Search Results (16)

Toxoplasma gondii is an Apicomplexan parasite that possesses a well-developed system of scavengers of reactive oxygen species (ROS). Among its components, T. gondii mitochondrial superoxide dismutase (TgSOD2) is essential, as predicted by the CRISPR phenotype index and evidenced by the non-viability of its constitutive knockouts. As an obligate intracellular parasite, TgSOD2 is upregulated during extracellular stages. Herein, we generated a viable TgSOD2 knockdown mutant using an inducible auxin–degron system to explore the biological role of TgSOD2 in T. gondii. Depletion of TgSOD2 led to impaired parasite growth and replication, reduced mitochondrial membrane potential (MMP), abnormalities in the distribution of ATP synthase within its mitochondrial electron transport chain (mETC), and increased susceptibility to mETC inhibitors. Through a proximal biotinylation approach, we identified the interactions of TgSOD2 with complexes IV and V of its mETC, suggesting that these sites are sensitive to ROS. Our study provides the first insights into the role of TgSOD2 in maintaining its mitochondrial redox homeostasis and subsequent parasite replication fitness. Significance: Toxoplasma gondii infects nearly a third of the world population and can cause fetal miscarriages or life-threatening complications in vulnerable patients. Current therapies do not eradicate the parasite from the human hosts, rendering them at risk of recurrence during their lifetimes. T. gondii has a single mitochondrion, which is well-known for its susceptibility to oxidative damage that leads to T. gondii’s death. Therefore, targeting T. gondii mitochondrion remains an attractive therapeutic strategy for drug development. T. gondii’s mitochondrial superoxide dismutase is an antioxidant protein in the parasite mitochondrion and is essential for its survival. Understanding its biological role could reveal mitochondrial vulnerabilities in T. gondii and provide new leads for the development of effective treatments for T. gondii infections. Full article

In production and transportation systems of the oil industry, brine solutions contain high concentrations of chloride and dissolved CO₂, which is a very corrosive medium to which carbon steel is exposed. Therefore, finding new effective and environmentally friendly corrosion inhibitors is of great importance. The effect of CeCl₃ (in concentrations from 5 mg dm⁻³ to 20 mg dm⁻³) and oleic acid imidazoline (IOA) (in concentrations from 5 mg dm⁻³ to 20 mg dm⁻³) separately and their mixtures (in concentrations from 5 mg dm⁻³ to 15 mg dm⁻³ of CeCl₃ and from 5 mg dm⁻³ to 20 mg dm⁻³ of IOA) as corrosion inhibitors of AISI 1018 carbon steel corrosion in simulated brine solution saturated with CO₂ at 60 °C were examined by means of weight-loss testing, electrochemical measurements (polarization resistance, linear polarization with Tafel extrapolation, electrochemical impedance spectroscopy) and surface analyses (scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses, Raman spectroscopy and X-ray diffraction). All test methods showed a higher efficiency of compounds′ mixtures (from 62.77% to 97.94%) and a higher degree of corrosion protection compared to the action of individual compounds (efficiency from 3.43% to 94.61% for IOA and from 57.58% to 96.27% for CeCl₃). Imidazoline, a common corrosion inhibitor in CO₂-saturated systems, most likely forms a surface film with voids via its adsorption on steel surface, while cerium carbonate tends to fill these voids by creating a more compact film. In this way, a denser and thicker surface film is formed. Full article

Hydrogen sulfide (H₂S), a known inhibitor of the electron transport chain, is endogenously produced in the periphery as well as in the central nervous system, where is mainly generated by glial cells. It affects, as a cellular signaling molecule, many different biochemical processes. In the central nervous system, depending on its concentration, it can be protective or damaging to neurons. In the study, we have demonstrated, in a primary mouse spinal cord cultures, that it is particularly harmful to motor neurons, is produced by glial cells, and is stimulated by inflammation. However, its role on glial cells, especially astrocytes, is still under-investigated. The present study was designed to evaluate the impact of H₂S on astrocytes and their phenotypic heterogeneity, together with the functionality and homeostasis of mitochondria in primary spinal cord cultures. We found that H₂S modulates astrocytes’ morphological changes and their phenotypic transformation, exerts toxic properties by decreasing ATP production and the mitochondrial respiration rate, disturbs mitochondrial depolarization, and alters the energetic metabolism. These results further support the hypothesis that H₂S is a toxic mediator, mainly released by astrocytes, possibly acting as an autocrine factor toward astrocytes, and probably involved in the non-cell autonomous mechanisms leading to motor neuron death. Full article

The plant vascular system is not only a transportation system for delivering nutrients but also a highway transport network for spreading viruses. Tomato spotted wilt orthotospovirus (TSWV) is among the most destructive viruses that cause serious losses in economically important crops worldwide. However, there is minimal information about the long-distance movements of TSWV in the host plant vascular system. In this this study, we confirm that TSWV virions are present in the xylem as observed by transmission electron microscopy (TEM). Further, a quantitative proteomic analysis based on label-free methods was conducted to reveal the uniqueness of protein expression in xylem sap during TSWV infection. Thus, this study identified and quantified 3305 proteins in two groups. Furthermore, TSWV infection induced three viral structural proteins, N, Gn and Gc, and 315 host proteins differentially expressed in xylem (163 up-regulated and 152 down-regulated). GO enrichment analysis showed up-regulated proteins significantly enriched in homeostasis, wounding, defense response, and DNA integration terms, while down-regulated proteins significantly enriched in cell wall biogenesis/xyloglucan metabolic process-related terms. KEGG enrichment analysis showed that the differentially expressed proteins (DEPs) were most strongly associated with plant-pathogen interaction, MAPK signaling pathway, and plant hormone signal transduction. Cluster analysis of DEPs function showed the DEPs can be categorized into cell wall metabolism-related proteins, antioxidant proteins, PCD-related proteins, host defense proteins such as receptor-like kinases (RLKs), salicylic acid binding protein (SABP), pathogenesis related proteins (PR), DNA methylation, and proteinase inhibitor (PI). Finally, parallel reaction monitoring (PRM) validated 20 DEPs, demonstrating that the protein abundances were consistent between label-free and PRM data. Finally, 11 genes were selected for RT-qPCR validation of the DEPs and label-free-based proteomic analysis concordant results. Our results contribute to existing knowledge on the complexity of host plant xylem system response to virus infection and provide a basis for further study of the mechanism underlying TSWV long-distance movement in host plant vascular system. Full article

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron degenerative disease that is associated with demyelination. The Wobbler (WR) mouse exhibits motoneuron degeneration, gliosis and myelin deterioration in the cervical spinal cord. Since male WRs display low testosterone (T) levels in the nervous system, we investigated if T modified myelin-relative parameters in WRs in the absence or presence of the aromatase inhibitor, anastrozole (A). We studied myelin by using luxol-fast-blue (LFB) staining, semithin sections, electron microscopy and myelin protein expression, density of IBA1⁺ microglia and mRNA expression of inflammatory factors, and the glutamatergic parameters glutamine synthetase (GS) and the transporter GLT1. Controls and WR + T showed higher LFB, MBP and PLP staining, lower g-ratios and compact myelin than WRs and WR + T + A, and groups showing the rupture of myelin lamellae. WRs showed increased IBA1⁺ cells and mRNA for CD11b and inflammatory factors (IL-18, TLR4, TNFαR₁ and P₂Y₁₂R) vs. controls or WR + T. IBA1⁺ cells, and CD11b were not reduced in WR + T + A, but inflammatory factors’ mRNA remained low. A reduction of GS⁺ cells and GLT-1 immunoreactivity was observed in WRs and WR + T + A vs. controls and WR + T. Clinically, WR + T but not WR + T + A showed enhanced muscle mass, grip strength and reduced paw abnormalities. Therefore, T effects involve myelin protection, a finding of potential clinical translation. Full article

Rare gastrointestinal stromal tumors (GISTs) are caused by mutations in the KIT and PDGFRA genes. Avapritinib (BLU-285) is a targeted selective inhibitor for mutated KIT and PDGFRA receptors that can be used to treat these tumors. However, there are subtypes of GISTs that exhibit resistance against BLU-285 and thus require other treatment strategies. This can be addressed by employing a drug delivery system that transports a combination of drugs with distinct cell targets. In this work, we present the synthesis of esterase-responsive polyglycerol-based nanogels (NGs) to overcome drug resistance in rare GISTs. Using inverse nanoprecipitation mediated with inverse electron-demand Diels–Alder cyclizations (iEDDA) between dPG-methyl tetrazine and dPG-norbornene, multi-drug-loaded NGs were formed based on a surfactant-free encapsulation protocol. The obtained NGs displayed great stability in the presence of fetal bovine serum (FBS) and did not trigger hemolysis in red blood cells over a period of 24 h. Exposing the NGs to Candida Antarctica Lipase B (CALB) led to the degradation of the NG network, indicating the capability of targeted drug release. The bioactivity of the loaded NGs was tested in vitro on various cell lines of the GIST-T1 family, which exhibit different drug resistances. Cell internalization with comparable uptake kinetics of the NGs could be confirmed by confocal laser scanning microscopy (CLSM) and flow cytometry for all cell lines. Cell viability and live cell imaging studies revealed that the loaded NGs are capable of intracellular drug release by showing similar IC₅₀ values to those of the free drugs. Furthermore, multi-drug-loaded NGs were capable of overcoming BLU-285 resistance in T1-α-D842V + G680R cells, demonstrating the utility of this carrier system. Full article

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH₂-DA). Non-fluorescent DCFH₂-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH₂) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH₂-DA is complicated by various protocol-related factors that mediate DCFH₂-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH₂-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH₂-DA solvent concentration. Conclusively, experiments with DCFH₂-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH₂-DA-DCF reaction and proper controls should always be built into the assay protocol. Full article

Phytocannabinoids represent a promising approach in glioblastoma therapy. Previous work has shown that a combined treatment of glioblastoma cells with submaximal effective concentrations of psychoactive Δ⁹-tetrahydrocannabinol (THC) and non-psychoactive cannabidiol (CBD) greatly increases cell death. In the present work, the glioblastoma cell lines U251MG and U138MG were used to investigate whether the combination of THC and CBD in a 1:1 ratio is associated with a disruption of cellular energy metabolism, and whether this is caused by affecting mitochondrial respiration. Here, the combined administration of THC and CBD (2.5 µM each) led to an inhibition of oxygen consumption rate and energy metabolism. These effects were accompanied by morphological changes to the mitochondria, a release of mitochondrial cytochrome c into the cytosol and a marked reduction in subunits of electron transport chain complexes I (NDUFA9, NDUFB8) and IV (COX2, COX4). Experiments with receptor antagonists and inhibitors showed that the degradation of NDUFA9 occurred independently of the activation of the cannabinoid receptors CB₁, CB₂ and TRPV1 and of usual degradation processes mediated via autophagy or the proteasomal system. In summary, the results describe a previously unknown mitochondria-targeting mechanism behind the toxic effect of THC and CBD on glioblastoma cells that should be considered in future cancer therapy, especially in combination strategies with other chemotherapeutics. Full article

Inflammation or injury to the somatosensory nervous system may result in chronic pain conditions, which affect millions of people and often cause major health problems. Emerging lines of evidence indicate that reactive oxygen species (ROS), such as superoxide anion or hydrogen peroxide, are produced in the nociceptive system during chronic inflammatory and neuropathic pain and act as specific signaling molecules in pain processing. Among potential ROS sources in the somatosensory system are NADPH oxidases, a group of electron-transporting transmembrane enzymes whose sole function seems to be the generation of ROS. Interestingly, the expression and relevant function of the Nox family members Nox1, Nox2, and Nox4 in various cells of the nociceptive system have been demonstrated. Studies using knockout mice or specific knockdown of these isoforms indicate that Nox1, Nox2, and Nox4 specifically contribute to distinct signaling pathways in chronic inflammatory and/or neuropathic pain states. As selective Nox inhibitors are currently being developed and investigated in various physiological and pathophysiological settings, targeting Nox1, Nox2, and/or Nox4 could be a novel strategy for the treatment of chronic pain. Here, we summarize the distinct roles of Nox1, Nox2, and Nox4 in inflammatory and neuropathic processing and discuss the effectiveness of currently available Nox inhibitors in the treatment of chronic pain conditions. Full article

The reduction-oxidation (redox) system consists of the coupling and coordination of various electron gradients that are generated thanks to serial reduction-oxidation enzymatic reactions. These reactions happen in every cell and produce radical oxidants that can be mainly classified into reactive oxygen species (ROS) and reactive nitrogen species (RNS). ROS and RNS modulate cell-signaling pathways and cellular processes fundamental to normal cell function. However, overproduction of oxidative species can lead to oxidative stress (OS) that is pathological. Oxidative stress is a main contributor to diabetic kidney disease (DKD) onset. In the kidney, the proximal tubular cells require a high energy supply to reabsorb proteins, metabolites, ions, and water. In a diabetic milieu, glucose-induced toxicity promotes oxidative stress and mitochondrial dysfunction, impairing tubular function. Increased glucose level in urine and ROS enhance the activity of sodium/glucose co-transporter type 2 (SGLT2), which in turn exacerbates OS. SGLT2 inhibitors have demonstrated clear cardiovascular benefits in DKD which may be in part ascribed to the generation of a beneficial equilibrium between oxidant and antioxidant mechanisms. Full article

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC₅₀ of 0.822 μM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites. Full article

Chattonella species, C. marina and C. ovata, are harmful raphidophycean flagellates known to have hemolytic effects on many marine organisms and resulting in massive ecological damage worldwide. However, knowledge of the toxigenic mechanism of these ichthyotoxic flagellates is still limited. Light was reported to be responsible for the hemolytic activity (HA) of Chattonella species. Therefore, the response of photoprotective, photosynthetic accessory pigments, the photosystem II (PSII) electron transport chain, as well as HA were investigated in non-axenic C. marina and C. ovata cultures under variable environmental conditions (light, iron and addition of photosynthetic inhibitors). HA and hydrogen peroxide (H₂O₂) were quantified using erythrocytes and pHPA assay. Results confirmed that% HA of Chattonella was initiated by light, but was not always elicited during cell division. Exponential growth of C. marina and C. ovata under the light over 100 µmol m⁻² s⁻¹ or iron-sufficient conditions elicited high hemolytic activity. Inhibitors of PSII reduced the HA of C. marina, but had no effect on C. ovata. The toxicological response indicated that HA in Chattonella was not associated with the photoprotective system, i.e., xanthophyll cycle and regulation of reactive oxygen species, nor the PSII electron transport chain, but most likely occurred during energy transport through the light-harvesting antenna pigments. A positive, highly significant relationship between HA and chlorophyll (chl) biosynthesis pigments, especially chl c2 and chl a, in both species, indicated that hemolytic toxin may be generated during electron/energy transfer through the chl c2 biosynthesis pathway. Full article

Layered double hydroxide (LDHs) coatings were developed for the corrosion protection of AZ31 Mg alloy. AZ31 is widely used in the transport industry due to its low mass density and good mechanical properties. LDH coatings were fabricated under co-precipitation conditions and applied under hydrothermal conditions. Two different systems Zn-Al LDH and Li-Al LDH were studied. Specimens were post-treated via immersion for 2 h at 45 °C in inhibitor aqueous baths. Na₂WO₄·H₂O and LiNO₃ inorganic inhibitors were used, respectively, to produce inhibitor-loaded systems: Zn-Al LDH(W) and Li-Al LDH(Li). The characterization of the coatings was carried out by field-emission scanning electron microscope (FESEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR). The corrosion process was studied by electrochemical impedance spectroscopy (EIS) and scanning vibrating electrode technique (SVET). The surface was also evaluated via the determination of water drop contact angle and the performance of a paint adhesion test using an epoxy primer. The characterization of the coating revealed two-layered coatings with a denser inner layer and a flaky outer layer. Both coatings improved the corrosion resistance of the AZ31 alloy. Loading with inhibitor further increased the corrosion resistance by one order of magnitude (bare substrate, Z_10mHz~10² Ω cm²; LDH, Z_10mHz~10^3–4 Ω cm²; LDH-inhibitor, Z_10mHz~10⁵ Ω cm²). Full article

Nitrous oxide (N₂O) emission-suppressing activity of some electron-transport inhibitors of the Hill reaction system was investigated. The Hill reaction inhibitors—paraquat, isouron, bromacil, diquat, and simazine—all of which have been or are currently being used as herbicides in farming activity are expected to inhibit the electron-transporting pathways of nitrate respiration in denitrifying bacteria. Using N₂O-emitting soil bed (5.0 g of fresh weight) from a continuously manured Andisol corn farmland in Hokkaido, Japan, which was autoclaved and further supplemented with an active N₂O-emitter, Pseudomonas sp. 5CFM15-6D, and 1 mL of 100 mM NH₄NO₃ or (NH₄)₂SO₄ solution as the sole nitrogen source (final concentration, 0.2 mM) in a 30 mL gas-chromatography vial, the effects of the five herbicides on N₂O emission were examined. Paraquat and isouron (each at 50 µM) showed a statistically significant suppression of N₂O emission in both the nitrification and the denitrification processes after a 7-day-incubation, whereas diquat at the same concentration accelerated N₂O emission in the presence of NO₃⁻. These results suggest that paraquat and isouron inhibited both the nitrification and the denitrification processes for N₂O generation, or its upstream stages, whereas diquat specifically inhibited N₂O reductase, an enzyme that catalyzes the reduction of N₂O to N₂ gas. Incomplete denitrifiers are the key players in the potent emission of N₂O from Andisol corn farmland soil because of the missing nosZ gene. The electron relay system-inhibiting herbicides—paraquat and isouron—possibly contribute to the prevention of denitrification-induced nitrogen loss from the farming soil. Full article

The roles of cytochrome P450 monooxygenases (CYPs) from Streptomyces spp. which are called the “treasure islands” for natural products for medicine and antibiotics are not well understood. Substrate specificity studies on CYPs may give a solution for elucidation of their roles. Based on homology sequence information, the CYP105D7 of a soluble cytochrome P450 known as heme protein from Streptomyces avermitilis MA4680 was expressed using the T7 promoter of the bacterial expression vector pET24ma, over-expressed in Escherichia coli system and characterized. An engineered whole cell system for daidzein hydroxylation was constructed using an exogenous electron transport system from ferredoxin reductase (PdR) and ferredoxin (Pdx). Also, an in vitro reaction study showed the purified CYP105D7 enzyme, using NADH-dependent-reducing equivalents of a redox partner from Pseudomonas putida, hydroxylated daidzein at the 3' position of the B ring to produce 7,3,'4' trihydroxyisoflavone. The hydroxylated position was confirmed by GC-MS analysis. The turnover number of the enzyme was 0.69 μmol 7,3,'4'-trihydroxyisoflavone produced per μmol P450 per min. This enzyme CYP105D7 represents a novel type of 3'-hydroxylase for daidzein hydroxylation. A P450 inhibitor such as coumarin significantly (ca.98%) inhibited the daidzein hydroxylation activity. Full article