Uncoupling Effect Research Articles

In isolated brown adipose tissue mitochondria, UCP1 is not essential for - nor involved in - the uncoupling effects of the classical uncouplers FCCP and DNP

Recent patch-clamp studies of mitoplasts have challenged the traditional view that classical chemical uncoupling (by e.g. FCCP or DNP) is due to the protonophoric property of these substances themselves. These studies instead suggest that in brown-fat mitochondria, FCCP- and DNP-induced uncoupling is mediated through activation of UCP1 (and in other tissues by activation of the adenine nucleotide transporter). These studies thus advocate an entirely new paradigm for the interpretation of standard bioenergetic experiments.To examine whether these patch-clamp results obtained in brown-fat mitoplasts are directly transferable to classical isolated brown-fat mitochondria studies, we investigated the effects of FCCP and DNP in brown-fat mitochondria from wildtype and UCP1 KO mice, comparing the FCCP and DNP effects with those of a fatty acid (oleate), a bona fide activator of UCP1.Whereas the sensitivity of brown-fat mitochondria to oleate was much higher in UCP1-containing than in UCP1 KO mitochondria, there was no difference in sensitivity to FCCP and DNP between these mitochondria, neither in oxygen consumption rate nor in membrane potential studies. Correspondingly, the UCP1-dependent ability of GDP to competitively inhibit activation by oleate was not seen with FCCP and DNP.It would thus be premature to abandon the established bioenergetic interpretation of chemical uncoupler effects in classical isolated brown-fat mitochondria—and probably also generally in this type of mitochondrial study. Understanding the molecular and structural reasons for the different outcomes of mitoplast and mitochondrial studies is a challenging task.

Common Mitochondrial Targets of Curcumin and Cinnamic Acid, the Membrane-Active Natural Phenolic Compounds

Background: Research has shown the multiple actions of curcumin on different cell systems, including enzymes and mitochondria. The detected effects of curcumin on mitochondria are diverse, ranging from protective to toxic. Objectives: In this present work, the influence of curcumin, as well as cinnamic acid, which is a microbial metabolite and a possible product of the microbial breakdown of curcumin, on isolated mitochondria, was investigated. Methods: Membrane potential, swelling, respiration, and calcium retention capacity were studied using selective electrodes, fluorescence and spectral methods. Results: It was found that curcumin at low concentrations (10–20 μM) activated the opening of the calcium-dependent permeability transition pore (mPTP) and decreased the calcium retention capacity and threshold concentrations necessary for the mPTP opening. Moreover, curcumin caused a concentration-dependent stepwise decrease in the membrane potential, accompanied by the activation of respiration and a decrease in oxidative phosphorylation, which indicates that curcumin is a typical mitochondrial uncoupler. The uncoupling effect strongly depended on the concentration of curcumin, which also increased, stepwise, from weak uncoupling at 25 µM to complete uncoupling at 75–100 µM. Cinnamic acid had similar effects, with the exception of the depolarizing effect, at concentrations that were an order of magnitude higher. Conclusions: Presumably, the uncoupling action of curcumin is a priming event that modulates any energy- and redox-dependent mitochondrial functions, from positive stimulation to toxic disorder. This effect can also underlie the curcumin-induced changes in different cellular processes and be achieved by targeted delivery of curcumin to certain cells, bypassing the microbiota.

Synthesis and anti-proliferative effect of novel 4-Aryl-1, 3-Thiazole-TPP conjugates via mitochondrial uncoupling process

With the advent of mitochondrial targeting moiety such as triphenlyphosphonium cation (TPP+), targeting mitochondria in cancer cells has become a promising strategy for combating tumors. Herein, a series of novel 4-aryl-1,3-thiazole derivatives linked to TPP+ moiety were designed and synthesized. The cytotoxicity against a panel of four cancer cell lines was evaluated by CCK-8 assay. Most of these compounds exhibited moderate to good inhibitory activity over HeLa, PC-3 and HCT-15 cells while MCF-7 cells were less sensitive to most compounds. Among them, compound 12a exhibited a significant anti-proliferative activity against HeLa cells, and prompted for further investigation. Specifically, 12a decreased mitochondrial membrane potential and enhanced levels of reactive oxygen species (ROS). The flow cytometry analysis revealed that compound 12a could induce apoptosis and cell cycle arrest at G0/G1 phase in HeLa cells. In addition, mitochondrial bioenergetics assay revealed that 12a displayed mild mitochondrial uncoupling effect. Taken together, these findings suggest the therapeutic potential of compound 12a as an antitumor agent targeting mitochondria.

Theoretical and Experimental Study of the Interaction of Protonophore Uncouplers and Decoupling Agents with Functionally Active Mitochondria.

The purpose of this work was to quantitatively characterize the effectiveness of oxidative phosphorylation uncouplers and decoupling agents in functionally active mitochondria, taking into account their content in the hydrophobic region of the inner membrane of these organelles. When conducting theoretical studies, it is accepted that uncouplers and decouplers occupy part of the volume of mitochondria to exhibit their activity, which is defined as the effective volume. The following quantities characterizing the action of these reagents are considered: (1) concentrations of reagents that cause double stimulation of mitochondrial respiration in state 4 ( ); (2) effective distribution coefficient ( ) - the ratio of the amount of reagents in the effective volume of mitochondria and the water volume; (3) the relative amount of reagents associated with the effective volume of mitochondria ( ); (4) specific activity of reagents localized in the effective volume of mitochondria ( ). We have developed methods for determining these values, based on an analysis of the dependence of the rate of mitochondrial respiration on the concentration of uncouplers and decoupling agents at two different concentrations of mitochondrial protein in the incubation medium. During experimental studies, we compared the effects of the classical protonophore uncouplers 2,4-dinitrophenol (DNP) and сarbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), the natural uncouplers lauric and palmitic acids, and the natural decouplers α,ω-tetradecanedioic (TDA) and α,ω-hexadecanedioic (HDA) acids that differ both in the structure of the molecule and in the degree of solubility in lipids. Using the developed methods, we have clarified the dependence of the degree of activity of these uncouplers and decoupling agents on the distribution of their molecules between the effective volume of mitochondria and the water volume.

A novel inhibitor of the mitochondrial respiratory complex I with uncoupling properties exerts potent antitumor activity

Cancer cells are highly dependent on bioenergetic processes to support their growth and survival. Disruption of metabolic pathways, particularly by targeting the mitochondrial electron transport chain complexes (ETC-I to V) has become an attractive therapeutic strategy. As a result, the search for clinically effective new respiratory chain inhibitors with minimized adverse effects is a major goal. Here, we characterize a new OXPHOS inhibitor compound called MS-L6, which behaves as an inhibitor of ETC-I, combining inhibition of NADH oxidation and uncoupling effect. MS-L6 is effective on both intact and sub-mitochondrial particles, indicating that its efficacy does not depend on its accumulation within the mitochondria. MS-L6 reduces ATP synthesis and induces a metabolic shift with increased glucose consumption and lactate production in cancer cell lines. MS-L6 either dose-dependently inhibits cell proliferation or induces cell death in a variety of cancer cell lines, including B-cell and T-cell lymphomas as well as pediatric sarcoma. Ectopic expression of Saccharomyces cerevisiae NADH dehydrogenase (NDI-1) partially restores the viability of B-lymphoma cells treated with MS-L6, demonstrating that the inhibition of NADH oxidation is functionally linked to its cytotoxic effect. Furthermore, MS-L6 administration induces robust inhibition of lymphoma tumor growth in two murine xenograft models without toxicity. Thus, our data present MS-L6 as an inhibitor of OXPHOS, with a dual mechanism of action on the respiratory chain and with potent antitumor properties in preclinical models, positioning it as the pioneering member of a promising drug class to be evaluated for cancer therapy.MS-L6 exerts dual mitochondrial effects: ETC-I inhibition and uncoupling of OXPHOS. In cancer cells, MS-L6 inhibited ETC-I at least 5 times more than in isolated rat hepatocytes. These mitochondrial effects lead to energy collapse in cancer cells, resulting in proliferation arrest and cell death. In contrast, hepatocytes which completely and rapidly inactivated this molecule, restored their energy status and survived exposure to MS-L6 without apparent toxicity.

Differences in the intestinal microbiota and association of host metabolism with hair coat status in cattle.

The hair coat status of cattle serves as an easily observed indicator of economic value in livestock production; however, the underlying mechanism remains largely unknown. Therefore, the objective of the current study was to determine differences in the intestinal microbiota and metabolome of cattle based on a division of with either slick and shining (SHC) or rough and dull (MHC) hair coat in Simmental cows. Eight SHC and eight MHC late-pregnancy Simmental cows (with similar parities, body weights, and body conditions) were selected based on their hair coat status, and blood samples (plasma) from coccygeal venipuncture and fecal samples from the rectum were collected. The intestinal microbiota (in the fecal samples) was characterized by employing 16S rRNA gene sequencing targeting the V3-V4 hypervariable region on the Illumina MiSeq PE300 platform, and plasma samples were subjected to LC-MS/MS-based metabolomics with Progenesis QI 2.3. Plasma macromolecular metabolites were examined for differences in the metabolism of lipids, proteins, mineral elements, and hormones. Notable differences between the SHC and MHC groups related to host hair coat status were observed in the host metabolome and intestinal microbiota (P < 0.05). The host metabolome was enriched in histidine metabolism, cysteine and methionine metabolism, and purine metabolism in the SHC group, and the intestinal microbiota were also enriched in histidine metabolism (P < 0.05). In the MHC group, the symbiotic relationship transitioned from cooperation to competition in the MHC group, and an uncoupling effect was present in the microbe-metabolite association of intestine microbiota-host interactions. The hubs mediating the relationships between intestinal microbiota and plasma metabolites were the intestinal bacterial genus g__norank_f__Eubacterium_coprostanoligenes_group, plasma inosine, triiodothyronine, and phosphorus, which could be used to differentiate cows' hair coat status (P < 0.05). Overall, the present study identified the relationships between the features of the intestinal microbiota and host hair coat status, thereby providing evidence and a new direction (intestine microbiota-host interplay) for future studies aimed at understanding the hair coat status of cattle.

Effects of electro-mechanical uncouplers, hormonal stimulation and pacing rate on the stability and function of cultured rabbit myocardial slices.

Introduction: Recent advances have enabled organotypic culture of beating human myocardial slices that are stable for weeks. However, human myocardial samples are rare, exhibit high variability and frequently originate from diseased hearts. Thus, there is a need to adapt long-term slice culture for animal myocardium. When applied to animal cardiac slices, studies in healthy or genetically modified myocardium will be possible. We present the culture of slices from rabbit hearts, which resemble the human heart in microstructure, electrophysiology and excitation-contraction coupling. Methods: Left ventricular myocardium from New Zealand White rabbits was cut using a vibratome and cultured in biomimetic chambers for up to 7days (d). Electro-mechanical uncoupling agents 2,3-butanedione monoxime (BDM) and cytochalasin D (CytoD) were added during initiation of culture and effects on myocyte survival were quantified. We investigated pacing rates (0.5Hz, 1Hz, and 2Hz) and hormonal supplements (cortisol, T3, catecholamines) at physiological plasma concentrations. T3 was buffered using BSA. Contractile force was recorded continuously. Glucose consumption and lactate production were measured. Whole-slice Ca2+ transients and action potentials were recorded. Effects of culture on microstructure were investigated with confocal microscopy and image analysis. Results: Protocols for human myocardial culture resulted in sustained contracture and myocyte death in rabbit slices within 24h, which could be prevented by transient application of a combination of BDM and CytoD. Cortisol stabilized contraction amplitude and kinetics in culture. T3 and catecholaminergic stimulation did not further improve stability. T3 and higher pacing rates increased metabolic rate and lactate production. T3 stabilized the response to β-adrenergic stimulation over 7d. Pacing rates above 1Hz resulted in progredient decline in contraction force. Image analysis revealed no changes in volume fractions of cardiomyocytes or measures of fibrosis over 7d. Ca2+ transient amplitudes and responsiveness to isoprenaline were comparable after 1d and 7d, while Ca2+ transient duration was prolonged after 7d in culture. Conclusions: A workflow for rabbit myocardial culture has been established, preserving function for up to 7d. This research underscores the importance of glucocorticoid signaling in maintaining tissue function and extending culture duration. Furthermore, BDM and CytoD appear to protect from tissue damage during the initiation phase of tissue culture.

Divergent Roles of Escherichia Coli Encoded Lon Protease in Imparting Resistance to Uncouplers of Oxidative Phosphorylation: Roles of marA, rob, soxS and acrB.

Uncouplers of oxidative phosphorylation dissipate the proton gradient, causing lower ATP production. Bacteria encounter several non-classical uncouplers in the environment, leading to stress-induced adaptations. Here, we addressed the molecular mechanisms responsible for the effects of uncouplers in Escherichia coli. The expression and functions of genes involved in phenotypic antibiotic resistance were studied using three compounds: two strong uncouplers, i.e., Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and 2,4-Dinitrophenol (DNP), and one moderate uncoupler, i.e., Sodium salicylate (NaSal). Quantitative expression studies demonstrated induction of transcripts encoding marA, soxS and acrB with NaSal and DNP, but not CCCP. Since MarA and SoxS are degraded by the Lon protease, we investigated the roles of Lon using a lon-deficient strain (Δlon). Compared to the wild-type strain, Δlon shows compromised growth upon exposure to NaSal or 2, 4-DNP. This sensitivity is dependent on marA but not rob and soxS. On the other hand, the Δlon strain shows enhanced growth in the presence of CCCP, which is dependent on acrB. Interestingly, NaSal and 2,4-DNP, but not CCCP, induce resistance to antibiotics, such as ciprofloxacin and tetracycline. This study addresses the effects of uncouplers and the roles of genes involved during bacterial growth and phenotypic antibiotic resistance. Strong uncouplers are often used to treat wastewater, and these results shed light on the possible mechanisms by which bacteria respond to uncouplers. Also, the rampant usage of some uncouplers to treat wastewater may lead to the development of antibiotic resistance.

The study of the protective effect of mitochondrial uncouplers during acute toxicity of the fungicide difenoconazole in different organs of mice.

Pesticides represent a serious problem for agricultural workers due to their neurotoxic effects. The aim of this study was to evaluate the ability of pharmacological oxidative phosphorylation uncouplers to reduce the effect of the difenoconazole fungicide on mitochondrial DNA (mtDNA) of various organs in mice. Injections of difenoconazole caused cognitive deficits in mice, and the protonophore 2,4-dinitrophenol (2,4-DNP) and Azur I (AzI), a demethylated metabolite of methylene blue (MB), prevented the deterioration of cognitive abilities in mice induced by difenoconazole. Difenoconazole increased the rate of reactive oxygen species (ROS) production, likely through inhibition of complex I of the mitochondrial respiratory chain. After intraperitoneal administration of difenoconazole lungs, testes and midbrain were most sensitive to the accumulation of mtDNA damage. In contrast, the cerebral cortex and hippocampus were not tolerant to the effects of difenoconazole. The protonophore 2,4-DNP reduced the rate of ROS formation and significantly reduced the amount of mtDNA damage caused by difenoconazole in the midbrain, and partially, in the lungs and testes. MB, an alternative electron carrier capable of bypassing inhibited complex I, had no effect on the effect of difenoconazole on mtDNA, while its metabolite AzI, a demethylated metabolite of MB, was able to protect the mtDNA of the midbrain and testes. Thus, mitochondria-targeted therapy is a promising approach to reduce pesticide toxicity for agricultural workers.

Mechanistic Aspects of Biphenyl Urea-Based Analogues in Triple-Negative Breast Cancer Cell Lines.

Triple-negative breast cancer (TNBC) poses significant challenges due to its aggressive nature and limited treatment options. In this study, we investigated the impact of urea-based compounds on TNBC cells to uncover their mechanisms of action and therapeutic potential. Notably, polypharmacology urea analogues were found to work via p53-related pathways, and their cytotoxic effects were amplified by the modulation of oxidative phosphorylation pathways in the mitochondria of cancer cells. Specifically, compound 1 demonstrated an uncoupling effect on adenosine triphosphate (ATP) synthesis, leading to a time- and concentration-dependent shift toward glycolysis-based ATP production in MDA-MB-231 cells. At the same time, no significant changes in ATP synthesis were observed in noncancerous MCF10A cells. Moreover, the unique combination of mitochondrial- and p53-related effects leads to a higher cytotoxicity of urea analogues in cancer cells. Notably, the majority of tested clinical agents, but sorafenib, showed significantly higher toxicity in MCF10A cells. To test our hypothesis of sensitizing cancer cells to the treatment via modulation of mitochondrial health, we explored the combinatorial effects of urea-based analogues with established chemotherapeutic agents commonly used in TNBC treatment. Synergistic effects were evident in most tested combinations in TNBC cell lines, while noncancerous MCF10A cells exhibited higher resistance to these combination treatments. The combination of compound 1 with SN38 displayed nearly 60-fold selectivity toward TNBC cells over MCF10A cells. Encouragingly, combinations involving compound 1 restored the sensitivity of TNBC cells to cisplatin. In conclusion, our study provides valuable insights into the mechanisms of action of urea-based compounds in TNBC cells. The observed induction of mitochondrial membrane depolarization, inhibition of superoxide dismutase activity, disruption of ATP synthesis, and cell-line-specific responses contribute to their cytotoxic effects. Additionally, we demonstrated the synergistic potential of compound 1 to enhance the efficacy of existing TNBC treatments. However, the therapeutic potential and underlying molecular mechanisms of urea-based analogues in TNBC cell lines require further exploration.

Hygrothermal analysis of a semi-infinite cylinder: an integral transform approach

PurposeThe purpose of this paper is to examine the impact of moisture and temperature changes on the behavior of a semi-infinite solid cylinder made of T300/5208 composite material. This study aims to provide analytical solutions for temperature, moisture and thermal stress through the de-coupling technique and the method of integral transforms. Both coupled and uncoupled cases are considered.Design/methodology/approachThis study investigates the hygrothermo-elastic response of a semi-infinite solid circular cylinder using an integral transform technique that includes Hankel and Fourier transforms. The cylinder is subjected to prescribed sources, and a numerical algorithm is developed for the numerical computation of the results. The goal is to understand how the cylinder responds to changes in temperature and moisture.FindingsThe paper presents an analytical solution for temperature, moisture and thermal stress in a semi-infinite solid cylinder obtained through the use of an integral transform technique. The study focuses on a graphite fiber-reinforced epoxy matrix composite material (T300/5208) and discusses the coupled and uncoupled effects of temperature, moisture and thermal stress on the material. The results of the transient response hygrothermo-elastic field are presented graphically to provide a visual representation of the findings.Research limitations/implicationsThe research presented in this article is primarily hypothetical and focused on the analysis of mathematical models.Originality/valueTo the authors' best knowledge, this study is the first to investigate the hygrothermal effect in a semi-infinite circular cylinder. Additionally, the material properties used in the analysis are both homogenous and isotropic and independent of both temperature and moisture. These unique aspects of the study make it a novel contribution to the field.

2,4-Dinitrophenol does not exert neuro-regenerative potential in experimental autoimmune neuritis

ObjectiveWe evaluated the potential neuro-regenerative effects of the mitochondrial uncoupler 2,4-Dinitrophenol in experimental autoimmune neuritis, an animal model for an acute autoimmune neuropathy. MethodsExperimental autoimmune neuritis was induced in Lewis rats. Different concentrations of 2,4-Dinitrophenol (1 mg/kg, 0.1 mg/kg and 0.01 mg/kg) were applied during the recovery phase of the neuritis (at days 18, 22 and 26) and compared to the vehicle. Any effects were assessed through functional, electrophysiological, and morphological analysis via electron microscopy of all groups at day 30. Additional immune-histochemical analysis of inflammation markers and remyelination of the sciatic nerves were performed for the dosage of 1 mg/kg and control. ResultsNo enhancement of functional or electrophysiological recovery was observed in all 2,4-Dinitrophenol-treated groups. Cellular inflammation markers of T cells (CD3+) were comparable to control, and an increase of macrophages (IbA1+) invasion in the sciatic nerves was observed. Treatment with 2,4-Dinitrophenol reduced axonal swelling in myelinated and unmyelinated fibers with an increased production of brain-derived neurotrophic factor. ConclusionOur findings do not support the hypothesis that repurposing of the mitochondrial uncoupler 2,4-Dinitrophenol exerts functionally relevant neuro-regenerative effects in autoimmune neuritis.

The mitochondrial uncoupling effects of nitazoxanide enhances cellular autophagy and promotes the clearance of α-synuclein: Potential role of AMPK-JNK pathway

Upregulation and aggregation of the pre-synaptic protein, α-synuclein plays a key role in Parkinson's disease (PD) and mitochondrial dysfunction was surmised to be an upstream event in the disease pathogenesis. Emerging reports identified the role of nitazoxanide (NTZ), an anti-helminth drug, in enhancing mitochondrial oxygen consumption rate (OCR) and autophagy. In the present study, we have examined the mitochondrial effects of NTZ in mediating cellular autophagy and subsequent clearance of both endogenous and pre-formed aggregates of α-synuclein in cellular model of PD. Our results demonstrate that the mitochondrial uncoupling effects of NTZ results in the activation of AMPK and JNK, which in-turn leads to the enhancement of cellular autophagy. Also,1-methyl-4-phenylpyridinium (MPP+) mediated decrease in autophagic flux with a concomitant increase in the α-synuclein levels were ameliorated in cells treated with NTZ. However, in cells lacking functional mitochondria (ρ0 cells), NTZ did not mitigate MPP+ mediated alterations in the autophagic clearance of α-synuclein, indicating that the mitochondrial effects of NTZ play a crucial role in the clearance of α-synuclein by autophagy. Also, the ability of AMPK inhibitor, compound C, in abrogating NTZ mediated enhancement in the autophagic flux and α-synuclein clearance highlight the pivotal role of AMPK in NTZ mediated autophagy. Further, NTZ per se enhanced the clearance of preformed α-synuclein aggregates that were exogenously added to the cells. Overall, the results of our present study suggest that NTZ activates macroautophagy in cells due to its uncoupling effects on mitochondrial respiration via activation of AMPK-JNK pathway resulting in the clearance of both endogenous and pre-formed α-synuclein aggregates. As NTZ happens to possess good bioavailability and safety profile, considering this drug for PD based on its mitochondrial uncoupling and autophagy enhancing properties for mitigating mitochondrial reactive oxygen species (ROS) and α-synuclein toxicity appears to be a promising therapeutic option.

Metabolic shift and the effect of mitochondrial respiration on the osteogenic differentiation of dental pulp stem cells.

Metabolism shifts from glycolysis to mitochondrial oxidative phosphorylation are vital during the differentiation of stem cells. Mitochondria have a direct function in differentiation. However, the metabolic shift and the effect of mitochondria in regulating the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain unclear. Human dental pulp stem cells were collected from five healthy donors. Osteogenic differentiation was induced by osteogenic induction medium. The activities of alkaline phosphatase, hexokinase, pyruvate kinase, and lactate dehydrogenase were analyzed by enzymatic activity kits. The extracellular acidification rate and the mitochondrial oxygen consumption rate were measured. The mRNA levels of COL-1, ALP, TFAM, and NRF1 were analyzed. The protein levels of p-AMPK and AMPK were detected by western blotting. Glycolysis decreased after a slight increase, while mitochondrial oxidative phosphorylation continued to increase when cells grew in osteogenic induction medium. Therefore, the metabolism of differentiating cells switched to mitochondrial respiration. Next, inhibiting mitochondrial respiration with carbonyl cyanide-chlorophenylhydrazone, a mitochondrial uncoupler inhibited hDPSCs differentiation with less ALP activity and decreased ALP and COL-1 mRNA expression. Furthermore, mitochondrial uncoupling led to AMPK activation. 5-Aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, simulated the effect of mitochondrial uncoupling by inhibiting osteogenic differentiation, mitochondrial biogenesis, and mitochondrial morphology. Mitochondrial uncoupling and activation of AMPK depressed mitochondrial oxidative phosphorylation and inhibited differentiation, suggesting that they may serve as regulators to halt osteogenic differentiation from impaired mitochondrial oxidative phosphorylation.

A study on L-threonine and L-serine uptake in Escherichia coli K-12.

In the current study, we report the identification and characterization of the yifK gene product as a novel amino acid carrier in E. coli K-12 cells. Both phenotypic and biochemical analyses showed that YifK acts as a permease specific to L-threonine and, to a lesser extent, L-serine. An assay of the effect of uncouplers and composition of the reaction medium on the transport activity indicates that YifK utilizes a proton motive force to energize substrate uptake. To identify the remaining threonine carriers, we screened a genomic library prepared from the yifK-mutant strain and found that brnQ acts as a multicopy suppressor of the threonine transport defect caused by yifK disruption. Our results indicate that BrnQ is directly involved in threonine uptake as a low-affinity but high-flux transporter, which forms the main entry point when the threonine concentration in the external environment reaches a toxic level. By abolishing YifK and BrnQ activity, we unmasked and quantified the threonine transport activity of the LIV-I branched chain amino acid transport system and demonstrated that LIV-I contributes significantly to total threonine uptake. However, this contribution is likely smaller than that of YifK. We also observed the serine transport activity of LIV-I, which was much lower compared with that of the dedicated SdaC carrier, indicating that LIV-I plays a minor role in the serine uptake. Overall, these findings allow us to propose a comprehensive model of the threonine/serine uptake subsystem in E. coli cells.

Mitochondrial ATP Synthase and Mild Uncoupling by Butyl Ester of Rhodamine 19, C4R1.

The homeostasis of the transmembrane potential of hydrogen ions in mitochondria is a prerequisite for the normal mitochondrial functioning. However, in different pathological conditions it is advisable to slightly reduce the membrane potential, while maintaining it at levels sufficient to produce ATP that will ensure the normal functioning of the cell. A number of chemical agents have been found to provide mild uncoupling; however, natural proteins residing in mitochondrial membrane can carry this mission, such as proteins from the UCP family, an adenine nucleotide translocator and a dicarboxylate carrier. In this study, we demonstrated that the butyl ester of rhodamine 19, C4R1, binds to the components of the mitochondrial ATP synthase complex due to electrostatic interaction and has a good uncoupling effect. The more hydrophobic derivative C12R1 binds poorly to mitochondria with less uncoupling activity. Mass spectrometry confirmed that C4R1 binds to the β-subunit of mitochondrial ATP synthase and based on molecular docking, a C4R1 binding model was constructed suggesting the binding site on the interface between the α- and β-subunits, close to the anionic amino acid residues of the β-subunit. The association of the uncoupling effect with binding suggests that the ATP synthase complex can provide induced uncoupling.

Repurposing nitazoxanide as a novel anti-atherosclerotic drug based on mitochondrial uncoupling mechanisms.

The anthelmintic drug nitazoxanide has a mitochondrial uncoupling effect. Mitochondrial uncouplers have been proven to inhibit smooth muscle cell proliferation and migration, inhibit NLRP3 inflammasome activation of macrophages and improve dyslipidaemia. Therefore, we aimed to demonstrate that nitazoxanide would protect against atherosclerosis. The mitochondrial oxygen consumption of cells was measured by using the high-resolution respirometry system, Oxygraph-2K. The proliferation and migration of A10 cells were measured by using Edu immunofluorescence staining, wound-induced migration and the Boyden chamber assay. Protein levels were measured by using the western blot technique. ApoE (-/-) mice were fed with a Western diet to establish an atherosclerotic model in vivo. The in vitro experiments showed that nitazoxanide and tizoxanide had a mitochondrial uncoupling effect and activated cellular AMPK. Nitazoxanide and tizoxanide inhibited serum- and PDGF-induced proliferation and migration of A10 cells. Nitazoxanide and tizoxanide inhibited NLRP3 inflammasome activation in RAW264.7 macrophages, the mechanism by which involved the AMPK/IκBα/NF-κB pathway. Nitazoxanide and tizoxanide also induced autophagy in A10 cells and RAW264.7 macrophages. The in vivo experiments demonstrated that oral administration of nitazoxanide reduced the increase in serum IL-1β and IL-6 levels and suppressed atherosclerosis in Western diet-fed ApoE (-/-) mice. Nitazoxanide inhibits the formation of atherosclerotic plaques in ApoE (-/-) mice fed on a Western diet. In view of nitazoxanide being an antiprotozoal drug already approved by the FDA, we propose it as a novel anti-atherosclerotic drug with clinical translational potential.

Toxic Effects of Xenobiotic Compounds on the Microbial Community of Activated Sludge

AbstractThe presence of xenobiotic compounds in biological wastewater treatment processes with activated sludge may reduce microbial communities, disrupt microbial diversity, and diminish system performance. Shock loads and unusual operating events in these biological systems have negative impacts on their efficiency and reliability for pollutant degradation, thereby posing high risk to microorganisms and water quality of receiving treated water bodies. The severity and characteristics of the occurring damage are determined by the toxic contaminant's degree, nature and mode of application. This review highlights the effects of metabolic uncouplers, heavy metals, carbon nanotubes, pharmaceuticals and personal care products, nanoparticles, and phenolic compounds stress on microbial biomass in activated sludge systems. The synergistic, antagonistic, and shock load toxic effects of hybrid substances exposure in an activated sludge sequential batch reactor (SBR) system on organic and nutrient removal, system efficiency, and toxicants biodegradation are discussed. The findings can be used to provide a theoretical foundation and professional assistance for optimizing the shock impacts of these toxic substances on biological wastewater treatment systems, which will help to reduce their negative effects on treatment system efficiency.

Toluidine blue O directly and photodynamically impairs the bioenergetics of liver mitochondria: a potential mechanism of hepatotoxicity.

Toluidine blue O (TBO) is a phenothiazine dye that, due to its photochemical characteristics and high affinity for biomembranes, has been revealed as a new photosensitizer (PS) option for antimicrobial photodynamic therapy (PDT). This points to a possible association with membranous organelles like mitochondrion. Therefore, here we investigated its effects on mitochondrial bioenergetic functions both in the dark and under photostimulation. Two experimental systems were utilized: (a) isolated rat liver mitochondria and (b) isolated perfused rat liver. Our data revealed that, independently of photostimulation, TBO presented affinity for mitochondria. Under photostimulation, TBO increased the protein carbonylation and lipid peroxidation levels (up to 109.40 and 119.87%, respectively) and decreased the reduced glutathione levels (59.72%) in mitochondria. TBO also uncoupled oxidative phosphorylation and photoinactivated the respiratory chain complexes I, II, and IV, as well as the FoF1-ATP synthase complex. Without photostimulation, TBO caused uncoupling of oxidative phosphorylation and loss of inner mitochondrial membrane integrity and inhibited very strongly succinate oxidase activity. TBO's uncoupling effect was clearly seen in intact livers where it stimulated oxygen consumption at concentrations of 20 and 40μM. Additionally, TBO (40μM) reduced cellular ATP levels (52.46%) and ATP/ADP (45.98%) and ATP/AMP (74.17%) ratios. Consequently, TBO inhibited gluconeogenesis and ureagenesis whereas it stimulated glycogenolysis and glycolysis. In conclusion, we have revealed for the first time that the efficiency of TBO as a PS may be linked to its ability to photodynamically inhibit oxidative phosphorylation. In contrast, TBO is harmful to mitochondrial energy metabolism even without photostimulation, which may lead to adverse effects when used in PDT.

Exploring optimal Taxol® CYP725A4 activity in Saccharomyces cerevisiae

BackgroundCYP725A4 catalyses the conversion of the first Taxol® precursor, taxadiene, to taxadiene-5α-ol (T5α-ol) and a range of other mono- and di-hydroxylated side products (oxygenated taxanes). Initially known to undergo a radical rebound mechanism, the recent studies have revealed that an intermediate epoxide mediates the formation of the main characterised products of the enzyme, being T5α-ol, 5(12)-oxa-3(11)-cyclotaxane (OCT) and its isomer, 5(11)-oxa-3(11)-cyclotaxane (iso-OCT) as well as taxadienediols. Besides the high side product: main product ratio and the low main product titre, CYP725A4 is also known for its slow enzymatic activity, massively hindering further progress in heterologous production of Taxol® precursors. Therefore, this study aimed to systematically explore the key parameters for improving the regioselectivity and activity of eukaryotic CYP725A4 enzyme in a whole-cell eukaryotic biocatalyst, Saccharomyces cerevisiae.ResultsInvestigating the impact of CYP725A4 and reductase gene dosages along with construction of self-sufficient proteins with strong prokaryotic reductases showed that a potential uncoupling event accelerates the formation of oxygenated taxane products of this enzyme, particularly the side products OCT and iso-OCT. Due to the harmful effect of uncoupling products and the reactive metabolites on the enzyme, the impact of flavins and irons, existing as prosthetic groups in CYP725A4 and reductase, were examined in both their precursor and ready forms, and to investigate the changes in product distribution. We observed that the flavin adenine dinucleotide improved the diterpenoids titres and biomass accumulation. Hemin was found to decrease the titre of iso-OCT and T5α-ol, without impacting the side product OCT, suggesting the latter being the major product of CYP725A4. The interaction between this iron and the iron precursor, δ-Aminolevulinic acid, seemed to improve the production of these diterpenoids, further denoting that iso-OCT and T5α-ol were the later products. While no direct correlation between cellular-level oxidative stress and oxygenated taxanes was observed, investigating the impact of salt and antioxidant on CYP725A4 further showed the significant drop in OCT titre, highlighting the possibility of enzymatic-level uncoupling event and reactivity as the major mechanism behind the enzyme activity. To characterise the product spectrum and production capacity of CYP725A4 in the absence of cell growth, resting cell assays with optimal neutral pH revealed an array of novel diterpenoids along with higher quantities of characterised diterpenoids and independence of the oxygenated product spectra from the acidity effect. Besides reporting on the full product ranges of CYP725A4 in yeast for the first time, the highest total taxanes of around 361.4 ± 52.4 mg/L including 38.1 ± 8.4 mg/L of T5α-ol was produced herein at a small, 10-mL scale by resting cell assay, where the formation of some novel diterpenoids relied on the prior existence of other diterpenes/diterpenoids as shown by statistical analyses.ConclusionsThis study shows how rational strain engineering combined with an efficient design of experiment approach systematically uncovered the promoting effect of uncoupling for optimising the formation of the early oxygenated taxane precursors of Taxol®. The provided strategies can effectively accelerate the design of more efficient Taxol®-producing yeast strains.