Bioluminescence burst caused by a process in carbohydrate metabolism in a luciferase reporter strain of Escherichia coli

2020 ◽  
Vol 532 (2) ◽  
pp. 185-189
Author(s):  
Kazuki Aiba ◽  
Yuto Kobayashi ◽  
Setsuyuki Aoki
Keyword(s):  
Escherichia Coli ◽  
Carbohydrate Metabolism ◽  
Luciferase Reporter ◽  
Reporter Strain

Posttranslationally caused bioluminescence burst of the Escherichia coli luciferase reporter strain

2015 ◽  
Vol 198 (1) ◽  
pp. 35-41 ◽  
Author(s):  
Yamato Ideguchi ◽  
Yuta Oshikoshi ◽  
Masashi Ryo ◽  
Shogo Motoki ◽  
Takashi Kuwano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Luciferase Reporter ◽  
Reporter Strain

scholarly journals Monitoring disease activity noninvasively in the mdx model of Duchenne muscular dystrophy

2018 ◽  
Vol 115 (30) ◽  
pp. 7741-7746 ◽  
Author(s):  
Antonio Filareto ◽  
Katie Maguire-Nguyen ◽  
Qiang Gan ◽  
Garazi Aldanondo ◽  
Léo Machado ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Disease Progression ◽  
Premature Death ◽  
Response To Therapy ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Muscle Degeneration ◽  
Reporter Strain ◽  
Novel Treatments

Duchenne muscular dystrophy (DMD) is a rare, muscle degenerative disease resulting from the absence of the dystrophin protein. DMD is characterized by progressive loss of muscle fibers, muscle weakness, and eventually loss of ambulation and premature death. Currently, there is no cure for DMD and improved methods of disease monitoring are crucial for the development of novel treatments. In this study, we describe a new method of assessing disease progression noninvasively in the mdx model of DMD. The reporter mice, which we term the dystrophic Degeneration Reporter strains, contain an inducible CRE-responsive luciferase reporter active in mature myofibers. In these mice, muscle degeneration is reflected in changes in the level of luciferase expression, which can be monitored using noninvasive, bioluminescence imaging. We monitored the natural history and disease progression in these dystrophic report mice and found that decreases in luciferase signals directly correlated with muscle degeneration. We further demonstrated that this reporter strain, as well as a previously reported Regeneration Reporter strain, successfully reveals the effectiveness of a gene therapy treatment following systemic administration of a recombinant adeno-associated virus-6 (rAAV-6) encoding a microdystrophin construct. Our data demonstrate the value of these noninvasive imaging modalities for monitoring disease progression and response to therapy in mouse models of muscular dystrophy.

scholarly journals Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture

2006 ◽  
Vol 50 (8) ◽  
pp. 2789-2796 ◽  
Author(s):  
Lucas D. Tilley ◽  
Orion S. Hine ◽  
Jill A. Kellogg ◽  
Jed N. Hassinger ◽  
Dwight D. Weller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Luciferase Reporter ◽  
Bacterial Cells ◽  
Luciferase Reporter Gene ◽  
E Coli ◽  
Specific Effects ◽  
Covalently Bonded ◽  
Serovar Typhimurium ◽  
Free Translation

ABSTRACT The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B isβ -alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targeted to luc inhibited luciferase activity 23 to 80% in growing cultures of Escherichia coli. In cell-free translation reactions, Luc RTRTRFLRRTXB-PMO inhibited luciferase synthesis significantly more than the other Luc peptide-PMOs or the Luc PMO not coupled to peptide. AcpP peptide-PMOs targeted to acpP inhibited growth of E. coli or Salmonella enterica serovar Typhimurium to various extents, depending on the strain. The concentrations of AcpP RFFRFFRFFXB-PMO, AcpP RTRTRFLRRTXB-PMO, AcpP KFFKFFKFFKXB-PMO, and ampicillin that reduced CFU/ml by 50% after 8 h of growth (50% inhibitory concentration [IC50]) were 3.6, 10.8, 9.5, and 7.5μ M, respectively, in E. coli W3110. Sequence-specific effects of AcpP peptide-PMOs were shown by rescuing growth of a merodiploid strain that expressed acpP with silent mutations in the region targeted by AcpP peptide-PMO. In Caco-2 cultures infected with enteropathogenic E. coli (EPEC), 10 μM AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO essentially cleared the infection. The IC50 of either AcpP RTRTRFLRRTXB-PMO or AcpP RFFRFFRFFXB-PMO in EPEC-infected Caco-2 culture was 3 μM. In summary, RFFRFFRFFXB, RTRTRFLRRTXB, or KFFKFFKFFXB, when covalently bonded to PMO, significantly increased inhibition of expression of targeted genes compared to PMOs without attached peptide.

scholarly journals Enhanced Expression of miR-34a Enhances Escherichia coli Lipopolysaccharide-Mediated Endometritis by Targeting LGR4 to Activate the NF-κB Pathway

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Xiaofei Ma ◽  
Baoyi Yin ◽  
Shuai Guo ◽  
Talha Umar ◽  
Junfeng Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Luciferase Reporter ◽  
Blue Staining ◽  
Histopathological Analysis ◽  
Dependent Manner ◽  
Luciferase Reporter Gene ◽  
Gram Staining

Background. Persistent endometritis caused by bacterial infections has lethal effects on the reproductive performance of dairy cattle, which compromises animal welfare and delays or prevents pregnancy. The microRNA (miRNA) miR-34 family plays a pivotal role in the inflammatory process; however, the precise mechanism of miR-34a in endometritis has not been thoroughly elucidated to date. Methods. In this study, the endometrium of cows diagnosed with endometritis was harvested for bacterial culture and Gram staining to evaluate bacterial contamination of the uterus. Based on this, a bovine endometrial epithelial cell (BEND) inflammation model and a mouse model stimulated with lipopolysaccharide (LPS) in vitro and in vivo were constructed. Cell viability was assessed by CCK-8, trypan blue staining, and flow cytometry. H&E was applied to histopathological analysis. Immunohistochemical, immunofluorescence, qRT-PCR, and western blot assays were performed to measure the mRNA and protein expression of relevant genes. Online databases, plasmid construction, and dual-luciferase reporter gene assays were used to predict and validate the interaction between miR-34a and its target gene LGR4. Finally, mice were injected vaginally with a local antagomir to validate the role of miR-34a in murine uterine inflammation. Results. In this study, we observed that Gram-negative bacteria, represented by Escherichia coli, are the predominant pathogenic agents responsible for the recurrent occurrence of endometritis in dairy cows. Further, miR-34a was found to repress the expression of LGR4 by targeting the 3 ′ untranslated region (3 ′ UTR) of LGR4. miR-34a was upregulated in bovine uterine tissues and bovine endometrial epithelial cells stimulated with LPS. miR-34a induced the release of the proinflammatory cytokines IL-1β, IL-6, and TNF-α by activating the phosphorylation of NF-κB p65. Furthermore, IL-1β upregulated miR-34a transcription and downregulated LGR4 expression in an IL-1β-dependent manner. Conclusions. Taken together, our study confirmed that miR-34a is regulated by IL-1β and suppresses the level of the LGR4 3 ′ UTR, which in turn exacerbates the inflammatory response. Thus, the knockdown of miR-34a might be a new direction for the treatment of endometritis.

scholarly journals Oligosaccharides increase the genotoxic effect of colibactin produced by pks+ Escherichia coli strains

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Manon Oliero ◽  
Annie Calvé ◽  
Gabriela Fragoso ◽  
Thibault Cuisiniere ◽  
Roy Hajjar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Ferrous Sulfate ◽  
Chromosomal Abnormalities ◽  
Double Strand Breaks ◽  
Luciferase Reporter ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
E Coli ◽  
The Impact

Abstract Background Colibactin is a genotoxin that induces DNA double-strand breaks that may lead to carcinogenesis and is produced by Escherichia coli strains harboring the pks island. Human and animal studies have shown that colibactin-producing gut bacteria promote carcinogenesis and enhance the progression of colorectal cancer through cellular senescence and chromosomal abnormalities. In this study, we investigated the impact of prebiotics on the genotoxicity of colibactin-producing E. coli strains Nissle 1917 and NC101. Methods Bacteria were grown in medium supplemented with 20, 30 and 40 mg/mL of prebiotics inulin or galacto-oligosaccharide, and with or without 5 μM, 25 μM and 125 μM of ferrous sulfate. Colibactin expression was assessed by luciferase reporter assay for the clbA gene, essential for colibactin production, in E. coli Nissle 1917 and by RT-PCR in E. coli NC101. The human epithelial colorectal adenocarcinoma cell line, Caco-2, was used to assess colibactin-induced megalocytosis by methylene blue binding assay and genotoxicity by γ-H2AX immunofluorescence analysis. Results Inulin and galacto-oligosaccharide enhanced the expression of clbA in pks+ E. coli. However, the addition of 125 μM of ferrous sulfate inhibited the expression of clbA triggered by oligosaccharides. In the presence of either oligosaccharide, E. coli NC101 increased dysplasia and DNA double-strand breaks in Caco-2 cells compared to untreated cells. Conclusion Our results suggest that, in vitro, prebiotic oligosaccharides exacerbate DNA damage induced by colibactin-producing bacteria. Further studies are necessary to establish whether oligosaccharide supplementation may lead to increased colorectal tumorigenesis in animal models colonized with pks+ E. coli.

scholarly journals Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

AMB Express ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Josué Daniel Mora-Garduño ◽  
Jessica Tamayo-Nuñez ◽  
Felipe Padilla-Vaca ◽  
Fátima Berenice Ramírez-Montiel ◽  
Ángeles Rangel-Serrano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Water Samples ◽  
Membrane Integrity ◽  
Analytical Techniques ◽  
Bacterial Cells ◽  
Reporter Strain ◽  
Dna Damaging Agents ◽  
Reporter Strains ◽  
High Concentrations

AbstractThe presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

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