoptosis

To study the growth inhibition and cell cycle changes in nasopharyngeal carcinoma (CNE1) cells after transfection with p53 gene. A mixture of nano-liposomes and plasmid containing p53 was used for transfecting CNE1 cells. Cellular apoptosis was examined after transfection using the CCK-8 reagent method with flow cytometry. The results showed that a ratio of nanoliposome/p-ORF-GFP of 3.5:1 showed the highest transfection efficiency in CNE1 cells. The cells transfected with a mixture of composites in this proportion showed significant apoptosis of up to 50–70%. In addition, we observed that cell cycle changes-measured using flow cytometry-as well as cellular apoptosis were accelerated after administration of composites. The CCK-8 kit was used to determine the viability of nano-liposome-encapsulated p53 transfected cells. In vitro experiments showed that the combination significantly inhibited the growth of CNE1 cells with an inhibition rate of approximately 63.8%. Therefore, the nanocomposites have a significant effect on inhibiting the growth of CNE1 cells. Through the investigation of apoptosis and cell cycle changes in CNE1 cells we found that the nanoliposome-encapsulated p53 gene can inhibit growth in these cells, and might therefore serve as a novel treatment strategy for adjuvant treatment of nasopharyngeal carcinoma and ca also reduce incompatibility issues with radiotherapy and chemotherapy. This method can also provide technical and theoretical support for the development of novel drugs.

scholarly journals Novel inhibitors of Mycobacterium tuberculosis GuaB2 identified by a target based high-throughput phenotypic screen

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Jonathan A. G. Cox ◽  
Grace Mugumbate ◽  
Laura Vela-Glez Del Peral ◽  
Monika Jankute ◽  
Katherine A. Abrahams ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
Phenotypic Screen

scholarly journals A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis

PLoS ONE ◽  
2019 ◽  
Vol 14 (1) ◽  
pp. e0205479 ◽  
Author(s):  
Juliane Ollinger ◽  
Anuradha Kumar ◽  
David M. Roberts ◽  
Mai A. Bailey ◽  
Allen Casey ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput ◽  
Whole Cell

scholarly journals Development of a whole‐cell high‐throughput phenotypic screen to identify inhibitors of mycobacterial amino acid biosynthesis

2019 ◽  
Vol 1 (4) ◽  
pp. 246-254 ◽  
Author(s):  
Christopher Burke ◽  
Katherine A. Abrahams ◽  
Emily J. Richardson ◽  
Nicholas J. Loman ◽  
Carlos Alemparte ◽  
...  
Keyword(s):  
Amino Acid ◽  
High Throughput ◽  
Amino Acid Biosynthesis ◽  
Acid Biosynthesis ◽  
Whole Cell ◽  
Phenotypic Screen

scholarly journals SYNTHESIS AND ANTITUBERCULAR ACTIVITY OF PIPERIDINE AND MORPHOLINE 1, 8 NAPHTHYRIDINE ANALOGUES

2016 ◽  
Vol 8 (12) ◽  
pp. 252 ◽  
Author(s):  
Muwaffag Badawneh ◽  
Jalal Aljamal
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Inhibitory Concentration ◽  
Antitubercular Activity ◽  
Antimycobacterial Activity ◽  
Significant Activity ◽  
Reference Drug ◽  
Novel Drugs ◽  
Elemental Analyses ◽  
Antimycobacterial Agents

<p><strong>Objective: </strong>The search for new, potentially useful antimycobacterial agents. In continuation with our previous screening for the discovery of novel drugs for tuberculosis, a new series of 1,8-naphthyridines derivatives were synthesized and evaluated <em>in vitro </em>for antimycobacterial activity against <em>Mycobacterium tuberculosis </em>H37Rv.</p><p><strong>Methods: </strong>Several 4-morpholinomethyl-1.8-naphthyridine derivatives have been synthesized in excellent yields. The synthesized compounds were characterized by spectroscopic methods as well as elemental analyses. They were screened for their antimycobacterial activity. The growth was monitored radiometrically in 7H12 broth with the BACTEC 460 TB system. The minimum inhibitory concentration (MIC) was determined for compounds that demonstrated ≥ 90% growth inhibition in the primary screening.</p><p><strong>Results: </strong>The obtained data suggested that all compounds showed significant activity against <em>Mycobacterium tuberculosis </em>H37Rv<em> </em>compared to the standard reference drug. Analogues (6-11) having heterocyclic groups in position 7 were the most potent of those we tested.</p><p><strong>Conclusion: </strong>These findings clearly identify the 1,8-naphthyridine analogue (10) with a 6-amino-2-(4'-methoxy benzylamine-4-morpholinomethyl-7-morpholino-substituent as promising anti-tubercular agents possessing significant activity against <em>Mycobacterium tuberculosis </em>H37Rv</p>

scholarly journals In vitro growth inhibition and bactericidal activity of spathulenol against drug-resistant clinical isolates of Mycobacterium tuberculosis

2019 ◽  
Vol 29 (6) ◽  
pp. 798-800 ◽  
Author(s):  
Angel de Jesús Dzul-Beh ◽  
Karlina García-Sosa ◽  
Andrés Humberto Uc-Cachón ◽  
Jorge Bórquez ◽  
Luis A. Loyola ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Growth Inhibition ◽  
Bactericidal Activity ◽  
Clinical Isolates ◽  
In Vitro Growth ◽  
Drug Resistant

scholarly journals Development of a Murine Mycobacterial Growth Inhibition Assay for Evaluating Vaccines against Mycobacterium tuberculosis

2009 ◽  
Vol 16 (7) ◽  
pp. 1025-1032 ◽  
Author(s):  
Marcela Parra ◽  
Amy L. Yang ◽  
JaeHyun Lim ◽  
Kristopher Kolibab ◽  
Steven Derrick ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Immune Responses ◽  
Growth Inhibition ◽  
In Vitro Assays ◽  
Functional Assay ◽  
Inhibition Assay ◽  
Growth Inhibition Assay ◽  
Vitro Assay ◽  
Mycobacterial Growth

ABSTRACT The development and characterization of new tuberculosis (TB) vaccines has been impeded by the lack of reproducible and reliable in vitro assays for measuring vaccine activity. In this study, we developed a murine in vitro mycobacterial growth inhibition assay for evaluating TB vaccines that directly assesses the capacity of immune splenocytes to control the growth of Mycobacterium tuberculosis within infected macrophages. Using this in vitro assay, protective immune responses induced by immunization with five different types of TB vaccine preparations (Mycobacterium bovis BCG, an attenuated M. tuberculosis mutant strain, a DNA vaccine, a modified vaccinia virus strain Ankara [MVA] construct expressing four TB antigens, and a TB fusion protein formulated in adjuvant) can be detected. Importantly, the levels of vaccine-induced mycobacterial growth-inhibitory responses seen in vitro after 1 week of coculture correlated with the protective immune responses detected in vivo at 28 days postchallenge in a mouse model of pulmonary tuberculosis. In addition, similar patterns of cytokine expression were evoked at day 7 of the in vitro culture by immune splenocytes taken from animals immunized with the different TB vaccines. Among the consistently upregulated cytokines detected in the immune cocultures are gamma interferon, growth differentiation factor 15, interleukin-21 (IL-21), IL-27, and tumor necrosis factor alpha. Overall, we have developed an in vitro functional assay that may be useful for screening and comparing new TB vaccine preparations, investigating vaccine-induced protective mechanisms, and assessing manufacturing issues, including product potency and stability.

An efficient fluorimetric method to measure the viability of intraerythrocytic Plasmodium falciparum

2008 ◽  
Vol 389 (12) ◽  
Author(s):  
Astrid Evers ◽  
Saskia Heppner ◽  
Matthias Leippe ◽  
Christoph Gelhaus
Keyword(s):  
Plasmodium Falciparum ◽  
Cost Effectiveness ◽  
Growth Inhibition ◽  
High Throughput ◽  
Test System ◽  
Antiplasmodial Activity ◽  
Routine Analysis ◽  
Fluorimetric Method

AbstractA range of various assays to measure chemosusceptibility ofPlasmodium falciparumhave been described in the literature. As the screening of a plethora of compounds for antiplasmodial activity is urgently needed and becomes a constantly increasing routine analysis, a test system has to fulfill the following requirements: sensitivity, reliability, simplicity of performance, high-throughput compatibility, and cost-effectiveness. Here, we describe an assay that fulfills all criteria and in which the fluorescent SYTOX®Green dye is introduced to determine growth inhibition ofPlasmodiainin vitrocultures.

scholarly journals Roles of SigB and SigF in the Mycobacterium tuberculosis Sigma Factor Network

2007 ◽  
Vol 190 (2) ◽  
pp. 699-707 ◽  
Author(s):  
Jong-Hee Lee ◽  
Petros C. Karakousis ◽  
William R. Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Sigma Factor ◽  
Ribosomal Proteins ◽  
Consensus Sequence ◽  
In Vitro Transcription ◽  
Transcription Analysis ◽  
Recombinant Strains ◽  
Regulatory Cascade

ABSTRACTTo characterize the roles of SigB and SigF in sigma factor regulation inMycobacterium tuberculosis, we used chemically inducible recombinant strains to conditionally overexpresssigBandsigF.Using whole genomic microarray analysis and quantitative reverse transcription-PCR, we investigated the resulting global transcriptional changes aftersigBinduction, and we specifically tested the relative expression of other sigma factor genes after knock-in expression ofsigBandsigF. Overexpression ofsigBresulted in significant upregulation of genes encoding several early culture filtrate antigens (ESAT-6-like proteins), ribosomal proteins, PE-PGRS proteins, the keto-acyl synthase, KasA, and the regulatory proteins WhiB2 and IdeR. Of note, the induction ofsigBdid not alter the expression of other sigma factor genes, indicating that SigB is likely to serve as an end regulator for at least one branch of theM. tuberculosissigma factor regulatory cascade. Analysis of the 5′-untranslated region (UTR) of SigB-dependent transcripts revealed a putative consensus sequence of NGTGG-N14-18-NNGNNG. This sequence appeared upstream of bothsigB(Rv2710) and the gene following it,ideR(Rv2711), and in vitro transcription analysis with recombinant SigB-reconstituted RNA polymerase confirmed SigB-dependent transcription from each of these promoters. Knock-in expression ofsigFrevealed that only thesigCgene was significantly upregulated 6 and 12 h aftersigFinduction. The previously identified SigF promoter consensus sequence AGTTTG-N15-GGGTTT was identified in the 5′ UTR of thesigCgene, and SigF-dependent in vitro transcription of the promoter upstream ofsigCwas confirmed by using recombinant SigF-reconstituted RNA polymerase. These two knock-in recombinant strains were tested in a macrophage model of infection which showed that overexpression ofsigBandsigFresulted in reduced rates ofM. tuberculosisintracellular growth. These results define the SigB promoter consensus recognition sequence and members of the SigB regulon. Moreover, the data suggest that, in addition to serving as an end regulator in a sigma factor cascade, SigB may auto-amplify its own expression under certain conditions.

A High-Throughput Crystallization Device to Study Biomineralization in Vitro

2005 ◽  
Vol 873 ◽  
Author(s):  
Alexander Becker ◽  
Matthias Epple
Keyword(s):  
Crystal Growth ◽  
Calcium Carbonate ◽  
Growth Inhibition ◽  
Aspartic Acid ◽  
High Throughput ◽  
Double Diffusion ◽  
Constant Composition ◽  
Crystal Growth Inhibition

AbstractA new crystallization device, based on a constant-composition double-diffusion setup, was constructed to study biomineralization in vitro. The device was tested with poly(aspartic acid) as a model additive in the precipitation of calcium carbonate, showing a complete crystal growth inhibition.

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