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A series of new gold(I) and silver(I) N-heterocyclic carbenes bearing a 1-thio-β-d-glucose tetraacetate moiety was synthesized and chemically characterized. The compounds' stability and solubility in physiological conditions were investigated employing a multitechnique approach. Interaction studies with biologically relevant proteins, such as superoxide dismutase (SOD) and human serum albumin (HSA), were conducted via UV-vis absorption spectroscopy and high-resolution ESI mass spectrometry. The biological activity of the compounds was evaluated in the A2780 and A2780R (cisplatin-resistant) ovarian cancer cell lines and the HSkMC (human skeletal muscle) healthy cell line. Inhibition studies of the selenoenzyme thioredoxin reductase (TrxR) were also carried out. The results highlighted that the gold complexes are more stable in aqueous environment and capable of interaction with SOD and HSA. Moreover, these carbenes strongly inhibited the TrxR activity. In contrast, the silver ones underwent structural alterations in the aqueous medium and showed greater antiproliferative activity.

Species of Leishmania and Trypanosoma genera are the causative agents of relevant parasitic diseases. Survival inside their hosts requires the existence of a potent antioxidant enzymatic machinery. Four iron superoxide dismutases have been described in trypanosomatids (FeSODA, FeSODB1, FeSODB2, and FeSODC) that hold a potential as therapeutic targets. Nonetheless, very few studies have been developed that make use of the purified enzymes. Moreover, FeSODC remains uncharacterised in Leishmania . In this work, for the first time, we describe the purification and enzymatic activity of recombinant versions of the four Leishmania FeSOD isoforms and establish an improved strategy for developing inhibitors. We propose a novel parameter [( V * cyt. c  -  V cyt. c )/ V cyt. c ] which, in contrast to that used in the classical cytochrome c reduction assay, correlates linearly with enzyme concentration. As a proof of concept, we determine the IC 50 values of two ruthenium carbosilane metallodendrimers against these isoforms.

Reactive oxygen species (ROS) are rapidly eliminated and reproduced in organisms, and they always play important roles in various biological functions and abnormal pathological processes. Evaluated ROS have frequently been observed in various cancers to activate multiple pro-tumorigenic signaling pathways and induce the survival and proliferation of cancer cells. Hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2 •- ) are the most important redox signaling agents in cancer cells, the homeostasis of which is maintained by dozens of growth factors, cytokines, and antioxidant enzymes. Therefore, antioxidant enzymes tend to have higher activity levels to maintain the homeostasis of ROS in cancer cells. Effective intervention in the ROS homeostasis of cancer cells by chelating agents or metal complexes has already developed into an important anti-cancer strategy. We can inhibit the activity of antioxidant enzymes using chelators or metal complexes; on the other hand, we can also use metal complexes to directly regulate the level of ROS in cancer cells via mitochondria. In this review, metal complexes or chelators with ROS regulation capacity and with anti-cancer applications are collectively and comprehensively analyzed, which is beneficial for the development of the next generation of inorganic anti-cancer drugs based on ROS regulation. We expect that this review will provide a new perspective to develop novel inorganic reagents for killing cancer cells and, further, as candidates or clinical drugs.

The visceral form of Leishmaniasis, also known as kala-azar, caused by Leishmania chagasi is the main etiological agent of this form in Brazil responsible for 30,000 annual deaths. Despite its epidemiological impact, treatment of the disease is limited by resistance, species-dependent efficacy and serious adverse effects. The application of computational tools to prioritize potential bioactive molecules based on 3D structural of biological target is a viable alternative. Among the L. chagasi validated targets, Fe + 2 superoxide dismutase B2 (LcFeSODB2) is the first parasite enzyme against oxidative stress and it is involved in essential metabolic processes for its survival. Due to substrate binding-site volume (superoxide ion) and consequent difficulty in its active site modulation for small molecules, the search for allosteric sites at LcFeSODB2 3D structure is a promising strategy. As there are no 3D structures of LcFeSODB2, comparative modeling was applied to build 3D models by SWISS-MODEL and MODELLER version 9.19. Next, the best 3D model was used in molecular dynamics (MD) routines with multiple probes on GROMACS version 5.1.2. In addition, potential allosteric sites predicted by FTMap and Metapocket web servers were used with probe occupancy maps from MD to select an allosteric binding site and propose a pharmacophore model. Next, it was used as a template in virtual screening by UNITY ® module available on SYBYL-X version 2.1.1 at Sigma-Aldrich CPR™ subset of ZINC12 database. The pharmacophore-based virtual screening resulted in the selection of two potential allosteric LcFeSOD compounds with partial pharmacophoric requirements, drug-like properties and commercial availability for enzymatic assays. Communicated by Ramaswamy H. Sarma.

Although the inhibition of acid ceramidase (AC) is known to induce antitumor effects in various cancers, there are few reports in pancreatic cancer, and the underlying mechanisms remain unclear. Moreover, there is currently no safe administration method of AC inhibitor. Here the effects of gene therapy using siRNA and shRNA for AC inhibition with its mechanisms for pancreatic cancer were investigated. The inhibition of AC by siRNA and shRNA using an adeno-associated virus 8 (AAV8) vector had antiproliferative effects by inducing apoptosis in pancreatic cancer cells and xenograft mouse model. Acid ceramidase inhibition elicits mitochondrial dysfunction, reactive oxygen species accumulation, and manganese superoxide dismutase suppression, resulting in apoptosis of pancreatic cancer cells accompanied by ceramide accumulation. These results elucidated the mechanisms underlying the antitumor effect of AC inhibition in pancreatic cancer cells and suggest the potential of the AAV8 vector to inhibit AC as a therapeutic strategy., (© 2021 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Relaxin, an ovarian polypeptide hormone, is found in the hypothalamic paraventricular nucleus (PVN) which is an important central integrative site for the control of blood pressure and sympathetic outflow. The aim of this study was to determine if superoxide anions modulate the effects of relaxin in the PVN. Experiments were performed in normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHRs). Relaxin mRNA and protein, and its receptor, relaxin family peptide receptor 1 (RXFP1) levels in PVN were 3.24, 3.17, and 3.64 times higher in SHRs than in WKY rats, respectively. Microinjection of relaxin-2 into the PVN dose-dependently increased mean arterial pressure (MAP), renal sympathetic nerve activity (RSNA) and heart rate (HR) in both WKY rats and SHRs, although the effects on MAP (16.87 ± 1.99 vs. 8.97 ± 1.48 mm Hg in 100 nmol), RSNA (22.60 ± 2.15 vs. 11.77 ± 1.43 % in 100 nmol) and HR (22.85 ± 3.13 vs. 12.62 ± 2.83 beats/min in 100 nmol) were greater in SHRs. Oxidative stress level was enhanced after relaxin-2 microinjection into the PVN. Pretreatment with superoxide anion scavengers or NADPH oxidase inhibitor blocked, and superoxide dismutase inhibitor potentiated the effects of relaxin-2 on MAP, RSNA and HR. RXFP1 knockdown significantly attenuated the blood pressure of SHRs, and inhibited the increases of atrial natriuretic peptide, brain natriuretic peptide, collagen I, collagen III and fibronectin in the heart of SHRs. These results demonstrated that relaxin is expressed in the PVN, and contributes to hypertension and sympathetic overdrive via oxidative stress. Down-regulation of RXFP1 in the PVN could attenuate hypertension and cardiac remodeling., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Using natural products as antioxidant agents has been beneficial to replace synthetic products. Efforts have been made to profile the antioxidant capacities of natural resources, such as medicinal plants. The polyphenol content of Himalayan rhubarb, Rheum emodi wall, was measured and the antioxidant activity was determined using DPPH and ABTS + assay, and the oxidative stress was assessed using SOD enzymatic assay. Five different solvent fractions, n -hexane, n -butanol, ethyl acetate, dichloromethane, and water, were used for screening the antioxidant capacity in effort to determine the optimum extraction solvent. The total phenolic contents for R. emodi fractions ranged from 27.76 to 209.21 mg of gallic acid equivalents (GAE)/g of dry weight. DPPH and ABTS + assay results are presented into IC 50 values, ranged from 21.52 to 2448.79 μg/mL and 90.25 to 1718.05 μg/mL, respectively. The ethyl acetate fraction had the highest antioxidant activity among other fractions. Also, n -butanol and water fractions showed significantly lower IC 50 values than the positive control in DPPH radical scavenging activity. The IC 50 values of SOD assay of fractions ranged from 2.31 to 64.78 μg/mL. A similar result was observed with ethyl acetate fraction showing the highest SOD radical scavenging activity. The study suggests that the ethyl acetate fraction of R. emodi possess the strongest antioxidant activity, thus the most efficient in extracting antioxidant contents. Moreover, a highly significant correlation was shown between total polyphenol content and antioxidant activity screening assays. The compounds related to the antioxidant activity of R. emodi were identified to myricitrin, myricetin 3-galloyl rhamnoside, and myricetin, which have not been reported in studies about R. emodi before.

We present a simple and efficient method based on ultrafiltration high-performance liquid chromatography coupled with a photodiode array detector and electrospray ionization mass spectrometry for the rapid screening and identification of ligands obtainable from the extract of Scutellaria baicalensis. Five major compounds (chrysin-6-C-arabinosyl-8-C-glucoside, chrysin-6-C-glucosyl-8-C-arabinoside, baicalin, oroxylin A-7-O-glucuronide, and wogonoside) were identified as potentially effective inhibitors of lipoxidase and superoxide dismutase. Subsequently, specific binding ligands were separated by high-speed countercurrent chromatography, using ethyl acetate/ethyl alcohol/water acetate (0.1%) (1.0:0.1:1.0, v/v/v) as the solvent system. To the best of our knowledge, this is the first report of S. baicalensis extracts containing potent lipoxidase and superoxide dismutase inhibitors. Our results demonstrate that the systematic isolation of bioactive components from the n-butyl alcohol layer of S. baicalensis guided by ultrafiltration high-performance liquid chromatography coupled with photodiode array detection and electrospray ionization mass spectrometry represents a feasible and efficient technique that could also be employed for the identification and isolation of other enzyme inhibitors., (© 2021 Wiley-VCH GmbH.)

Nanoparticles that functionally mimic the activity of metal-containing enzymes (metallo-nanozymes) are of therapeutic importance for treating various diseases. However, it is still not clear whether such nanozymes can completely substitute the function of natural enzymes in living cells. In this work, we show for the first time that a cerium vanadate (CeVO 4 ) nanozyme can substitute the function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the neuronal cells even when the natural enzyme is down-regulated by specific gene silencing. The nanozyme prevents the mitochondrial damage in SOD1- and SOD2-depleted cells by regulating the superoxide levels and restores the physiological levels of the anti-apoptotic Bcl-2 family proteins. Furthermore, the nanozyme effectively prevents the mitochondrial depolarization, leading to a significant improvement in the cellular levels of ATP under oxidative stress., (© 2020 Wiley-VCH GmbH.)

Neocryptolepine (5-methyl-5H-indolo[2,3-b] quinoline) analogs were synthesized and evaluated in vitro and in vivo for their effect versus Ehrlich ascites carcinoma (EAC). The analogs showed stronger cytotoxic activity against EAC cells than the reference drug. The in vivo evaluation of the target compounds against EAC-induced solid tumor in the female albino Swiss mice revealed a remarkable decrease in the tumor volume (TV) and hepatic lipid peroxidation. A noticeable increase of both superoxide dismutase (SOD) and catalase (CAT) levels was reported ( p < 0.001), which set-forth proof of their antioxidant effect. In addition, the in vitro antioxidant activity of the neocryptolepine analogs was screened out using the DPPH method and showed promising activities activity. The histopathological investigations affirmed that the tested analogs have a remarkable curative effect on solid tumors with minimal side-effect on the liver. The study also includes illustrated mechanism of the antitumor activity at the cell level by flow cytometry. The cell cycle analysis showed that the neocryptolepine analogs extensively increase the aggregation of tumor cells in three phases of the cell cycle (G0/G1, S and G2/M) with the emergence of a hypo-diploid DNA content peak (sub-G1) in the cell cycle experiments, which is a clear-cut for the apoptotic cell population. Furthermore, the immunological study manifested a significant elevation in splenic lymphocyte count ( p < 0.001) with the elevation of the responsiveness of lymphocytes to phytohemagglutinin (PHA). These results indicate that these naturally-based neocryptolepine alkaloids exhibit marked antitumor activity in vivo and represent an important lead in the development of natural-based anticancer drugs.

Cancer chemotherapeutic drugs exert cytotoxic effects by modulating intracellular reactive oxygen species (ROS) levels. However, whether ROS modulates the efficacy of targeted therapeutics remains poorly understood. Previously, we reported that upregulation of the anti-apoptotic protein, BCL-X L , by KRAS activating mutations was a potential target for KRAS-mutant colorectal cancer (CRC) treatment. Here, we demonstrated that the BCL-X L targeting agent, ABT-263, increased intracellular ROS levels and targeting antioxidant pathways augmented the therapeutic efficacy of this BH3 mimetic. ABT-263 induced expression of genes associated with ROS response and increased intracellular ROS levels by enhancing mitochondrial superoxide generation. The superoxide dismutase inhibitor, 2-methoxyestradiol (2-ME), exhibited synergism with ABT-263 in KRAS-mutant CRC cell lines. This synergistic effect was attributed to the inhibition of mTOR-dependent translation of the anti-apoptotic MCL-1 protein via caspase 3-mediated cleavage of AKT and S6K. In addition, combination treatment of ABT-263 and 2-ME demonstrated a synergistic effect in in vivo patient-derived xenografts harboring KRAS mutations. Our data suggest a novel role for ROS in BH3 mimetic-based targeted therapy and provide a novel strategy for treatment of CRC patients with KRAS mutations., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: We previously synthesized two DNA intercalative Pyrimido[4',5':4,5]thieno(2,3-b) quinolines (PTQ), 9-hydroxy-4-(3-diethylaminopropylamino)pyrimido[4',5':4,5]thieno(2,3-b) quinolines (Hydroxy- DPTQ) and 8-methoxy-4-(3-diethylaminopropylamino) pyrimido[4',5':4,5]thieno(2,3-b) quinolines (Methoxy-DPTQ), and reported their cytotoxicity against cancer cell lines., Methods: In the present study, we sought to analyze the antitumor activity of Hydroxy-DPTQ and Methoxy-DPTQ on Ehrlich's ascites carcinoma in vivo models, along with other pharmacological activities and toxicity., Results: In this study, both the test molecules studied possess potent in vivo antitumor activity without any hematological, biochemical or nephrotoxicity. Significant tumor regression was observed after treatment with both the test molecules, which is suggested by the decrease in the bodyweight of tumour-bearing mice. Mean survival time of mice with tumor was increased from 16 days to 25 and 29 days after 40 and 80 mg/kg Hydroxy- DPTQ treatment, respectively, with a similar result for Methoxy-DPTQ. A dose-dependent increase in lifespan up to 80-85% was also displayed by both Hydroxy-DPTQ and Methoxy-DPTQ. Reduction in the tumor volume of mice, upon treatment with molecules also confirmed their antitumor activity. These molecules also exhibited pharmacological activities such as antioxidant, anti-inflammatory and analgesic activities. Administration of Hydroxy-DPTQ and Methoxy-DPTQ not only reduced the level of lipid peroxidation in tumor bearing mice but also restored the superoxide dismutase, glutathione, and catalase levels to normal, substantiating the antioxidant property. Also, treatment of Hydroxy-DPTQ and Methoxy-DPTQ inhibited the pain to approximately 60-80% and 19-33%, respectively. Further, the treatment with Hydroxy-DPTQ and Methoxy-DPTQ reversed the abnormality in the RBC, WBC and haemoglobin levels, and gentamicin induced nephrotoxicity., Conclusion: Hydroxy-DPTQ and Methoxy-DPTQ are good antitumor molecules with pharmacological properties., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Di-2-ethylhexyl phthalate (DEHP) has been widely used as a plasticizer in industry and can affect memory; however, the underlying mechanism remains unclear. In the present study, mouse HT22 cells, an immortalized hippocampal neuronal cell line, was utilized as an in vitro model. We showed that DEHP dramatically inhibited cell viability and increased lactate dehydrogenase (LDH) release from the cells in a dose-dependent manner, suggesting that DEHP could cause cytotoxicity of mouse HT22 cells. The protein levels of cleaved Caspase-8, cleaved Caspase-3, and Bax markedly increased in the DEHP-treated cells, whereas there was a significant decrease in the Bcl-2 protein level, implying that DEHP could induce apoptosis of mouse HT22 cells. DEHP exposure significantly increased the content of malondialdehyde, whereas it markedly decreased the level of glutathione and the activities of glutathione peroxidase and superoxide dismutase, suggesting that DEHP induced oxidative stress of the cells. Compared with the DEHP-treated group, the inhibition of cell viability and the release of LDH were rescued in the N -acetyl-l-cysteine plus DEHP group. Furthermore, inhibition of oxidative stress could rescue the induction of apoptosis by DEHP. Collectively, our results indicated that DEHP could induce apoptosis of mouse HT22 cells via oxidative stress.

It is well known that the disruption of the mitochondrial respiratory components prolongs lifespan in many species. The mitochondrial stress response can lead to an increased survival rate through the restoration of the cellular homeostasis. Therefore, developing pharmacological interventions that induce mitochondrial stress response may be desirable to delay the onset of age-related diseases and promote a healthy life. In this study, we present chemical compounds, revealed by systematic screening of chemical libraries, which inhibit mitochondrial ATP synthesis in mammalian cells. Our study demonstrates that these compounds alter the body length and promote the oxidative stress response which leads to an increased longevity in Caenorhabditis elegans. Thus, our study identifies chemical compounds that may have potential therapeutic applications through affecting the mitochondrial function., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Structural modification of active natural compoundswhichwereoriginated fromTraditional Chinese Medicine (TCM) have showedgreat advantagesin thedevelopmentof new drugs. In TCM, "Huangqin-Huanglian" is a classic "medicine couple"thathas been used to treat intestinal diseases for thousands ofyears, while baicalinand berberine are the major active compoundsof Huangqin and Huanglianrespectively. Based onthis"medicine couple",wedesignedand synthesizeda newbaicalin and berberine hybrid compound (BBH).Its molecular structure wasconfirmedby spectroscopy.The antibacterial activity of BBH was detected in vitro.Results indicatedthat the new hybrid compound exhibited the best antibacterial activity forproteobacteria as compared with its original synthetic materials (baicalin andberberine). In vivo, the effect of BBHon ulcerative colitiswas alsoinvestigated.BBH treatment significantly ameliorated the disease symptoms andpreventedthe colon damage of ulcerative colitis. Furthermore, BBH showed asignificant anti-inflammatory effect through regulating activities of SOD, MPOandexpressions of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in colontissue. Data also suggested that BBH was more superior than baicalin and berberine inameliorating colonic damage. This indicated that the new hybrid compound BBHshowed enhanced efficacy in treating ulcerative colitis., (Copyright © 2020. Published by Elsevier Ltd.)

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic and fibrotic lung disease of unknown causes. Given the crucial role of oxidative stress in the progression of IPF, antioxidant therapy may be speculated to be an efficient therapeutic approach. Therefore, the present study aimed to evaluate the protective effects of Crocin as a potent, natural antioxidant against Bleomycin-induced PF in male Wistar rats., Methods: Forty male Wistar rats were randomly divided into four groups. Group 1 received intratracheal saline on day 7 and oral gavage of saline for 28 days. Group 2 received a single dose of Bleomycin on day 7 and oral gavage of saline for 28 days. Groups 3 received a single dose of Bleomycin on day 7, accompanied with oral administration of Crocin for 28 days. Group 4 orally received Crocin for 28 days. Finally, the lungs were removed for measuring the biochemical and histopathological markers., Results: The results showed that Crocin therapy remarkably decreased TNF-α, MDA and NO levels in the lungs of Bleomycin-exposed rats. Furthermore, a significant increase was seen in lung GSH content, catalase, and GPx activities in the Crocin/Bleomycin-treated group as compared with Bleomycin-treated group. However, Crocin could not markedly change the lung index and SOD activity. Histopathological changes, fibrosis and hydroxyproline content of lungs also significantly decreased by Crocin therapy in the Crocin/Bleomycin-treated group., Conclusion: In sum, Crocin therapy could modulate biochemical and histological changes induced by Bleomycin; therefore, it might be considered as an effective therapeutic approach against IPF.

Acute myeloid leukemia (AML) is a difficult therapeutic hematological tumor. It is urgent to find a non-toxic natural drug to treat AML. Herein, the selenium nanoparticles (SeNPs) embedded in nanotubes consisted of triple helix β-(1, 3)-d-glucan (BFP) from the black fungus that were wrapped to form stable inclusion complex BFP-Se, which was self-assembled and exhibited high stability in water. In vitro, the BFP-Se significantly inhibited the proliferation of AML cells and increased the cytotoxicity on AML cells. On single-cell levels, the U937 cells were gradually swelled and lysed with BFP-Se treatment on optofluidics chips. Further, the blood and bone marrow analysis indicated the anti-leukemia effects of BFP-Se in vivo. Moreover, BFP-Se increased the total antioxidant capacity of AML cells and decreased the expression of c-Jun activation domain-binding protein 1 and thioredoxin 1. Our results suggest that this biocompatible polysaccharide nanotube containing Se nanoparticles would provide a novel strategy for AML therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Notopterol (NOT) is a major bioactive ingredient extracted from the rhizomes of either Notopterygium incisum Ting ex H. T. Chang or N. forbesii Boiss (Qianghuo in Chinese), a botanical drug that was adopted as a traditional Chinese medicine. NOT is suggested to show analgesic and anti-inflammatory effects in clinical practice. The inhibitory effects of NOT on human cytochrome P450 enzymes were investigated in the present study. Our results indicate that NOT inhibited the activity of CYP2D6 in a time-, concentration- and NADPH-dependent manner. The values of K I and k inact were 10.8 μM and 0.62 min -1 , respectively. The calculated k obs at 10 μM was 0.29 min -1 , above the 0.02 min -1 risk level. After incubation with NOT at 10 μM for 9 min, approximately 92% of CYP2D6 activity was inhibited. Such loss of enzyme activity was not restored through dialysis, which indicates that the observed enzyme inhibition was irreversible. Partition ratio of the inactivation was approximately 29. Quinidine, a competitive CYP2D6 inhibitor, demonstrated protection on enzymes against the NOT-induced inactivation, but such protection was not found in incubation systems fortified with glutathione or catalase/superoxide dismutase. Additionally, CYP3A4 was observed to function as an enzyme mainly involved in the biotransformation of NOT. Taken together, these findings indicate that NOT served as a mechanism-based inactivator of CYP2D6, meanwhile, those observed effects may induce the latent drug-drug interactions. The metabolic activation of NOT may be the key to trigger the inactivation of the enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Pure compounds belonging to phenolic family were studied for their biological potential such as 5,8-dihydroxy-1,4-naphthoquinone (M1), rutin hydrate (M2), 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone (M3), taxifolin (M4), myricetin (M5), plumbagin (M6), silibinin (M7), dihydromyricetin (M8), shikonin (M9), quercetin 3-β-D-glucoside (M10), (±)-taxifolin hydrate (M11), cardamonin (M12),(-)-epicatechin (M13), 9-chloro-10-hydroxy-anthracene-1,4-dione (M14), 9-chloro-10-hydroxy-2,3-dimethyl-anthracene-1,4-dione (M15), 2-chloro-3-(2-hydroxy-5-methylanilino)-1,4-naphthoquinone (M16), 2-chloro-3-(4-hydroxy-phenylamino)-(1,4) naphthoquinone (M17), 2-chloro-3-(3,5-di-tert-butyl-4-hydroxy-phenyl)-(1,4)-naphtoquinone (M18), and myricitrin dihydrate (M19). These molecules were chosen based on two reasons; the limited or total absence of their exploitation in several studied activities and the use of other tests for the same activity. The evaluation of the in vitro anti-acetyl-cholinesterase (AChE), anti-5-lipoxygenase (5-LOX), anti-xanthine oxidase (XOD), anti-alpha glucosidase, anti-superoxide dismutase (SOD), anti-oxidant (DPPH (1, 1-diphenyl-2-picrylhydrazyl) and ABTS (2, 2- azinobis-3-ethylbenzothiazoline-6-sulphonate)), and anticancer activities of mentioned 19 molecules was explored during this work. M3, M14, M15, M16, M17, M18, M19 were exploited for the first time for such purposes. Tested compounds were shown to have interesting radical scavenging abilities against DPPH radicals, and the highest molecules among them were M19 and M5 (IC50 = 12.0 and 15.5 µM, respectively), and M4, M19 and M2 against ABTS (IC50= 1.9, 4.3 and 4.3 µM, respectively). Moreover, the majority of products showed very important cytotoxic activity since IC50 values were ranging between (IC50= 0.2 µM (M1) and 79 µM (M8)) against HCT116 cell line, and values of IC50= 0.2 µM for M1 against MCF7 cell line. All new molecules (non studied before) were shown to have great cytotoxic effect against both cancer cell lines.Furthermore, molecule M5 was shown to have anti-inflammatory potential via the inhibition of 5-LOX enzyme (65% at 100 µM). In addition, M19 showed important anti XOD activity with 47% of inhibition at 100 µM. Also, it has been found that compound M3 had the best anti alpha glucosidase activity with 43.8 % of inhibition at 100 µM, the highest anti-AChE effect (IC50= 14.5 µM), and the best effect towards SOD (IC50= 10.0 µM).  A structure-activity relationship study was also performed.

Globally, more antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance (AMR). The development of novel ionophores, a class of antimicrobials used exclusively in animals, holds promise as a strategy to replace or reduce essential human antimicrobials in veterinary practice. PBT2 is a zinc ionophore with recently demonstrated antibacterial activity against several Gram-positive pathogens, although the underlying mechanism of action is unknown. Here, we investigated the bactericidal mechanism of PBT2 in the bovine mastitis-causing pathogen, Streptococcus uberis In this work, we show that PBT2 functions as a Zn 2+ /H + ionophore, exchanging extracellular zinc for intracellular protons in an electroneutral process that leads to cellular zinc accumulation. Zinc accumulation occurs concomitantly with manganese depletion and the production of reactive oxygen species (ROS). PBT2 inhibits the activity of the manganese-dependent superoxide dismutase, SodA, thereby impairing oxidative stress protection. We propose that PBT2-mediated intracellular zinc toxicity in S. uberis leads to lethality through multiple bactericidal mechanisms: the production of toxic ROS and the impairment of manganese-dependent antioxidant functions. Collectively, these data show that PBT2 represents a new class of antibacterial ionophores capable of targeting bacterial metal ion homeostasis and cellular redox balance. We propose that this novel and multitarget mechanism of PBT2 makes the development of cross-resistance to medically important antimicrobials unlikely. IMPORTANCE More antimicrobials are used in food-producing animals than in humans, and the extensive use of medically important human antimicrobials poses a significant public health threat in the face of rising antimicrobial resistance. Therefore, the elimination of antimicrobial crossover between human and veterinary medicine is of great interest. Unfortunately, the development of new antimicrobials is an expensive high-risk process fraught with difficulties. The repurposing of chemical agents provides a solution to this problem, and while many have not been originally developed as antimicrobials, they have been proven safe in clinical trials. PBT2, a zinc ionophore, is an experimental therapeutic that met safety criteria but failed efficacy checkpoints against both Alzheimer's and Huntington's diseases. It was recently found that PBT2 possessed potent antimicrobial activity, although the mechanism of bacterial cell death is unresolved. In this body of work, we show that PBT2 has multiple mechanisms of antimicrobial action, making the development of PBT2 resistance unlikely., (Copyright © 2020 Harbison-Price et al.)

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5' UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.

Objective: The aim of this study was to clarify the potential effect of zoledronic acid on alleviating oxidative stress and promoting bone marrow mesenchymal stem cells (BMSCs) osteogenesis through the SIRT3/SOD2 pathway, thus alleviating the progression of osteoporosis., Materials and Methods: Relative expression levels of osteogenesis-related genes (ALP, RUNX2, and Bglap) were determined. Meanwhile, ALP activity and capacity of mineralization in BMSCs treated with different doses of zoledronic acid were measured. Subsequently, viability and ROS level in H2O2-induced BMSCs influenced by zoledronic acid treatment were assessed. The regulatory effect of zoledronic acid on the SIRT3/SOD2 pathway was detected by Western blot. Furthermore, the involvement of the SIRT3/SOD2 pathway in zoledronic acid-mediated BMSCs osteogenesis was evaluated., Results: Zoledronic acid treatment significantly up-regulated the levels of ALP, RUNX2, and Bglap. Meanwhile, it improved ALP activity and capacity of mineralization in BMSCs dose-dependently. H2O2 induction markedly suppressed viability and enhanced ROS level in BMSCs, which were reversed by zoledronic acid treatment. Besides, zoledronic acid protected H2O2-induced SIRT3 down-regulation and AC-SOD2/SOD2 up-regulation in BMSCs. In addition, silence of SIRT3 reversed the protective effects of zoledronic acid on osteogenesis of BMSCs., Conclusions: Zoledronic acid alleviates the progression of osteoporosis. Meanwhile, it accelerates BMSCs osteogenesis by inhibiting oxidative stress via the SIRT3/SOD2 pathway.

Aim and Objective: Humans continuously use pesticides in the field to control the pest population and weeds for considerable agricultural productivity. Side-by species like grazinganimals, insects and other species are adversely affected by or become resistant to pesticides. Insects, birds and cattle are highly abundant dwellers of the agriculture-field and represent three distinct phyla having versatile physiological features. Besides higher agricultural-productivity, protection to several species will maintain ecological/environmental balance. Studies on the effect of widely used pesticides on their DNA-stability and important enzymatic-activities are insufficient., Materials and Methods: Antioxidant-activity (Superoxide-dismutase; SOD/Catalase- by gelzymogram- assay) and DNA-stability (fragmentation-assay) in hepatic/gut tissues were studied after in vitro exposure of Chlorpyrifos, Fenvalerate, Nimbecidine or Azadirachtin to goat/cow/poultry-hen/insect., Results: In general, all pesticides were found to impair enzymatic-activities. However, lower organisms were affected more than higher vertebrates by azadirachtin-treatment. DNA fragmentation was found more in insects/poultry-birds than that of the cattle in hepatic/gut tissues. Inversely, toxicity/antioxidant marker-enzymes were more responsive in insect gut-tissues. However, mitochondrialtoxicity revealed variable effects on different species. It has been noticed that chlorpyrifos is the most toxic pesticide, followed by Fenvalerate/Nimbecidine (Azadirachtin, AZT). Nevertheless, AZT revealed its higher DNA-destabilizing effects on the field-insects as compared to the other animals., Conclusion: Field-insects are highly integrated into the ecosystem and the local bio-geo-chemical cycle, which may be impaired. Pesticides may have toxic effects on higher vertebrates and may sustain in the soil after being metabolized into their different derivatives. Some of the sensitive biochemical parameters of this organism may be used as a biomarker for pesticide toxicity., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Applying the single-cell gel electrophoresis (comet) assay, we show that the widely used organophosphorus pesticide malathion is cytotoxic, genotoxic, and induces oxidative stress in human lymphocytes., Competing Interests: Declaration of Competing Interest None declared., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The cytotoxic activity of the pyridazin-3-one derivative LCS-1 was previously suggested to be due to the inhibition of superoxide dismutase 1 (SOD1). However, no direct evidence was provided that LCS-1 inhibits SOD1 within cells., Methods: In this study, we investigated the cytotoxic activity of LCS-1 against bloodstream forms of Trypanosoma brucei , a protozoan parasite that does not express copper/zinc-containing SOD1, but an iron-containing superoxide dismutase (FeSOD)., Results: At 250 µM, LCS-1 did not inhibit the activity of FeSOD in cell lysates of bloodstream forms of T. brucei , confirming that the compound is a specific inhibitor of SOD1. However, LCS-1 displayed substantial trypanocidal activity with a minimum inhibitory concentration of 10 µM and a half-maximal effective concentration of 1.36 µM, indicating that the cytotoxic action of the compound cannot solely be due to inhibition of SOD1., Conclusion: The results of this study is an important finding as it shows that LCS-1 has more than one cytotoxic mode of action., Competing Interests: The authors declare no conflict of interests., (© Georg Thieme Verlag KG Stuttgart · New York.)

Leishmaniasis is considered as one of the major neglected tropical diseases due to its magnitude and wide geographic distribution. Leishmania braziliensis, responsible for cutaneous leishmaniasis, is the most prevalent species in Brazil. Superoxide dismutase (SOD) belongs to the antioxidant pathway of the parasites and human host. Despite the differences between SOD of Leishmania braziliensis and human make this enzyme a promising target for drug development efforts. No medicinal chemistry effort has been made to identify LbSOD inhibitors. Herein, we show that thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) can be employed as primary and secondary screens to achieve this goal. Moreover, we show that thiazole derivatives bind to LbSOD with micromolar affinity.

Computational techniques are widely used to reduce costs associated with new drug development with the ability to bind a specific molecular target. These studies need a Brookhaven protein data bank structure sample of the enzyme interaction with an inhibitor of adequate size. In this context, a new computational methodology is postulated to be used when there are no published samples fulfilling this requirements. In this study, 7 compounds, which showed anti-T. cruzi, L. donovani and L. infantum properties, and proved to be inhibitors of their Fe-SOD enzymes, have been theoretically evaluated against related parasites Fe-SOD enzymes, which have been proposed as targets for antiparasitic drugs. This methodology may be applied to similar cases and also to generate starting structures to be used with different CADD methods., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Leishmaniasis is a widespread neglected tropical disease complex that is responsible of one million new cases per year. Current treatments are outdated and pose many problems that new drugs need to overcome. With the goal of developing new, safe, and affordable drugs, we have studied the in vitro activity of 12 different 5-nitroindazole derivatives that showed previous activity against different strains of Trypanosoma cruzi in a previous work. T. cruzi belongs to the same family as Leishmania spp., and treatments for the disease it produces also needs renewal. Among the derivatives tested, compounds 1, 2, 9, 10, 11, and 12 showed low J774.2 macrophage toxicity, while their effect against both intracellular and extracellular forms of the studied parasites was higher than the ones found for the reference drug Meglumine Antimoniate (Glucantime®). In addition, their Fe-SOD inhibitory effect, the infection rates, metabolite alteration, and mitochondrial membrane potential of the parasites treated with the selected drugs were studied in order to gain insights into the action mechanism, and the results of these tests were more promising than those found with glucantime, as the leishmanicidal effect of these new drug candidates was higher. The promising results are encouraging to test these derivatives in more complex studies, such as in vivo studies and other experiments that could find out the exact mechanism of action., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Effect of metal oxide nanoparticles on calli of two wheat varieties: Parabola (stress tolerant) and Raweta (sensitive) was studied. ZnO induced 10% larger membrane damage in Raweta calli. TiO2, Al2O3, and ZrO2 caused nearly 30% greater lactate dehydrogenase leakage for Raweta compared to Parabola. UV-irradiation of samples containing ZnO particles intensified this effect. Membrane lipid peroxidation in ZnO treated Raweta calli was twice as high as in Parabola and further increased after UV-irradiation. TiO2, Al2O3, and ZrO2 nanoparticles caused a 4-fold increase in malondialdehyde concentration in Raweta calli in comparison to Parabola calli. The nanoparticles studied damaged the cellular defense system by inactivating the antioxidative enzymes.

The current chemotherapy against Chagas disease is inadequate and insufficient. A series of ten Mannich base-type derivatives have been synthesized to evaluate their in vitro antichagasic activity. After a preliminary screening, compounds 7 and 9 were subjected to in vivo assays in a murine model. Both compounds caused a substantial decrease in parasitemia in the chronic phase, which was an even better result than that of the reference drug benznidazole. In addition, compound 9 also showed better antichagasic activity during the acute phase. Moreover, metabolite excretion, effect on mitochondrial membrane potential and the inhibition of superoxide dismutase (SOD) studies were also performed to identify their possible mechanism of action. Finally, docking studies proposed a binding mode of the Fe-SOD enzyme similar to our previous series, which validated our design strategy. Therefore, the results suggest that these compounds should be considered for further preclinical evaluation as antichagasic agents., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Oleuropein (OP) is a bioactive compound derived from plants of the genus Oleaceae exhibiting antitumor properties in several human cancers, including non-small-cell lung cancer (NSCLC). Recent evidence suggests that OP has proapoptotic effects on NSCLC cells via the mitochondrial apoptotic pathway. However, the exact molecular mechanisms behind the apoptogenic action of OP in NSCLC are still largely unknown. Glyoxalase 2 (Glo2) is an ancient enzyme belonging to the glyoxalase system involved in the detoxification of glycolysis-derived methylglyoxal. However, emerging evidence suggests that Glo2 may have also nonenzymatic roles in some malignant cells. In the present study, we evaluated whether and how Glo2 participated in the proapoptotic effects of OP in NSCLC A549 cells. Our results indicate that OP is able to induce apoptosis in A549 cells through the upregulation of mitochondrial Glo2 (mGlo2), mediated by the superoxide anion and Akt signaling pathway. Moreover, our data shows that the proapoptotic role of mGlo2, observed following OP exposure, occurs via the interaction of mGlo2 with the proapoptotic Bax protein. Conversely, OP does not alter the behavior of nonmalignant human BEAS-2B cells or mGlo2 expression, thus suggesting a specific anticancer role for this bioactive compound in NSCLC. Our data identify a novel pathway through which OP exerts a proapoptotic effect in NSCLC and suggest, for the first time, a novel, nonenzymatic antiapoptotic role for this ancient enzyme in NSCLC., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper.

Purpose: To investigate the mitochondria-related mechanism of Gynura segetum (GS)-induced apoptosis and the protective effect of phosphocreatine (PCr), a mitochondrial respiration regulator. Methods: First, the mechanism was explored in human hepatocyte cell line. The mitochondrial oxidative stress was determined by fluorescence assay. The level of sirtuin 3 (SIRT3), acetylated superoxide dismutase 2 (Ac-SOD2), SOD2, and apoptosis were detected by Western blotting. Mito-TEMPO and cell lines of viral vector-mediated overexpression of SIRT3 and SIRT3 H248Y were used to further verify the mechanism of GS-induced apoptosis. GS-induced liver injury mice models were built by GS through intragastric administration and interfered by PCr through intraperitoneal injection. A total of 30 C57BL/6J mice were assigned to 5 groups and treated with either saline, PCr (100 mg/kg), GS (30 g/kg), or PCr (50 or 100 mg/kg)+GS (30 g/kg). Liver hematoxylin and eosin (HE) staining, immunohistochemical analysis, and blood biochemical evaluation were performed. Results: GS induced hepatocyte apoptosis and elevated levels of mitochondrial ROS in L-02 cells. The expression of SIRT3 was decreased. Downregulation of SIRT3 was associated with increased levels of Ac-SOD2, which is the inactivated enzymatic form of SOD2. Conversely, when overexpressing SIRT3 in GS-treated cells, SOD2 activity was restored, and mitochondrial ROS levels and hepatocyte apoptosis declined. Upon administration of PCr to GS-treated cells, they exhibited a significant upregulation of SIRT3 and were protected against apoptosis. In animal experiments, serum ALT level and mitochondrial ROS of the mice treated with GS and 50 mg/kg PCr were significantly attenuated compared with only GS treated. The changes in SIRT3 expression were also consistent with the in vitro results. In addition, immunohistochemical analysis of the mouse liver showed that Ac-SOD2 was decreased in the PCr and GS co-treated group compared with GS treated group. Conclusion: GS caused liver injury by dysregulating mitochondrial ROS generation via a SIRT3-SOD2 pathway. PCr is a potential agent to treat GS-induced liver injury by mitochondrial protection., Competing Interests: The authors report no conflicts of interest in this work.

Mitochondria are major sites of energy metabolism that influence numerous cellular events, including immunity and cancer development. Previously, we reported that the mitochondrion-specific antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), has dual roles in early- and late-carcinogenesis stages. However, how defective MnSOD impacts the chain of events that lead to cell transformation in pathologically normal epidermal cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD leading to mitochondrial injury and mitophagy. In keratinocytes, exposure to UVB radiation decreased mitochondrial oxidative phosphorylation, increased glycolysis and the expression of autophagy-related genes, and enhanced AKT Ser/Thr kinase (AKT) phosphorylation and cell growth. Interestingly, UVB initiated a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the mTOR complex 2 (mTORC2) pathway. Knockdown of rictor but not raptor abrogated UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that ROS-mediated mTOC2 activation in mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with the MnSOD mimic MnTnBuOE-2-PyP 5+ (MnP) attenuates mTORC2 activation and suppresses UVB-induced mitophagy. UVB radiation exposure also increased cell growth as assessed by soft-agar colony survival and cell growth assays, and pretreatment with MnP or the known autophagy inhibitor 3-methyladenine abrogated UVB-induced cell growth. These results indicate that MnSOD is a major redox regulator that maintains mitochondrial health and show that UVB-mediated MnSOD inactivation promotes mitophagy and thereby prevents accumulation of damaged mitochondria., (© 2019 Dhar et al.)

Despite an increase in the survival rate of patients with cancer owing to the use of current chemotherapeutic agents, adverse effects of cancer therapies remain a concern. Combination therapies have been developed to increase efficacy, reduce adverse effects, and overcome drug resistance. Genipin is a natural product derived from Gardenia jasminoides, which has been associated with anti-inflammatory, anti-angiogenic, and anti-proliferative effects; hypertension; and anti-ischemic brain injuries. However, the enhancement of oxaliplatin sensitivity by genipin remains unexplored. Our study showed that a combination of genipin and oxaliplatin exerts synergistic antitumor effects in vitro and in vivo in colorectal cancer cell lines through the reactive oxygen species (ROS)/endoplasmic reticulum (ER) stress/BIM pathway. Importantly, the combination did not affect normal colon cells. BIM knockdown markedly inhibited apoptosis induced by the combination. In addition, genipin induced ROS by inhibiting superoxide dismutase 3 activity. These findings suggest that genipin may be a novel agent for increasing the sensitivity of oxaliplatin against colorectal cancer. The combination of oxaliplatin and genipin hold significant therapeutic potential with minimal adverse effects., (©2019 American Association for Cancer Research.)

Biogenic magnetic nanoparticles (BMNPs) can be formed by numerous microorganisms. However, the significance of their formation and their possible functions have not been explored in detail. To explore a possible function of Fe 3 O 4 NPs in Burkholderia sp. strain YN01, we investigated their catalytic abilities in the elimination of intracellular reactive oxygen species (ROS). Changes in ROS content under different conditions were assessed and showed that low oxygen and high iron concentrations in the growth medium promoted ROS production. However, the levels of ROS gradually decreased with BMNP formation, suggesting that these particles possess intrinsic superoxide dismutase (SOD)-like activity and catalase (CAT)-like activity, as proven in this study. To ensure that the observed ROS decrease was not due to antioxidase overexpression caused by the oxidative stress response, SOD and CAT were inhibited in vivo to analyse the ROS variation and BMNP yield under microoxic and high-iron conditions respectively. The results demonstrated that the formation of these intracellular iron nanoparticles was required for the efficient scavenging of excess ROS, which was dependent on their antioxidase-like properties. This result reveals a novel physiological function of biogenic intracellular Fe 3 O 4 nanoparticles., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Isoquercitrin (ISO), one of phytochemical isolated from aerial parts of Aster yomena, has been reported to have antifungal activity. However, the synergistic effect and the mechanism of ISO in combination with conventional antifungal agents are poorly understood. Therefore, synergistic antifungal effect between ISO and conventional antifungal agents was investigated. ISO at non-antifungal concentration interacts synergistically with amphotericin B (AMB) and fluconazole (FLC), but the combination with flucytosine (5-FC) showed no interaction. ISO disrupted an antioxidant system by inhibiting the activity of superoxide dismutase. This redox imbalance was shown to induce intracellular reactive oxygen species (ROS) accumulation and oxidative stress. ISO combined with FLC caused metacaspase activation and DNA condensation, markers of apoptosis, higher than the combination with ISO/AMB. In contrast, ISO with AMB synergistically stimulated membrane permeabilization compared to ISO/FLC. Scavenging ROS consequently reduced the synergy-induced apoptosis and membrane permeabilization, indicating combinations induced ROS were associated with the synergy effect of ISO. In conclusion, AMB and FLC enhanced the antifungal potency of ISO through oxidative stress when used in synergy. © 2018 IUBMB Life, 71(1):283-292, 2019., (© 2018 International Union of Biochemistry and Molecular Biology.)

FlowNMR has the aim of continuously monitoring processes that occur in conditions that are not compatible with being carried out within a closed tube. However, it is sample intensive and not suitable for samples, such as proteins or living cells, that are often available in limited volumes and possibly low concentrations. We here propose a dialysis-based modification of a commercial flowNMR setup that allows for recycling the medium while confining the sample (proteins and cells) within the active volume of the tube. This approach is demonstrated in the specific cases of in-cell NMR and protein-based ligand studies., (Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to investigate the in vitro effect of an antifungal fraction obtained from Jacquinia macrocarpa plant (JmAF) in the generation of reactive oxygen species (ROS) and the activity of the catalase (CAT) and superoxide dismutase (SOD) enzymes from Fusarium verticillioides , as well as their influence in the viability of the fungus spores. The compounds present in the JmAF were determined by gas chromatography/quadrupole time-of-flight mass spectrometry (GC/QTOF-MS). The effect of the exposition to JmAF on the generation of ROS, as well as in the CAT and SOD activities in F. verticillioides , was determined. The main compounds detected were γ-sitosterol, stephamiersine, betulinol and oleic acid. JmAF showed very high ability in inhibiting the spore viability of F. verticillioides , and their capacity to cause oxidative stress by induction of ROS production. JmAF induced the highest ROS concentration and also inhibited CAT and SOD activities. The results obtained in this study indicate that JmAF is worthy of being considered for the fight against phytopathogenic fungi.

The spectrum and efficacy of available antileishmanial drugs is limited. In the present work we evaluated in vitro the antiproliferative activity of 11 compounds based on tetradentate polyamines compounds against three Leishmania species ( L. braziliensis , L. donovani and L. infantum ) and the possible mechanism of action. We identified six compounds ( 3 , 5 , 6 , 7 , 8 and 10 ) effective against all three Leishmania spp both on extracellular and intracellular forms. These six most active leishmanicidal compounds also prevent the infection of host cells. Nevertheless, only compound 7 is targeted against the Leishmania SOD. Meanwhile, on the glucose metabolism the tested compounds have a species-specific effect on Leishmania spp.: L. braziliensis was affected mainly by 10 and 8 , L. donovani by 7 , and L. infantum by 5 and 3 . Finally, the cellular ultrastructure was mainly damaged by 11 in the three Leishmania spp. studied. These identified antileishmania candidates constitute a good alternative treatment and will be further studied.

This study aimed to better understand in situ cumulative effects of anthropogenic stressors on the health of St. Lawrence River (QC, Canada) yellow perch populations using high-throughput transcriptomics and a multi-biological level approach. Fish were collected in the upstream fluvial Lake Saint-François (LSF) with low degree of environmental perturbations; Lake Saint-Louis (LSL) considered having a moderate degree of anthropogenic stressors, and Lake Saint-Pierre (LSP) a sector where the perch population has been severely declining. Morphometric results indicated that fish from the downstream LSP showed lower body condition compared to LSF and LSL. Liver transcriptomic responses were assessed by RNA-sequencing. Two hundred and eighty genes were over-transcribed in LSP perch while 200 genes were under-transcribed compared to LSF and LSL. In LSP fish, genes transcripts related to reproduction, retinol, iron, thyroid hormones, oxidative stress, lipid metabolism and immune functions were among the most abundant suggesting that multiple metabolic and physiological pathways were impacted by environmental stressors at this site. Inhibition of liver superoxide dismutase, catalase and glutathione S-transferase activities were also observed at the cellular level. Overall, identified impacted biological pathways in perch from LSP may help understand the precarious state of this population and identify the factors inhibiting its recovery., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Several studies have demonstrated that the mitochondrial membrane switches from selective to non-selective permeability because of its improved matrix Ca 2+ accumulation and oxidative stress. This process, known as permeability transition, evokes severe dysfunction in mitochondria through the opening of a non-specific pore, whose chemical nature is still under discussion. There are some proposals regarding the components of the pore structure, e.g., the adenine nucleotide translocase and dimers of the F1 Fo-ATP synthase. Our results reveal that Ca 2+ induces oxidative stress, which not only increases lipid peroxidation and ROS generation but also brings about both the collapse of the transmembrane potential and the membrane release of cytochrome c. Additionally, it is shown that Ca 2+ increases the binding of the probe eosin-5-maleimide to adenine nucleotide translocase. Interestingly, these effects are diminished after the addition of ADP. It is suggested that pore opening is caused by the binding of Ca 2+ to the adenine nucleotide translocase.

Circulation of cancer cells in the bloodstream is a vital step for distant metastasis, during which cancer cells are exposed to hemodynamic shear stress (SS). The actions of SS on tumor cells are complicated and not fully understood. We previously reported that fluidic SS was able to promote migration of breast cancer cells by elevating the cellular ROS level. In this study, we further investigated the mechanisms regulating SS-promoted cell migration and identified the role of MnSOD in the related pathway. We found that SS could enhance tumor cell adhesion to extracellular matrix and endothelial monolayer, and MnSOD also regulated this process. Briefly, SS stimulates the generation of mitochondrial superoxide in tumor cells. MnSOD then converts superoxide into hydrogen peroxide, which activates ERK1/2 to promote tumor cell migration and activates FAK to promote tumor cell adhesion. Combining our previous and present studies, we present experimental evidence on the pro-metastatic effects of hemodynamic SS and reveal the underlying mechanism. Our findings provide new insights into the nature of cancer metastasis and the understanding of tumor cell responses to external stresses and have valuable implications for cancer therapy development., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We tested a hypothesis that superoxide, by inducing Sp3, increases renal renin activity, renal angiotensin II type 1 receptor (AT1R) function, and blood pressure (BP) in rats. Group 1 rats were treated with vehicle, saline. Group 2 rats were treated with superoxide dismutase (SOD) inhibitor diethylthiocarbamate (DETC). Group 3 rats were treated with DETC and an SOD mimetic, tempol. Group 4 rats were treated with tempol only. All four groups of rats were treated for 2 wk then anesthetized, and BP was recorded. Thereafter, diuresis and natriuresis in response to AT1R blocker candesartan were determined. When compared with vehicle rats, BP increased in DETC rats. The increased BP in DETC rats decreased with tempol. Diuresis and natriuresis in response to candesartan increased in controls, and this further increased in DETC rats and decreased with tempol. A second set of four groups of rats underwent the same treatment as above and were anesthetized, and their kidneys were obtained for biochemical studies. The levels of superoxide but not hydrogen peroxide increased, whereas SOD activities decreased further in the renal cortical tissues of DETC rats than vehicle rats. These effects were attenuated with tempol in DETC rats. Moreover, tissue renin activity and abundance of membranous AT1R proteins increased more in DETC rats than vehicle rats, and decreased with tempol in DETC rats. Furthermore, the levels of lysine-acetylated, but not serine-phosphorylated, Sp3 increased more in the nuclei of DETC rats than vehicle rats. The increased levels of Sp3 lysine acetylation decreased in DETC rats with tempol. Taken together, our results suggest that superoxide activates renal Sp3 via lysine acetylation increasing renin activity, AT1R function, and BP in rats.

Background Aortic valve sclerosis ( AVS c), the early asymptomatic presentation of calcific aortic valve (AV) disease, affects 25% to 30% of patients aged >65 years. In vitro and ex vivo experiments with antioxidant strategies and antagonists of osteogenic differentiation revealed that AVS c is reversible. In this study, we characterized the underlying changes in the extracellular matrix architecture and valve interstitial cell activation in AVSc and tested in vitro and in vivo the activity of a clinically approved SOD (superoxide dismutase) mimic and redox-active drug MnTnBu OE -2-PyP 5+ ( BMX -001). Methods and Results After receiving informed consent, samples from patients with AVS c, AV stenosis, and controls were collected. Uniaxial mechanical stimulation and in vitro studies on human valve interstitial cells were performed. An angiotensin II chronic infusion model was used to impose AV thickening and remodeling. We characterized extracellular matrix structures by small-angle light scattering, scanning electron microscopy, histology, and mass spectrometry. Diseased human valves showed altered collagen fiber alignment and ultrastructural changes in AVS c, accumulation of oxidized cross-linking products in AV stenosis, and reversible expression of extracellular matrix regulators ex vivo. We demonstrated that MnTnBu OE -2-PyP 5+ inhibits human valve interstitial cell activation and extracellular matrix remodeling in a murine model (C57 BL /6J) of AVS c by electron microscopy and histology. Conclusions AVS c is associated with architectural remodeling despite marginal effects on the mechanical properties in both human and mice. MnTnBu OE -2-PyP 5+ controls AV thickening in a murine model of AVS c. Because this compound has been approved recently for clinical use, this work could shift the focus for the treatment of calcific AV disease, moving from AV stenosis to an earlier presentation ( AVS c) that could be more responsive to medical therapies.

Chagas disease, a neglected tropical disease caused by infection with the protozoan parasite Trypanosoma cruzi, is a potentially life-threatening illness that affects 5-8 million people in Latin America, and more than 10 million people worldwide. It is characterized by an acute phase, which is partly resolved by the immune system, but then develops as a chronic disease without an effective treatment. There is an urgent need for new antiprotozoal agents, as the current standard therapeutic options based on benznidazole and nifurtimox are characterized by limited efficacy, toxicity, and frequent failures in treatment. In vitro and in vivo assays were used to identify some new low-cost 5-nitroindazoles as a potential antichagasic therapeutic alternative. Compound 16 (3-benzyloxy-5-nitro-1-vinyl-1H-indazole) showed improved efficiency and lower toxicity than benznidazole in both in vitro and in vivo experiments, and its trypanocidal activity seems to be related to its effect at the mitochondrial level. Therefore, compound 16 is a promising candidate for the development of a new anti-Chagas agent, and further preclinical evaluation should be considered., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Two tetradentated pyridine-based manganese complexes (Cpd2 and Cpd3) were previously reported to inhibit efficiently the growth of Trypanosoma cruzi in vitro and in vivo. Cpd3 was also shown to be a potent inhibitor of trypanosomal iron superoxide dismutase (Fe-SOD) and its trypanocidal activity linked to the inhibition of this enzyme. Here we investigated the anti-trypanosomal activity of the two compounds against bloodstream forms of Trypanosoma brucei. Both compounds displayed potent trypanocidal activity against T. brucei bloodstream forms with minimum inhibitory concentrations (MICs) and 50% growth inhibition (GI 50 ) values of 1 μM and 0.2-0.3 μM, respectively. Cpd2 and Cpd3 also showed cytotoxicity against HL-60 cells but based on GI 50 values the human cells were 14 and 87 times less sensitive indicating moderate selectivity. In contrast to previous observation, Cpd3 did not inhibit Fe-SOD within trypanosomes and Cpd2 inhibited the enzyme only by 34%. As Fe-SOD together with ornithine decarboxylase play vital roles in the antioxidant defence in bloodstream forms of T. brucei, inhibition of both enzymes should be synergistically. Therefore, the interaction of Cpd2 and Cpd3 with the ornithine decarboxylase inhibitor eflornithine was determined. Both compounds were found in combination with eflornithine to produce only an additive effect. Thus, the observed lack of synergy between Cpd2/Cpd3 and eflornithine can be regarded as further indication that both compounds are not very strong inhibitors of trypanosomal Fe-SOD. Nevertheless, tetradentated pyridine-based manganese complexes are interesting compounds with promising anti-trypanosomal activity., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Schistosomiasis - caused by trematodes from the genus Schistosoma - affects more than 200 million people worldwide. Growing resistance to therapy with praziquantel (PZQ) has encouraged the search for novel treatments against this neglected disease. The compound 7-epiclusianone (7-epi) - isolated from 'bacupari' (the fruit of the Gracinia brasiliensis tree) - has promising activity against Schistosoma mansoni in vitro, damaging the parasite's tegument. However, the target and mechanism of action of 7-epi have not been identified. Here, we examined the possibility that 7-epi harms the tegument by inhibiting parasite superoxide dismutase (SOD), which protects the tegument from damage by reactive oxygen species produced by host immune cells. Molecular docking analysis in silico suggested strong interactions between 7-epi and S. mansoni cytosolic superoxide dismutase (SmCtSOD) at allosteric cavities. Schistosoma mansoni couples were cultivated ex vivo with 12.44-198.96 μm 7-epi for 24 h, and then parasite extracts were tested for lipid peroxidation (as a surrogate for oxidative stress), and SOD activity and expression. Lipid peroxidation levels increased after incubation with concentrations ≥99.48 μm 7-epi, and this compound reduced SOD activity at concentrations ≥24.87 μm. However, contact with 7-epi did not alter SOD expression, by quantitative real-time polymerase chain reaction (qRT-PCR). Our results show that the inhibition of SmCtSOD is partly responsible for the tegument detachment observed after incubation with 7-epi, but is not the only cause of the antiparasitic action of this compound in vitro.

A significant factor in the development of hypertension may be excessive vasoconstriction within the renal medulla. This study therefore investigated the role of superoxide dismutase (SOD) in the regulation of renal medullary and cortical blood perfusion (MBP and CBP, respectively) in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar rats. CBP and MBP were measured before and after intra-renal infusion of the SOD inhibitor, diethyldithio-carbamic acid (DETC). Under basal conditions, mean arterial pressure was significantly greater in SHRSP than Wistar rats, but both MBP and heart rate (HR) were significantly lower in SHRSP relative to Wistar rats (P<0.05, n=7 in both groups). Infusion of DETC (2 mg/kg/min) into the cortico-medullary border area of the kidney significantly decreased MBP in the SHRSPs (by 28+/-3 %, n=7, P<0.05), indicating a greater vasoconstriction within this vascular bed. However, DETC also significantly decreased MBP in Wistar rats to a similar extent (24+/-4 %, n=7, P<0.05). These results suggest that superoxide anions play a significant role in reducing renal vascular compliance within the renal medulla in both normotensive and hypertensive animals, although the responses are not greater in the hypertensive relative to the control animals.

Growing evidences have demonstrated alternative splicing makes great contribution to tumor metastasis since multiple protein isoforms from a single gene that often display different functions in the cell. Human manganese superoxide dismutase (hMnSOD) was revealed dysregulation in progress of tumor metastasis, while the effect of hMnSOD isoforms on metastasis remained unclear. In this study, we showed a novel truncated hMnSOD isoform hMnSOD183, which lacked 39 amino acids compared with hMnSOD222. We expressed two hMnSOD protein isoforms in Escherichia coli, respectively, and found that the MnSOD activity of truncated hMnSOD isoform was especially weaker. In 95-D cells, mRNA levels of hMnSOD variants and MnSOD activity were significantly increased than that in A549 cells. Furthermore, the hMnSODc exhibited lower mRNA level than hMnSODa/b in A549 and 95-D cells. Additionally, the effects of two isoforms were assessed about cell invasion, overexpression of hMnSOD222 but not hMnSOD183 promoted 95-D cells metastasis, and hMnSOD knockdown significantly reduced cells invasive behavior. Overexpression of hMnSOD isoforms also caused changes of metastasis associated proteins, such as up-regulation of MMPs, VEGF and Vimentin and down-regulation of E-cadherin. Moreover, overexpression of hMnSOD183 had weaker effect on metastasis related signaling proteins, such as Akt, JNK and IKKβ, compared to hMnSOD222. In conclusion, our study identified that hMnSOD isoforms induced lung cancer cells invasion through Akt-JNK-IKKβ signaling pathways and the hMnSOD183 isoform played a weaker role than hMnSOD222. Characterization of hMnSOD isoforms is useful for future investigation on metastasis of lung cancer cells., (© 2018 International Federation for Cell Biology.)