scholarly journals Exposure of HepaRG Cells to Sodium Saccharin Underpins the Importance of Including Non-Hepatotoxic Compounds When Investigating Toxicological Modes of Action Using Metabolomics

Metabolites ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 265
Author(s):  
Matthias Cuykx ◽  
Charlie Beirnaert ◽  
Robim Marcelino Rodrigues ◽  
Kris Laukens ◽  
Tamara Vanhaecke ◽  
...  
Keyword(s):  
Cell Death ◽  
Experimental Design ◽  
Mode Of Action ◽  
Liver Toxicity ◽  
Data Interpretation ◽  
Reference Compound ◽  
Metabolic Alterations ◽  
Modes Of Action ◽  
Heparg Cells ◽  
Sodium Saccharin

Metabolites represent the most downstream information of the cellular organisation. Hence, metabolomics experiments are extremely valuable to unravel the endogenous pathways involved in a toxicological mode of action. However, every external stimulus can introduce alterations in the cell homeostasis, thereby obscuring the involved endogenous pathways, biasing the interpretation of the results. Here we report on sodium saccharin, which is considered to be not hepatotoxic and therefore can serve as a reference compound to detect metabolic alterations that are not related to liver toxicity. Exposure of HepaRG cells to high levels of sodium saccharin (>10 mM) induced cell death, probably due to an increase in the osmotic pressure. Yet, a low number (n = 15) of significantly altered metabolites were also observed in the lipidome, including a slight decrease in phospholipids and an increase in triacylglycerols, upon daily exposure to 5 mM sodium saccharin for 72 h. The observation that a non-hepatotoxic compound can affect the metabolome underpins the importance of correct experimental design and data interpretation when investigating toxicological modes of action via metabolomics.

scholarly journals Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1098
Author(s):  
Federica Calderaro ◽  
Loes E. Bevers ◽  
Marco A. van den Berg
Keyword(s):  
Experimental Design ◽  
Mode Of Action ◽  
Data Interpretation ◽  
Electron Donors ◽  
Cellulolytic Enzymes ◽  
Inhibiting Factors ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Power Tools ◽  
Reliable Methods

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

scholarly journals Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple negative breast cancer cells

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Yahui Ding ◽  
Xiaoping Chen ◽  
Can Liu ◽  
Weizhi Ge ◽  
Qin Wang ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Death ◽  
Small Molecule ◽  
Mode Of Action ◽  
Rna Silencing ◽  
Breast Cancer Cells ◽  
Triple Negative ◽  
Parent Compound ◽  
Aggressive Breast Cancer

Abstract Background TNBC is the most aggressive breast cancer with higher recurrence and mortality rate than other types of breast cancer. There is an urgent need for identification of therapeutic agents with unique mode of action for overcoming current challenges in TNBC treatment. Methods Different inhibitors were used to study the cell death manner of DMOCPTL. RNA silencing was used to evaluate the functions of GPX4 in ferroptosis and apoptosis of TNBC cells and functions of EGR1 in apoptosis. Immunohistochemical assay of tissue microarray were used for investigating correlation of GPX4 and EGR1 with TNBC. Computer-aided docking and small molecule probe were used for study the binding of DMOCPTL with GPX4. Results DMOCPTL, a derivative of natural product parthenolide, exhibited about 15-fold improvement comparing to that of the parent compound PTL for TNBC cells. The cell death manner assay showed that the anti-TNBC effect of DMOCPTL mainly by inducing ferroptosis and apoptosis through ubiquitination of GPX4. The probe of DMOCPTL assay indicated that DMOCPTL induced GPX4 ubiquitination by directly binding to GPX4 protein. To the best of our knowledge, this is the first report of inducing ferroptosis through ubiquitination of GPX4. Moreover, the mechanism of GPX4 regulation of apoptosis is still obscure. Here, we firstly reveal that GPX4 regulated mitochondria-mediated apoptosis through regulation of EGR1 in TNBC cells. Compound 13, the prodrug of DMOCPTL, effectively inhibited the growth of breast tumor and prolonged the lifespan of mice in vivo, and no obvious toxicity was observed. Conclusions These findings firstly revealed novel manner to induce ferroptosis through ubiquitination of GPX4 and provided mechanism for GPX4 inducing mitochondria-mediated apoptosis through up-regulation of EGR1 in TNBC cells. Moreover, compound 13 deserves further studies as a lead compound with novel mode of action for ultimate discovery of effective anti-TNBC drug.

scholarly journals Measurement Error and Resolution in Quantitative Stable Isotope Probing: Implications for Experimental Design

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Ella T. Sieradzki ◽  
Benjamin J. Koch ◽  
Alex Greenlon ◽  
Rohan Sachdeva ◽  
Rex R. Malmstrom ◽  
...  
Keyword(s):  
Measurement Error ◽  
Experimental Design ◽  
Stable Isotope ◽  
Statistical Power ◽  
Quantitative Estimation ◽  
Cost Benefit ◽  
Data Interpretation ◽  
Stable Isotope Probing ◽  
Isotope Enrichment ◽  
Community Interactions

ABSTRACT Quantitative stable isotope probing (qSIP) estimates isotope tracer incorporation into DNA of individual microbes and can link microbial biodiversity and biogeochemistry in complex communities. As with any quantitative estimation technique, qSIP involves measurement error, and a fuller understanding of error, precision, and statistical power benefits qSIP experimental design and data interpretation. We used several qSIP data sets—from soil and seawater microbiomes—to evaluate how variance in isotope incorporation estimates depends on organism abundance and resolution of the density fractionation scheme. We assessed statistical power for replicated qSIP studies, plus sensitivity and specificity for unreplicated designs. As a taxon’s abundance increases, the variance of its weighted mean density declines. Nine fractions appear to be a reasonable trade-off between cost and precision for most qSIP applications. Increasing the number of density fractions beyond that reduces variance, although the magnitude of this benefit declines with additional fractions. Our analysis suggests that, if a taxon has an isotope enrichment of 10 atom% excess, there is a 60% chance that this will be detected as significantly different from zero (with alpha 0.1). With five replicates, isotope enrichment of 5 atom% could be detected with power (0.6) and alpha (0.1). Finally, we illustrate the importance of internal standards, which can help to calibrate per sample conversions of %GC to mean weighted density. These results should benefit researchers designing future SIP experiments and provide a useful reference for metagenomic SIP applications where both financial and computational limitations constrain experimental scope. IMPORTANCE One of the biggest challenges in microbial ecology is correlating the identity of microorganisms with the roles they fulfill in natural environmental systems. Studies of microbes in pure culture reveal much about their genomic content and potential functions but may not reflect an organism’s activity within its natural community. Culture-independent studies supply a community-wide view of composition and function in the context of community interactions but often fail to link the two. Quantitative stable isotope probing (qSIP) is a method that can link the identity and functional activity of specific microbes within a naturally occurring community. Here, we explore how the resolution of density gradient fractionation affects the error and precision of qSIP results, how they may be improved via additional experimental replication, and discuss cost-benefit balanced scenarios for SIP experimental design.

scholarly journals Oxidative burst and cell death induced by b-quercinin and zoospores of Phytophthora quercina in tobacco cell cultures

2017 ◽  
Vol 38 (SI 2 - 6th Conf EFPP 2002) ◽  
pp. 446-448 ◽  
Author(s):  
J. Koehl ◽  
E.F. Elstner ◽  
W. Oßwald ◽  
I. Heiser
Keyword(s):  
Cell Death ◽  
Cell Suspension ◽  
Cell Cultures ◽  
Oxidative Burst ◽  
Mode Of Action ◽  
Cell Suspension Cultures ◽  
Suspension Cultures ◽  
Tobacco Cell ◽  
Tobacco Cell Suspension

Mode of action of β-quercinin, a novel elicitin on tobacco cell suspension cultures (cvs. Bel B and Bel W3) was investigated by measuring the oxidative burst and cell death in these cell cultures. β-quercinin induced an oxidative burst comparable to that excited by zoospores from P. quercina. Adding superoxidedismutase, catalase and diphenyleneiodonium to elicited cell cultures, it could be demonstrated, that the induction of cell death in tobacco cell cultures is not correlated to the oxidative burst.

scholarly journals The use of valinomycin, nigericin and trichlorocarbanilide in control of the protonmotive force in Escherichia coli cells

1983 ◽  
Vol 212 (1) ◽  
pp. 105-112 ◽  
Author(s):  
S Ahmed ◽  
I R Booth
Keyword(s):  
Escherichia Coli ◽  
Membrane Potential ◽  
Mode Of Action ◽  
Ph Gradient ◽  
Protonmotive Force ◽  
Modes Of Action ◽  
Escherichia Coli Cells ◽  
Low Concentrations ◽  
High K ◽  
Specific Control

Valinomycin, nigericin and trichlorocarbanilide were assessed for their ability to control the protonmotive force in Escherichia coli cells. Valinomycin, at high K+ concentrations, was found to decrease the membrane potential delta phi and indirectly to decrease the pH gradient delta pH. Nigericin was found to have two modes of action. At low concentrations (0.05-2 microM) it carried out K+/H+ exchange and decreased delta pH. At higher concentrations (50 microM) it carried out a K+-dependent transfer of H+, decreasing both delta phi and delta pH. In EDTA-treated cells only the latter mode of action was evident, whereas in a mutant sensitive to deoxycholate both types of effect were observed. Trichlorocarbanilide is proposed as an alternative to nigericin for the specific control of delta pH, and it can be used in cells not treated with EDTA.

Pharmacology of TMS measures

2012 ◽  
Author(s):  
Ulf Ziemann
Keyword(s):  
Motor Cortex ◽  
Mode Of Action ◽  
Cortical Excitability ◽  
Drug Effects ◽  
Main Mode ◽  
Multiple Modes ◽  
Modes Of Action ◽  
Health And Disease ◽  
Cortical Physiology ◽  
Chronic Drug Effects

This article discusses various aspects of the pharmacology of transcranial magnetic stimulator (TMS) measures. TMS measures reflect axonal, or excitatory or inhibitory synaptic excitability in distinct interneuron circuits. TMS measures can be employed to study the effects of a drug with unknown or multiple modes of action, and hence to determine its main mode of action at the systems level of the motor cortex. TMS experiments can also study acute drug effects that may be different from chronic drug effects. TMS or repetitive TMS may induce changes in endogenous neurotransmitter or neuromodulator systems. This allows for the study of neurotransmission along defined neuronal projections in health and disease. This article describes pharmacological experiments that have characterized the physiology of TMS measures of motor cortical excitability. Pharmacological challenging of TMS measures has opened a broad window into human cortical physiology.

scholarly journals Further Characterization ofBacillus subtilisAntibiotic Biosensors and Their Use for Antibacterial Mode-of-Action Studies

2011 ◽  
Vol 55 (4) ◽  
pp. 1784-1786 ◽  
Author(s):  
Katherine R. Mariner ◽  
Nicola Ooi ◽  
Deborah Roebuck ◽  
Alex J. O'Neill ◽  
Ian Chopra
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Mode Of Action ◽  
Trna Synthetase ◽  
Dna Intercalators ◽  
Peptidoglycan Synthesis ◽  
Modes Of Action ◽  
Polymerase Inhibitors

ABSTRACTWe further examined the usefulness of previously reportedBacillus subtilisbiosensors for antibacterial mode-of-action studies. The biosensors could not detect the tRNA synthetase inhibitors mupirocin, indolmycin, and borrelidin, some inhibitors of peptidoglycan synthesis, and most membrane-damaging agents. However, the biosensors confirmed the modes of action of several RNA polymerase inhibitors and DNA intercalators and provided new insights into the possible modes of action of ciprofloxacin, anhydrotetracycline, corralopyronin, 8-hydroxyquinoline, and juglone.

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