Superoxide In Response Research Articles

A Tumor-Specific Cascade-Activating Smart Prodrug System for Enhanced Targeted Therapy.

Developing intelligently targeted drugs with low side effects is urgent for cancer treatment. Toward this goal, a tumor-specific cascade-activating smart prodrug system consisting of a G-quadruplex(G4)-modulated tumor-targeted DNA vehicle and a well-designed cellular stimuli-responsive ligand-drug conjugates (LDCs) is proposed. An original "donor-acceptor" binary fluorescent ligand, with ultrahigh affinity, brightness, and photostability, is engineered to tightly bind G4 structures and significantly improve the nuclease resistance of the DNA vehicle, which serves as a bridge contributing to the construction of the prodrug system, named ApG4/LDCs. Sodium nitroprusside and doxorubicin are loaded into ApG4/LDCs in one pot and generate nitric oxide and superoxide anion in response to cancer cellular environments, which in cascade generates peroxynitrite to cause DNA damage while promoting the self-monitored drug release to achieve enhanced targeted therapy. Such a cascade activation and self-reinforcement process is executed only when the prodrug system targets the tumor tissue followed by cell uptake, showing significant antitumor efficacy and greatly weakening the damage to normal tissues. Given the unique features, the innovative strategy for prodrug design may open a new door to precision disease treatment.

Acori Tatarinowii Rhizoma prevents the fluoxetine-induced multiple-drug resistance of Escherichia coli against antibiotics

BackgroundIn treating depression, the residual anti-depressant in gut interacts with the microbiome, leading to the appearance of multiple drug resistant (MDR) mutants, which poses a challenge for the treatment of infectious complications. Strategy is needed to combat this issue. Acori Tatarinowii Rhizoma (ATR, rhizome of Acorus tatarinowii Schott, Araceae), a traditional Chinese medicine, has been widely used for treatment of neurological disorders and gastrointestinal digestive disease in China. Here, ATR was demonstrated an excellent MDR-preventing effect in fluoxetine-induced Escherichia coli (E. coli). Aim of the studyThis study aimed to reveal the effective role of ATR and its signaling cascades involved in preventing fluoxetine-induced MDR. Materials and methodsThe water extract of ATR was co-applied with sub-minimum inhibitory concentration (100 mg/l) of fluoxetine in E. coli to evaluate its anti-MDR potential. Formation of reactive oxygen species (ROS) and expression of MDR-related genes in bacteria were measured by dichloro-dihydro-fluorescein diacetate assay and real-time PCR, respectively. Two fluorescent dyes, 1-N-phenylnapthylamine and 3,3′-dipropylthiadicarbocyanine were used to analyze the outer membrane permeability and inner membrane depolarization of E. coli. The accumulation of fluoxetine in the treated E. coli was determined via HPLC. The active fraction of ATR was identified. ResultsThe water extract of ATR significantly decreased the number of MDR mutants induced by fluoxetine and had half effective concentrations (EC50) of 55.5 μg/ml and 16.8 μg/ml for chloramphenicol and tetracycline, respectively. ATR robustly reversed the fluoxetine-induced superoxide response and membrane damage in E. coli. In addition, the inclusion of ATR significantly reduced the accumulation of fluoxetine in E. coli. After further fractionation, the polysaccharide of ATR was demonstrated as the fraction with the most significant anti-MDR activity. ConclusionsThis is the first report to investigate the MDR-preventing effect of ATR. The results of this study proposed ATR as an excellent herbal product to prevent MDR issues, as induced by fluoxetine, with the potential to reduce the side effects during the drug therapy of depression.

Genomic organization and transcription of superoxide dismutase genes (sod1, sod2, and sod3b) and response to diazinon toxicity in platyfish (Xiphophorus maculatus) by using SOD enzyme activity

The aim of this study is to determine the effects of 50% of 96 h LC50 (5.25 ppm) diazinon on the expression of superoxide dismutase (SOD) enzyme genes (sod1, sod2, and sod3b) and SOD enzyme activity at the end of 24, 48, 72, and 96 h in platyfish liver and gill tissues. To this end, we determined the tissue-specific distribution of sod1, sod2, and sod3b genes and performed in silico analyses in platyfish (Xiphophorus maculatus). It was determined that malondialdehyde (MDA) level and SOD enzyme activity were increased in the liver [(43.90 EU mg protein−1 (control), 62.45 EU mg protein−1 (24 h), 73.17 EU mg protein−1 (48 h), 82.18 EU mg protein−1 (72 h), 92.93 EU mg protein−1 (96 h)] and gill [(16.44 EU mg protein−1 (control), 33.47 EU mg protein−1 (24 h), 50.38 EU mg protein−1 (48 h), 64.62 EU mg protein−1 (72 h), 74.04 EU mg protein−1 (96 h)] tissues of platyfish exposed to diazinon, while the expression of the sod genes was down-regulated. The tissue-specific distribution of the sod genes varied, with the tissues and the sod genes expression were being predominant in the liver (628.32 in sod1, 637.59 in sod2, 888.5 in sod3b). Thus, the liver was considered a suitable tissue for further gene expression studies. Based on the phylogenetic analyses, platyfish sod genes can be reported to be orthologs of sod/SOD genes from other vertebrates. Identity/similarity analyses supported this determination. Conserved gene synteny proved that there are conserved sod genes in platyfish, zebrafish, and humans.

Functional interactions between NADPH oxidase 5 and actin.

NADPH oxidase 5 (NOX5) is a transmembrane oxidative signaling enzyme which produces superoxide in response to intracellular calcium flux. Increasing evidence indicates that NOX5 is involved in a variety of physiological processes as well as human disease, however, details of NOX5 signaling pathways and targets of NOX5 mediated oxidative modifications remain poorly resolved. Actin dynamics have previously been shown to be modulated by oxidative modification, however, a direct connection to NOX5 expression and activity has not been fully explored. Here we show that NOX5 and actin interact in the cell, and each modulate the activity of the other. Using actin effector molecules jasplakinolide, cytochalasin D and latrunculin A, we show that changes in actin dynamics affect NOX5 superoxide production. In tandem, NOX5 oxidatively modifies actin, and shifts the ratio of filamentous to monomeric actin. Finally, we show that knockdown of NOX5 in the pancreatic cancer cell line PSN-1 impairs cell migration. Together our findings indicate an important link between actin dynamics and oxidative signaling through NOX5.

Natural variation in macrophage polarization and function impact pneumocyte senescence and susceptibility to fibrosis.

Radiation-induced pulmonary fibrosis (RIPF), a late adverse event of radiation therapy, is characterized by infiltration of inflammatory cells, progressive loss of alveolar structure, secondary to the loss of pneumocytes and accumulation of collagenous extracellular matrix, and senescence of alveolar stem cells. Differential susceptibility to lung injury from radiation and other toxic insults across mouse strains is well described but poorly understood. The accumulation of alternatively activated macrophages (M2) has previously been implicated in the progression of lung fibrosis. Using fibrosis prone strain (C57L), a fibrosis-resistant strain (C3H/HeN), and a strain with intermediate susceptibility (C57BL6/J), we demonstrate that the accumulation of M2 macrophages correlates with the manifestation of fibrosis. A comparison of primary macrophages derived from each strain identified phenotypic and functional differences, including differential expression of NADPH Oxidase 2 and production of superoxide in response to M2 polarization and activation. Further, the sensitivity of primary AECII to senescence after coculture with M2 macrophages was strain dependent and correlated to observations of sensitivity to fibrosis and senescence in vivo. Taken together, these data support that the relative susceptibility of different strains to RIPF is closely related to distinct senescence responses induced through pulmonary M2 macrophages after thoracic irradiation.

Uncovering the source of mitochondrial superoxide in pro-inflammatory macrophages: Insights from immunometabolism

Mitochondrial-derived reactive oxygen species are important as antimicrobial agents and redox signals in pro-inflammatory macrophages. Macrophages produce superoxide in response to the TLR4 ligand LPS. However, the mechanism of LPS-induced superoxide generation is not fully understood. Superoxide is produced at complex I and complex III of the electron transport chain. Production of superoxide at either of these sites is highly dependent on the metabolic state of the cell which is dramatically altered by TLR4-induced metabolic reprogramming. This review will outline how metabolism impacts superoxide production in LPS-activated macrophages downstream of TLR4 signalling and address outstanding questions in this field.

Mediterranean G6PD variant rats are protected from Angiotensin II-induced hypertension and kidney damage, but not from inflammation and arterial stiffness

RationaleEpidemiological studies suggest that individuals in the Mediterranean region with deficiency of glucose-6-phosphate dehydrogenase (G6PD) are less susceptible to cardiovascular diseases. However, our knowledge regarding the effects of G6PD deficiency on pathogenesis of vascular diseases caused by factors, like angiotensin II (Ang-II), which stimulate synthesis of inflammatory cytokines and vascular inflammation, is lacking. Furthermore, to-date the effect of G6PD deficiency on vascular health has been controversial and difficult to experimentally prove due to a lack of good animal model. ObjectiveTo determine the effect of Ang-II-induced hypertension (HTN) and stiffness in a rat model of the Mediterranean G6PD (G6PDS188F) variant and in wild-type (WT) rats. Methods and resultsOur findings revealed that infusion of Ang-II (490 ng/kg/min) elicited less HTN and medial hypertrophy of carotid artery in G6PDS188F than in WT rats. Additionally, Ang-II induced less glomerular and tubular damage in the kidneys – a consequence of elevated pressure – in G6PDS188F than WT rats. However, Ang-II-induced arterial stiffness increased in G6PDS188F and WT rats, and there were no differences between the groups. Mechanistically, we found aorta of G6PDS188F as compared to WT rats produced less sustained contraction and less inositol-1,2,3-phosphate (IP3) and superoxide in response to Ang-II. Furthermore, aorta of G6PDS188F as compared to WT rats expressed lower levels of phosphorylated extracellular-signal regulated kinase (ERK). Interestingly, the aorta of G6PDS188F as compared to WT rats infused with Ang-II transcribed more (50-fold) myosin heavy chain-11 (MYH11) gene, which encodes contractile protein of smooth muscle cell (SMC), and less (2.3-fold) actin-binding Rho-activating gene, which encodes a protein that enhances SMC proliferation. A corresponding increase in MYH11 and Leiomodin-1 (LMOD1) staining was observed in arteries of Ang-II treated G6PDS188F rats. However, G6PD deficiency did not affect the accumulation of CD45+ cells and transcription of genes encoding interleukin-6 and collagen-1a1 by Ang-II. ConclusionsThe G6PDS188F loss-of-function variant found in humans protected rats from Ang-II-induced HTN and kidney damage, but not from vascular inflammation and arterial stiffness.

Identification of protein targets for NADPH oxidase 5 mediated oxidative modification

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to calcium binding. NOX5 has been identified as a key player for a variety of physiological and pathological conditions, yet a comprehensive understanding of its role in the cell has been lacking. Here we present a workflow to identify NOX5 protein partners as well as proteins which become oxidatively modified in a NOX5 dependent manner. These protein candidates can then be assessed for their role in mediating NOX5 dependent signaling in the cell which is involved in cellular differentiation, angiogenesis, and human disease. Investigating these results will allow us to understand how NOX5 carries out its physiological roles as well as provide indications for how dysregulation can lead to the initiation and progression of disease.

Bromodomain Inhibition Is Effective Against Acute Myeloid Leukemia Cells Under Hypoxia and Induces Genome-Dependent Metabolic Perturbations

Inhibition of bromodomain-containing (BRD) proteins showed anti-tumor activity against different subtypes of acute myeloid leukemia (AML; Dawson et al. Nature 2011; Zuber et al. Nature 2011; Dawson et al. Leukemia 2013; Chen et al. Cancer Cell 2014; Gröschel et al. Cell 2014; Zhao et al. Cell Reports 2016). However, epigenetic and functional changes occurring under hypoxic conditions may affect BRD protein activity and the consequences of its inhibition. Moreover, the levels of c-MYC, a main target of bromodomain inhibitors, are decreased at low oxygen concentrations mimicking the bone marrow microenvironment which harbor leukemia stem cells, the final target of successful therapies.We treated AML cell lines representative of diverse genetic background (KG1; MLL-driven: MOLM-13, NOMO-1, NPM1 and DNMT3A mutated: OCI-AML3; t(8;21): Kasumi-1; c-MYC-amplified: HL-60) for 48h with the bromodomain inhibitor GSK1215101A (250 µM or 500 µM) under hypoxic conditions (1% O2). GSK1215101A reduced cell viability in a dose-dependent manner in all the cell lines (15%-35% reduction at 250 µM and 25%-65% reduction at 500 µM) except for HL60, with Kasumi-1 being the most sensitive model. A significant increase of Annexin V+ apoptotic cells was detected in NOMO-1 and Kasumi-1 cells, while OCI-AML3, Kasumi-1, HL-60 and KG-1 showed a marked decrease in cell proliferation with reduction of the percentage of cells in S phase and arrest into G0/G1 phase.To better understand GSK1215101A effects on AML cells, we performed gene expression profiling of actively translated mRNAs isolated by polysome profiling, along with western blot analysis of OCI-AML3, Kasumi-1 (drug-sensitive) and HL-60 (drug-resistant) cell lines after 16 hour of treatment (in order to allow cell adaption to the selective pressure induced by the drug, while avoiding massive cell death). GSK1215101A enforced hypoxia-induced c-MYC downregulation in all the cell lines and induced HIF-1a upregulation specifically in drug-sensitive ones, which was confirmed by immuofluorescence.Moreover, since c-MYC is a known regulator of cancer cell metabolism, we analyzed the metabolic changes induced by GSK1215101A by Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (Metabolon, Inc). GSK1215101A treatment resulted in changes in metabolite levels that are consistent with alterations in energy metabolism and cell component biosynthesis, with marked differences between the OCI-AML3 and Kasumi-1 models. Lactate levels were significantly reduced in hypoxic Kasumi-1 cells after treatment, which may be consistent with a drug-dependent decrease in lactate dehydrogenase activity, while no differences were observed at transcriptional and translational level. Little effects were observed on OCI-AML3 lactate levels. In parallel, OCI-AML3 showed a significant increase of both the reduced (3.9-fold) and oxidized (2.6-fold) forms of glutathione, which plays an important role in antioxidant defense, redox-homeostasis and protein folding. Consistently, the translational profile of OCI-AML3 cells showed enrichment of deregulated genes involved in superoxide metabolic process and response to oxidative stress (NCF1/2, ADNP2, XBP1, HSPA1A/B).Taken together, these data indicate that inhibition of bromodomain-containing proteins is effective against AML cells under hypoxic conditions and offer insights into the drug effects on leukemic cell metabolism, which is highly dependent on the genomic backgrounds. Moreover, the results suggest potential targets for novel combination therapies interfering with metabolic pathways or HIF-1a activity.Supported by: EHA research fellowship award, ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. DisclosuresMartinelli:ARIAD/INCYTE: Consultancy; PFIZER: Consultancy; ROCHE: Consultancy; JOHNSON & JOHNSON: Consultancy; CELGENE: Consultancy; AMGEN: Consultancy.

Nitric oxide and heme-NO stimulate superoxide production by NADPH oxidase 5

Nitric oxide (NO) is a ubiquitous cell signaling molecule which mediates widespread and diverse processes in the cell. These NO dependent effects often involve activation (e.g. NO binding to the heme group of soluble guanylyl cyclase for cGMP production) or inactivation (e.g. S-nitrosation) of protein targets. We studied the effect of NO and heme-NO on the transmembrane signaling enzyme NADPH oxidase 5 (NOX5), a heme protein which produces superoxide in response to increases in intracellular calcium. We found that treatment with NO donors increases NOX5 activity through heme-dependent effects, and that this effect could be recapitulated by the addition of heme-NO. This work adds to our understanding of NOX5 regulation in the cell but also provides a framework for understanding how NO could cause widespread changes in hemeprotein activity based on different affinities for heme v. heme-NO, and helps explain the opposing roles NO plays in activation and inactivation of hemeprotein targets.

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency

AbstractRho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G→A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways.

Dynamic regulation of NADPH oxidase 5 by intracellular heme levels and cellular chaperones

NADPH oxidase 5 (NOX5) is a transmembrane signaling enzyme that produces superoxide in response to elevated cytosolic calcium. In addition to its association with numerous human diseases, NOX5 has recently been discovered to play crucial roles in the immune response and cardiovascular system. Details of NOX5 maturation, and specifically its response to changes in intracellular heme levels have remained unclear. Here we establish an experimental system in mammalian cells that allows us to probe the influence of heme availability on ROS production by NOX5. We identified a mode of dynamic regulatory control over NOX5 activity through modulation of its heme saturation and oligomeric state by intracellular heme levels and Hsp90 binding. This regulatory mechanism allows for fine-tuning and reversible modulation of NOX5 activity in response to stimuli.

Redox-Based Synthetic Biology Enables Electrochemical Detection of the Herbicides Dicamba and Roundup via Rewired Escherichia coli.

Synthetic biology is typically exploited to endow bacterial cells with new biosynthetic capabilities. It can also serve to create "smart" bacteria such as probiotics that detect and treat disease. Here, we show how minimally rewiring the genetic regulation of bacterial cells can enable their ability to recognize and report on chemical herbicides, including those routinely used to clear weeds from gardens and crops. In so doing, we demonstrate how constructs of synthetic biology, in this case redox-based synthetic biology, can serve as a vector for information flow mediating molecular communication between biochemical systems and microelectronics. We coupled the common genetic reporter, β-galactosidase, with the E. coli superoxide response regulon promoter pSoxS, for detection of the herbicides dicamba and Roundup. Both herbicides activated our genetic construct in a concentration dependent manner. Results indicate robust detection using spectrophotometry, via the Miller assay, and electrochemistry using the enzymatic cleavage of 4-aminophenyl β-d-galactopyranoside into the redox active molecule p-aminophenol. We found that environmental components, in particular, the availability of glucose, are important factors for the cellular detection of dicamba. Importantly, both herbicides were detected at concentrations relevant for aquatic toxicity.

Redox regulation in human endothelial cells: a critical role for the glycocalyx in mechanotransduction of fluid shear stress

Redox status in endothelial cells (EC) is focally regulated by fluid shear stress (FSS) forces acting on the vessel lumen. Notably, endogenous antioxidant genes targeted by the redox sensitive transcription factor nuclear factor E2-related factor 2 (Nrf2) are upregulated by unidirectional (US) FSS. The glycocalyx (GCX), a meshwork of glycosaminoglycans, is prominent in atheroprotected EC but scarce in atherosusceptible EC regions, and a prerequisite for FSS induced nitric oxide and reactive oxygen species generation. In this study, we investigate the role of the GCX as a modulator of redox signaling in human EC (HUVEC) subjected to either US (15 dynes/cm2) or oscillatory (±5 dynes/cm2, 1 Hz) shear for 48 h in microfluidic slides (Ibidi GmbH). The sialic acid (SA) component of the GCX and expression of heme oxygenase-1 (HO-1) were differentially regulated by US and OS compared to static cultures. Removal of SA with neuraminidase (2U/ml, 30 min) prior to US exposure abrogated HO-1 induction and enhanced mitochondrial superoxide in response to FSS. These findings demonstrate that FSS-sensitive Nrf2 signaling is dependent on mechanotransduction through the GCX, and restoration of the GCX may normalise redox homeostasis in atheroprone vascular regions.

Benzophenone-4 Promotes the Growth of a Pseudomonas sp. and Biogenic Oxidation of Mn(II).

Interactions between microbes and micropollutants (MPs) play a crucial role in water purification or treatment. Current studies have generally focused on the direct degradation or cometabolism of MPs. Considering the increasing interest in and importance of the roles of MPs in microbial metabolism, we adopted an Mn(II)-oxidizing Pseudomonas sp. QJX-1 using tyrosine (Tyr) as the sole carbon and nitrogen source to investigate the effects of seven MPs on its growth and function. Six MPs exhibited an inhibition effect on bacterial growth and Mn(II) oxidation. Only benzophenone-4 (BP-4) promoted the growth of QJX-1 and biogenic oxidation Mn(II), but its concentration was not directly coupled to growth, which was unexpected. RNA-seq data suggested that the addition of BP-4 did not significantly change the basic metabolic function of QJX-1, but stimulated the upregulation of the pyruvate and gluconeogenesis metabolic pathways of Tyr for QJX-1 growth. Furthermore, protein identification and extracellular superoxide detection indicated that Mn(II) oxidation was largely driven by the formation of superoxide in response to Tyr starvation; the acceleration of superoxide production, due to BP-4 accelerating Tyr consumption, was responsible for the promotion effect of BP-4 on QJX-1 Mn(II) oxidation. Our findings highlight the dual effects that MPs can have on the growth and function of a single strain in aquatic ecosystem, i.e., the coexistence of inhibition and promotion.

100 - The Role of N-Acetylcysteine and 3H-1,2-Dithiole-3-Thione in Maintaining Glutathione Status and Protection Against Advanced Glycation End Product Induced Neurite Degeneration in SH-SY5Y Cells

Diabetes mellitus, a disease hallmarked by hyperglycemia, is one of the most common chronic diseases worldwide and new diagnoses are on an upward trajectory. Peripheral neuropathy (PN) affects at least 50% of diabetic patients. Unfortunately, medications available for patients with PN treat the symptoms but not the cellular damage, and come with severe side effects leading many to discontinue their use. The overall objective of this study was to gain a better understanding of the mechanism of action and potential impact of therapeutic substances, such as N-acetylcysteine (NAC), and preventive dietary components, such as the cruciferous vegetable constituent 3H-1,2-dithiole-3-thione (D3T), which is critically important for patient health. Previous studies in our lab have shown that when SH-SY5Y cells, a validated model for PN, are treated with advanced glycation end products (AGE), there is increased reactive oxygen species (ROS) generation, a significant decrease in glutathione (GSH) concentrations, and neurite loss. NAC was shown to protect against AGE-induced cell death, GSH depletion and neurite loss, indicating that maintaining GSH was important for neurite protection. Our lab has also shown that D3T, a potent inducer of nuclear factor (erythroid-derived 2)- like (Nrf2), can significantly increase SH-SY5Y cellular GSH concentrations and protect against H2O2 –induced cell death. Paradoxically, D3T conferred no protection against AGE-induced cell death or neurite degeneration. We report here follow up studies. The first aim of this study was to test whether NAC conferred protection through maintaining GSH, its intrinsic antioxidant properties, or both. Using buthionine sulfoximine (BSO) as a tool to evaluate the mechanistic role of NAC in neurite protection, we found that at high concentrations of NAC (2 mM) both intrinsic and GSH-maintaining properties were observed. At lower concentrations (1 mM), GSH-maintaining properties predominated. The second aim was to elucidate a mechanism for the paradoxical effect of D3T focusing on the role of NADPH-mediated ROS generation. NADPH is an electron donor for the oxidase enzyme that generates superoxide in response to the activation of the receptor for AGE (RAGE). D3T increased ROS generation and depleted GSH only in the presence of AGE, suggesting its role as a NADPH generating agent may promote oxidative stress rather than protect against it under these conditions. Inhibition of NADPH generation via glucose-6-phosphate dehydrogenase (G6PD), the protein product of a Nrf2-responsive gene, with dehydro-epiandrosterone (DHEA) was found to protect against AGE-induced loss of cell viability. These results suggest that the source of ROS is important to consider in evaluating the extent to which adaption to oxidative stress provides protection.

The mechanism of ROS regulation of antibiotic resistance and antimicrobial lethality.

Misuse and overuse of antibiotics have led to serious resistance problems that pose a grave threat to human health. How to solve the increasing antibiotic resistance problem is a huge challenge. Besides the traditional strategy of developing novel antimicrobial agents, exploring ways to enhance the lethal activity of antibiotics currently available is another feasible approach to fight against resistance. Recent studies showed that ROS plays an important role in regulating both antibiotic resistance and antimicrobial lethality. ROS produced by sublethal levels of antibiotic induces antibiotic resistance through activating drug efflux pumps via MarR(Multiple antibiotic resistance repressor)-MarA(Multiple antibiotic resistance activator), triggers the protective function against stress via SoxR (Superoxide response transcriptional regulator)-SoxS (Superoxide response transcription factor), and promotes mutagenesis by induction of SOS system. On the contrary, ROS triggered by lethal levels of antibiotic promotes bacterial killing and suppresses resistance. In addition to the concentration of antibiotic, the role of ROS in mediating antimicrobial resistance and bacterial killing is also regulated by a series of genetic regulators (e.g. MazEF, Cpx, SoxR, MarRAB). Thus, how ROS contribute to antimicrobial resistance and bacterial killing is complex. In this review, we summarized the mechanism of ROS in regulating antibiotic resistance and antimicrobial lethality, which may provide references and guidance for finding new ways to enhance antimicrobial lethality of currently available antimicrobials and battling antibiotic resistance.

TcI Isolates of Trypanosoma cruzi Exploit the Antioxidant Network for Enhanced Intracellular Survival in Macrophages and Virulence in Mice.

Trypanosoma cruzi species is categorized into six discrete typing units (TcI to TcVI) of which TcI is most abundantly noted in the sylvatic transmission cycle and considered the major cause of human disease. In our study, the TcI strains Colombiana (COL), SylvioX10/4 (SYL), and a cultured clone (TCC) exhibited different biological behavior in a murine model, ranging from high parasitemia and symptomatic cardiomyopathy (SYL), mild parasitemia and high tissue tropism (COL), to no pathogenicity (TCC). Proteomic profiling of the insect (epimastigote) and infective (trypomastigote) forms by two-dimensional gel electrophoresis/matrix-assisted laser desorption ionization-time of flight mass spectrometry, followed by functional annotation of the differential proteome data sets (≥2-fold change, P < 0.05), showed that several proteins involved in (i) cytoskeletal assembly and remodeling, essential for flagellar wave frequency and amplitude and forward motility of the parasite, and (ii) the parasite-specific antioxidant network were enhanced in COL and SYL (versus TCC) trypomastigotes. Western blotting confirmed the enhanced protein levels of cytosolic and mitochondrial tryparedoxin peroxidases and their substrate (tryparedoxin) and iron superoxide dismutase in COL and SYL (versus TCC) trypomastigotes. Further, COL and SYL (but not TCC) were resistant to exogenous treatment with stable oxidants (H2O2 and peroxynitrite [ONOO(-)]) and dampened the intracellular superoxide and nitric oxide response in macrophages, and thus these isolates escaped from macrophages. Our findings suggest that protein expression conducive to increase in motility and control of macrophage-derived free radicals provides survival and persistence benefits to TcI isolates of T. cruzi.

SS-31 attenuates TNF-α induced cytokine release from C2C12 myotubes

TNF-α is a key inflammatory mediator and is proposed to induce transcriptional responses via the mitochondrial generation of Reactive Oxygen Species (ROS). The aim of this study was to determine the effect of TNF-α on the production of myokines by skeletal muscle. Significant increases were seen in the release of IL-6, MCP-1/CCL2, RANTES/CCL5 and KC/CXCL1 and this release was inhibited by treatment with Brefeldin A, suggesting a golgi-mediated release of cytokines by muscle cells. An increase was also seen in superoxide in response to treatment with TNF-α, which was localised to the mitochondria and this was also associated with activation of NF-κB. The changes in superoxide, activation of NF-kB and release of myokines were attenuated following pre-treatment with SS-31 peptide indicating that the ability of TNF-α to induce myokine release may be mediated through mitochondrial superoxide, which is, at least in part, associated with activation of the redox sensitive transcription factor NF-kB.

Three members of polyamine modulon under oxidative stress conditions: two transcription factors (SoxR and EmrR) and a glutathione synthetic enzyme (GshA).

Members of polyamine modulon whose synthesis is enhanced at the level of translation were looked for under oxidative stress conditions caused by 0.6 μM K2TeO3. When an Escherichia coli polyamine-requiring mutant MA261 was cultured in the presence of K2TeO3, the degree of polyamine stimulation of cell growth was greater than in cells cultured in the absence of K2TeO3. Under these conditions, synthesis of SoxR, a transcriptional factor for expression of the superoxide response regulon, EmrR, a negative transcriptional factor for expression of the genes for drug excretion proteins, EmrA and EmrB, and of GshA, γ-glutamylcysteine synthetase necessary for glutathione (GSH) synthesis, were stimulated by polyamines at the level of translation. Polyamine stimulation of SoxR and EmrR synthesis was dependent on the existence of an unusually located Shine-Dalgarno (SD) sequence in soxR and emrR mRNAs. Polyamine stimulation of GshA synthesis was due to the existence of the inefficient initiation codon UUG instead of AUG. Polyamine stimulation of the synthesis of EmrR was mainly observed at the logarithmic phase of growth, while that of the synthesis of SoxR and GshA was at the stationary phase. These results strongly suggest that polyamines are involved in easing of oxidative stress through stimulation of synthesis of SoxR, EmrR and GshA together with RpoS, previously found as a member of polyamine modulon at the stationary phase.