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Evaluation of PTD-Luc in the in vivo ATP assay. HeLa cells were diluted two-fold serially to give from 0 to 100,000 cells per well and incubated in a 96-well plate in DMEM with 10% FBS. The luminescence signal (p/s) was recorded 5 min after treatment with 40 nM Luc or PTD-Luc. The luminescence signal is plotted against the cell number for 50 to 1,000 cells per well (A) and more than 1,000 cells per well (B). Symbols represent mean values, and lines represent the linear data fit. Pearson's correlation coefficient (R) is shown above the plot.
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Luciferase is a sensitive, reliable biological sensor used for measuring ATP. However, its widespread application in drug discovery and toxicology studies has been limited due to unavoidable cell extraction processes, which cause inaccurate measurements of intracellular ATP and obstruct the application of homogenous high-throughput screening. Recen...
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... luminescence signal of PTD-Luc was 12.6 times that of Luc in 1,000 HeLa cells, and PTD-Luc could measure the intracellular luminescence of as few as 50 cells. Strong correlations were found between the luminescence signal and cell number over a wide range of cell numbers, from high and low (Figure 4, R = 0.985, p < 0.001, for >1,000 cells; R = 0.960, p < 0.001, for <1,000 cells). These results are consistent with many reports using cell extracts to show that the amount of ATP bioluminescence is directly proportional to the number of cells [2,11,12]. ...Similar publications
A survey was conducted at eight U.S. drinking water plants, that spanned a wide range of water qualities and treatment/disinfection practices. Plants that treated heavily-wastewater-impacted source waters had lower trihalomethane to dihaloacetonitrile ratios due to the presence of more organic nitrogen and HAN precursors. As the bromide to total or...
Matrix metalloproteinase-13 (MMP-13) degrades collagen and other matrix components, thus playing a critical role in the development of osteoarthritis (OA). The expression level of microRNA‑9 (miR‑9) is significantly depressed in cartilage tissues of OA patients. Furthermore, bioinformatics analysis demonstrated complementary binding sites between m...
Removal of effluent organic matter (EfOM) from a wastewater secondary effluent by aluminum sulfate (alum) coagulation and its effects on haloacetic acid (HAA) formation were studied in the range of alum dose 0–120 mg/L and the pH range 4.0–9.0. Surrogate parameters, such as dissolved organic carbon (DOC), UV254, specific UV absorbance (SUVA), and f...
We investigated whether different doses of undenatured type II collagen (undenatured collagen, UC-II) help improve monosodium iodoacetate (MIA)-induced (osteoarthritis) OA in young and old rats. A total of 70 rats were divided into five groups: (1) control; (2) MIA (a single intra-articular injection of MIA); (3)–(5) MIA+ Undenatured Collagen with...
ATP sulfurylase (ATPS) is an enzyme of primary importance for photosynthetic organisms since it catalyzes the first reaction of the sulfate assimilation pathway. ATPS was highly studied in plants and fungi, but little is known about the algal enzyme and no attempts have been done yet to study its possible redox regulation. We used a bioinformatic a...
Citations
... ATP levels are also used as a surrogate for cell viability since lethal cell damage and a loss of membrane integrity results in an inability to synthesize ATP and a corresponding reduction in cellular ATP levels [43,44]. A decline in ATP levels can match the IC 50 values generated by MTT and/or LDH assays in undifferentiated and differentiated SH-SY5Y cells in response to different neurotoxicants [27,32]. ...
Mefloquine (MQ) is a quinoline-based anti-malarial drug used for chemoprophylaxis or as a treatment in combination with artesunate. Although MQ has clear anti-Plasmodium falciparum properties, it can induce neurotoxicity and undesired neuropsychiatric side effects in humans. Hence, this study aimed to characterize the neurotoxicity of MQ using human neuroblastoma SH-SY5Y cells. The effects of MQ on neuronal toxicity and cell viability were investigated over a concentration range of 1-100 µM using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The influence of MQ on cellular bioenergetics was examined by measuring cellular ATP levels and from the induction of reactive oxygen species (ROS). An in silico approach was used to assess the potential neurotoxicity of MQ mediated via binding to the active sites of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and then experimentally validated via in vitro enzymatic assays. MQ was cytotoxic to neuronal cells in a concentration and exposure duration dependent manner and induced a significant reduction in viability at concentrations of ≥25 µM after a 24 h exposure. MQ adversely impacted cellular bioenergetics and significantly depleted ATP production at concentrations of ≥1 µM after 24 h. MQ-induced cellular ROS production, which was correlated with the induction of apoptosis, as revealed by flow cytometry. In silico studies suggested that MQ was a dual cholinesterase inhibitor and one with remarkably potent binding to BuChE. Modelling data were supported by in vitro studies which showed that MQ inhibited both human AChE and BuChE enzymes. In summary, MQ is an antimalarial drug that may induce neurotoxicity by impacting cellular bioenergetics and perturbing the activity of cholinesterases at exposure concentrations relevant to human dosage.
... The measurement of cellular adenosine 5 -triphosphate (ATP) content using firefly luciferase enzyme was used to indicate mitochondrial function and associated cellular bioenergetic activity of the cells [56]. In the case of loss of cell membrane integrity by injury, ATP synthesis in the cells is decreased, and endogenous ATPase is rapidly released to deplete any remaining ATP from the cytoplasm [56]. ...
... The measurement of cellular adenosine 5 -triphosphate (ATP) content using firefly luciferase enzyme was used to indicate mitochondrial function and associated cellular bioenergetic activity of the cells [56]. In the case of loss of cell membrane integrity by injury, ATP synthesis in the cells is decreased, and endogenous ATPase is rapidly released to deplete any remaining ATP from the cytoplasm [56]. Briefly, SH-SY5Y cells were seeded at 1 × 10 5 cells/well in 6-well plates, grown to 80-90% confluency, and then treated with L-Glu or acai berry extracts or both as detailed in Section 2.2.4. ...
... The levels of ROS induced after L-Glu exposure to SHSY-5Y cells were quantified using a DCFDA assay. Treatment of cells with L-Glu for 3 h induced ROS in a concentrationdependent way, which reached significance at concentrations of 11.1, 33.3, and 100 mM (56,50, and 91% increase from controls, respectively) ( Figure 5A). In general, an incubation with acai berry extracts for 3 h showed no increases in ROS production except for the 0.01 and 1000 µg/mL concentrations of the ethanolic extract ( Figure 5B). ...
Aberrant accumulation of the neurotransmitter L-glutamate (L-Glu) has been implicated as a mechanism of neurodegeneration, and the release of L-Glu after stroke onset leads to a toxicity cascade that results in neuronal death. The acai berry (Euterpe oleracea) is a potential dietary nutraceu-tical. The aim of this research was to investigate the neuroprotective effects of acai berry aqueous and ethanolic extracts to reduce the neurotoxicity to neuronal cells triggered by L-Glu application. L-Glu and acai berry effects on cell viability were quantified using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays, and effects on cellular bioenergetics were assessed via quantitation of the levels of cellular ATP, mitochondrial membrane potential (MMP), and production of reactive oxygen species (ROS) in neuroblastoma cells. Cell viability was also evaluated in human cortical neuronal progenitor cell culture after L-Glu or/and acai berry application. In isolated cells, activated currents using patch-clamping were employed to determine whether L-Glu neurotoxicity was mediated by ionotropic L-Glu-receptors (iGluRs). L-Glu caused a significant reduction in cell viability, ATP, and MMP levels and increased ROS production. The co-application of both acai berry extracts with L-Glu provided neuroprotection against L-Glu with sustained cell viability, decreased LDH production, restored ATP and MMP levels, and reduced ROS levels. Whole-cell patch-clamp recordings showed that L-Glu toxicity is not mediated by the activation of iGluRs in neuroblastoma cells. Fractionation and analysis of acai berry extracts with liquid chromatography-mass spectrometry identified several phytochemical antioxidants that may have provided neuroprotective effects. In summary, the acai berry contains nutraceuticals with antioxidant activity that may be a beneficial dietary component to limit pathological deficits triggered by excessive L-Glu accumulations.
... The presence of a greater proportion of extracellular ATP indicates a less healthy population. Several methods have been developed to measure the ATP levels, which include colori-metric, fluorescent, and bioluminescent methods, as well as liquid chromatography and mass spectrometry [118,[120][121][122][123][124][125]. The bioluminescent ATP assays are widely used due to their high sensitivity and rapid detection. ...
... The extracellular ATP is usually measured in the cell supernatants or in the cell cultures by using the standard bioluminescence luciferine/luciferase assay [118]. In contrast, the intracellular ATP is determined using live cell ATP assays that can penetrate the cell membrane without interfering with cellular metabolism or cell integrity, and the luciferase activity is proportional to the intracellular ATP concentration [122]. A major advantage of ATP monitoring is that it does not require culturing microbes for a lengthy period and can provide rapid test results. ...
Nitrification is a major challenge in chloraminated drinking water systems, resulting in undesirable loss of disinfectant residual. Consequently, heterotrophic bacteria growth is increased, which adversely affects the water quality, causing taste, odour, and health issues. Regular monitoring of various water quality parameters at susceptible areas of the water distribution system (WDS) helps to detect nitrification at an earlier stage and allows sufficient time to take corrective actions to control it. Strategies to monitor nitrification in a WDS require conducting various microbiological tests or assessing surrogate parameters that are affected by microbiological activities. Additionally, microbial decay factor (Fm) is used by water utilities to monitor the status of nitrification. In contrast, approaches to manage nitrification in a WDS include controlling various factors that affect monochloramine decay rate and ammonium substrate availability, and that can inhibit nitrification. However, some of these control strategies may increase the regulated disinfection-by-products level, which may be a potential health concern. In this paper, various strategies to monitor and control nitrification in a WDS are critically examined. The key findings are: (i) the applicability of some methods require further validation using real WDS, as the original studies were conducted on laboratory or pilot systems; (ii) there is no linkage/formula found to relate the surrogate parameters to the concentration of nitrifying bacteria, which possibly improve nitrification monitoring performance; (iii) improved methods/monitoring tools are required to detect nitrification at an earlier stage; (iv) further studies are required to understand the effect of soluble microbial products on the change of surrogate parameters. Based on the current review, we recommend that the successful outcome using many of these methods is often site-specific, hence, water utilities should decide based on their regular experiences when considering economic and sustainability aspects.
... (c) Adenosine triphosphate assay: All living cells contain adenosine triphosphate as their source of energy and as they die the existing adenosine triphosphate pool starts declining. Therefore, measurement of the intracellular adenosine triphosphate content is widely used as a marker for the analysis of the degree of cellular toxicity (Lee et al. 2012). The most common technique for the determination of adenosine triphosphate is the bioluminescent method in which the enzyme luciferase, obtained from the firefly, catalyses the conversion of the luciferin to oxyluciferin in the presence of adenosine triphosphate. ...
Cancer is one of the most fatal diseases causing deaths of millions of people worldwide. Since actual cancer treatments are rarely efficient and often toxic, there is a demand for innocuous and more effective anticancer drugs. For instance, metal/metal oxide nanoparticles allow controlled drug release and drug delivery to specific targets. In particular, medicinal plant-based metal nanoparticles appear as safer and more effective. Here, we present the synthesis of metal nanoparticles using medicinal plant extracts, and the toxicity of these nanoparticles for cancerous cells. The anticancer effect is explained by several possible mechanisms such as generation of reactive oxygen species, cell cycle arrest, antioxidant property, apoptosis and autophagy. We present plant compounds that induce cytotoxicity against cancerous cells, and assays for the measurement of cell toxicity.
... As the universal energy donor molecules of living cells, ATP and ADP are representative of cellular energy and health. There are several methods that have been used to measure the concentration of ATP, ADP, and AMP, including the direct measurement of adenine nucleotide fluorescence from cell extract by HPLC (Bhatt et al., 2012), monitoring of single-wavelength genetically encoded fluorescent sensors for imaging of extracellular and cytosolic ATP (Lobas et al., 2019), and enzyme-mediated assays of cell extracts (Lee et al., 2012). The direct measurement of adenine nucleotide fluorescence includes several analytical procedures utilizing either isocratic or gradient reversed-phase, ion-exchange, or ion-pairing HPLC in combination with UV or fluorescence detection (Bhatt et al., 2012). ...
As the principal co‐factors of many metabolic pathways, the measurement of both adenine nucleotides and nicotinamide adenine dinucleotide provides important information about cellular energy metabolism. However, given their rapid and reversible conversion as well as their relatively low concentration ranges, it is difficult to measure these compounds. Here, we describe a highly sensitive and selective ion‐pairing HPLC method with fluorescence detection to quantify adenine nucleotides in plants. In addition, nicotinamide adenine dinucleotide is a crucially important redox‐active substrate for multiple catabolic and anabolic reactions with the ratios of NAD ⁺ /NADH and NADP ⁺ /NADPH being suggested as indicators of the general intracellular redox potential and hence metabolic state. Here, we describe highly sensitive enzyme cycling−based colorimetric assays (with a detection limit in the pmol range) performed subsequent to a simple extraction procedure involving acid or base extraction to allow the measurement of the cellular levels of these metabolites. © 2020 The Authors.
Basic Protocol 1 : Preparation of plant material for the measurement
Basic Protocol 2 : Measurement of ATP, ADP, and AMP via HPLC
Basic Protocol 3 : NAD ⁺ /NADP ⁺ measurements
Basic Protocol 4 : NADH/NADPH measurements
Basic Protocol 5 : Data analysis and quality control approaches
... Furthermore, cell injury results in not only reduced ATP synthesis but also immediate diminution of endogenous ATP levels which is caused by the escape of ATP-converting enzymes (e.g., ATPase) [65]. Therefore, quantification of the intracellular ATP content is crucial for the assessment of the degree of cellular toxicity [66]. ...
Cell division is a phenomenon that is encountered in all cells in nature. While normal cell division results in proliferation in single-celled organisms, and development and repair in multicellular organisms, aberrant and untimely cell division results in tumor formation. Therefore, the understanding of the cell division is hidden in identifying the details of the molecular mechanisms that govern cellular division at the exact time and under right conditions. Sometimes these molecular mechanisms are distorted by both intrinsic and extracellular factors, and the division process halts or deviates to an abnormal pathway. At this point, it is essential that the abnormal cells are removed from the tissue by an appropriate mechanism. In this context, in this review, general and specific information about cell division and its molecular control mechanisms were discussed, and different types of cell death mechanisms were mentioned accordingly. In addition, chemical, biological, and physical cytotoxic agents that negatively affect cell division and their mechanisms of action are explained. Finally, a brief review of the principles of different cytotoxicity (cell viability and proliferation) test systems has been performed to provide a source of information for investigators who study cell viability, proliferation , or different types of cellular death pathways.
... The in vivo visualization of luciferase activity using the BLI technique was conducted according to our previous reports (LeeHer, 2013;Lee et al., 2012a;Lee et al., 2011;Lee et al., 2010;Lee et al., 2012b). In brief, all mice received an intraperitoneal injection of 100 mg/kg D-luciferin dissolved in Dulbecco's PBS and then were anesthetized in an induction chamber with 2.5% isoflurane in 100% oxygen at a flow rate of 1.0 L/min for 10 min. ...
Bioluminescence imaging has proven to be a highly sensitive technique for assessing in vitro transcriptional activity toward understanding gene regulation patterns; however, application of this technique is limited for brain research. In particular, the poor spatiotemporal resolution is a major hurdle for monitoring the dynamic changes of transcriptional activity in specific regions of the brain during longitudinal analysis of living animals. To overcome this limitation, in this study, we modified a lentivirus-based luciferase glucocorticoid receptor (GR) reporter by inserting destabilizing sequence genes, and then the reporter was stereotaxically injected in the mouse infralimbic prefrontal cortex (IL-PFC). Using this strategy, we could successfully pin-point and monitor the dynamic changes in GR activity in IL-PFC during normal stress adaptation. The modified reporter showed a 1.5-fold increase in temporal resolution for monitoring GR activity compared to the control, with respect to the intra-individual coefficients of variation. This novel in vivo method has broad applications, as it is readily adaptable to different types of transcription factor arrays as well spanning wide target regions of the brain to other organs and tissues.
... A genetically encoded photoswitchable luciferase provides an additional tool for the cellular detection of ATP concentrations at different locations and different time points. [16] In order to efficiently identify amino acids in potentially allosteric regions of FLuc useful for the incorporation of AzoF without the need for extensive mutational analysis, computational structural modeling and protein stability calculations were carried out for the three different conformational states of firefly luciferase in the two-step chemiluminescence reaction: the open inactive state (state A), the closed active state for the generation of luciferyl-AMP (state B), and the closed active state for the generation of oxyluciferin (state C). ...
The genetic encoding of three different azobenzene phenylalanines with different photochemical properties was achieved in human cells by using an engineered pyrrolysyl tRNA/tRNA synthetase pair. In order to demonstrate reversible light control of protein function, azobenzenes were site‐specifically introduced into firefly luciferase. Computational strategies were applied to guide the selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. In addition, the new azobenzene analogues provide enhanced thermal stability, high photoconversion, and responsiveness to visible light. These small‐molecule photoswitches can reversibly photocontrol protein function with excellent spatiotemporal resolution, and preferred sites for incorporation can be computationally determined, thus providing a new tool for investigating biological processes.
... Several strategies focus on sensors that directly image the ATP concentrations inside living cells. One strategy is to internalize a variant of the luciferase protein fused to a protein transduction domain (PTD-Luc), to ensure a controlled and homogenous cellular uptake of the sensor [15]. Another strategy is the development of bioluminescent sensors based on genetically encoded fluorescent reporter proteins fused to ATP binding proteins as Parceval (GFP-GlnK1) [16,17] or A-Team (CFP-F 1 -Epsilon-YFP) [18]. ...
Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL(+) into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL(+) to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.
... Cellular level of ATP, as critical parameter of energy status, was evaluated using bioluminescent ATP Determination Kit (Taylor et al., 1998), based on the conversion of luciferin to oxyluciferin in the presence of ATP and oxygen in reaction catalysed by luciferase with emission of bioluminescence (max. at 560 nm) proportional to cellular ATP content (Lee et al., 2012). ...
In a search for the safe vitamin carrier the PAMAM G3 dendrimer covalently substituted with 9 and 10 molecules of vitamin B7 (biotin) and B6 (pyridoxal), respectively (BC-PAMAM) was investigated. Dendrimer substitution with B-group vitamins significantly alters its biological properties as compared to native form. Observed effects on investigated cell parameters including morphology, adhesion, migration and ATP level were different for normal human fibroblasts (BJ) and squamous cell carcinoma (SCC-15) cell lines. BC-PAMAM revealed significantly less pronounced effects on investigated parameters, particularly at higher concentrations (5–50 μM), which is relevant with its lower positive surface charge, as compared with native form. The bioconjugate, up to 50 μM concentration, appeared to be a safe vitamin carrier to normal fibroblasts, without significant effect on their adhesion, shape and migration as well as on intracellular ATP level. In SCC-15 cells BC-PAMAM, at low concentrations (0.1–0.5 μM), altered the cell shape and increase adhesion, whereas at higher concentrations opposite effects were seen. Measurements of cellular level of ATP showed that higher resistance of cancer cells to toxic effects of native PAMAM dendrimers may be due to higher energy supply of cancer cells.
