Multiplexing fluorogenic esterase-based viability assay with luciferase assays

Ohgane, Kenji; Yoshioka, Hirosuke; Hashimoto, Yuichi

doi:10.1016/j.mex.2019.09.008

Cited by 5 publications

(2 citation statements)

References 18 publications

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“…While it may be impossible to directly interpret 16S-RNAseq results for accurate viability quantifications, the thoughts here are promising in terms of qualitative assessment: determining which microbes are generally more active in a (or similar) environmental source. Other potential improvements might include the combination of other microbiological experiments, such as metabolic capacity measurements that directly capture the metabolism activities [55][56][57], biochemical colorimetry that based in the membrane integrity of viable cells [58], or to explore alternative activity markers from mRNA transcribed from protein-coding genes. A small number of protein coding genes have also been proposed for microbial community viability profiling in previous work, such rpoB, gyrB, and cpn60 [59][60][61].…”

Section: Discussionmentioning

confidence: 99%

RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities

et al. 2023

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Background Characterization of microbial activity is essential to the understanding of the basic biology of microbial communities, as the function of a microbiome is defined by its biochemically active (“viable”) community members. Current sequence-based technologies can rarely differentiate microbial activity, due to their inability to distinguish live and dead sourced DNA. As a result, our understanding of microbial community structures and the potential mechanisms of transmission between humans and our surrounding environments remains incomplete. As a potential solution, 16S rRNA transcript-based amplicon sequencing (16S-RNA-seq) has been proposed as a reliable methodology to characterize the active components of a microbiome, but its efficacy has not been evaluated systematically. Here, we present our work to benchmark RNA-based amplicon sequencing for activity assessment in synthetic and environmentally sourced microbial communities. Results In synthetic mixtures of living and heat-killed Escherichia coli and Streptococcus sanguinis, 16S-RNA-seq successfully reconstructed the active compositions of the communities. However, in the realistic environmental samples, no significant compositional differences were observed in RNA (“actively transcribed — active”) vs. DNA (“whole” communities) spiked with E. coli controls, suggesting that this methodology is not appropriate for activity assessment in complex communities. The results were slightly different when validated in environmental samples of similar origins (i.e., from Boston subway systems), where samples were differentiated both by environment type as well as by library type, though compositional dissimilarities between DNA and RNA samples remained low (Bray–Curtis distance median: 0.34–0.49). To improve the interpretation of 16S-RNA-seq results, we compared our results with previous studies and found that 16S-RNA-seq suggests taxon-wise viability trends (i.e., specific taxa are universally more or less likely to be viable compared to others) in samples of similar origins. Conclusions This study provides a comprehensive evaluation of 16S-RNA-seq for viability assessment in synthetic and complex microbial communities. The results found that while 16S-RNA-seq was able to semi-quantify microbial viability in relatively simple communities, it only suggests a taxon-dependent “relative” viability in realistic communities.

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Section: Discussionmentioning

confidence: 99%

RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities

et al. 2023

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“…Luciferase Analysis [20] Luciferase activity was assayed using a dual luciferase reporter kit (Promega Luciferase Assay System E1501). A reporter gene plasmid containing the 3-UTR of the target protein was constructed according to the manufacturer's protocol and transferred into 293T cells using Lipofectamine 2000 (Invitrogen) to test whether miR-181b could bind to the predicted target sequence.…”

Section: Western Blot Analysismentioning

confidence: 99%

Cx43-Delivered miR-181b Negatively Regulates Sirt1/FOXO3a Signalling Pathway-Mediated Apoptosis on Intestinal Injury in Sepsis

Zou

Huang

et al. 2023

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Introduction: Gap junctions can transmit signals between cells, including miRNAs, leading to the amplification of adjacent cell damage. No previous study has addressed gap junctions and miRNAs in sepsis because the internal mechanism of sepsis-induced intestinal injury is complex. Therefore, we studied the relationship between connexin43 (Cx43) and miR-181b and provided a research direction for further study of sepsis. Methods: A mouse caecal ligation and puncture method was used to construct a mouse sepsis model. Firstly, damage to intestinal tissues at different time points was analysed. The levels of Cx43, miR-181b, Sirt1, and FOXO3a in intestinal tissues and the transcription and translation of the apoptosis-related genes Bim and puma, which are downstream of FOXO3a were analysed. Secondly, the effect of Cx43 levels on miR-181b and Sirt1/FOXO3a signalling pathway activity was explored by using the Cx43 inhibitor heptanol. Finally, luciferase assays were used to determine miR-181b binding to the predicted target sequence. Results: The results show that during sepsis, intestinal injury becomes increasingly worse with time, and the expression of Cx43 and miR-181b increase. In addition, we found that heptanol could significantly reduce intestinal injury. This finding indicates that inhibiting Cx43 regulates the transfer of miR-181b between adjacent cells, thereby reducing the activity of the Sirt1/FOXO3a signalling pathway and reducing the degree of intestinal injury during sepsis. Conclusions: In sepsis, the enhancement of Cx43 gap junctions leads to an increase in miR-181b intercellular transfer, affects the downstream SIRT1/FOXO3a signalling pathway and causes cell and tissue damage.

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Luciferase-based HMG-CoA reductase degradation assay for activity and selectivity profiling of oxy(lano)sterols

Sagimori

Yoshioka

Hashimoto

et al. 2020

Bioorganic & Medicinal Chemistry

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Multiplexing fluorogenic esterase-based viability assay with luciferase assays

Abstract: Graphical abstract

Cited by 5 publications

References 18 publications

RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities

RNA-based amplicon sequencing is ineffective in measuring metabolic activity in environmental microbial communities

Cx43-Delivered miR-181b Negatively Regulates Sirt1/FOXO3a Signalling Pathway-Mediated Apoptosis on Intestinal Injury in Sepsis

Luciferase-based HMG-CoA reductase degradation assay for activity and selectivity profiling of oxy(lano)sterols

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