Rapid Reactive Oxygen Species Research Articles

Rice roots avoid asymmetric heavy metal and salinity stress via an RBOH-ROS-auxin signaling cascade

Root developmental plasticity is crucial for plants to adapt to a changing soil environment, where nutrients and abiotic stress factors are distributed heterogeneously. How plant roots sense and avoid heterogeneous abiotic stress in soil remains unclear. Here, we show that, in response to asymmetric stress of heavy metals (cadmium, copper, or lead) and salt, rice roots rapidly proliferate lateral roots (LRs) in the stress-free area, thereby remodeling root architecture to avoid localized stress. Imaging and quantitative analyses of reactive oxygen species (ROS) showed that asymmetric stress induces a ROS burst in the tips of the exposed roots and simultaneously triggers rapid systemic ROS signaling to the unexposed roots. Addition of a ROS scavenger to either the stressed or stress-free area abolished systemic ROS signaling and LR proliferation induced by asymmetric stress. Asymmetric stress also enhanced cytosolic calcium (Ca2+) signaling; blocking Ca2+signaling inhibited systemic ROS propagation and LR branching in the stress-free area. We identified two plasma-membrane-localized respiratory burst oxidase homologs, OsRBOHA and OsRBOHI, as key players in systemic ROS signaling under asymmetric stress. Expression of OsRBOHA and OsRBOHI in roots was upregulated by Cd stress, and knockout of either gene reduced systemic ROS signaling and LR proliferation under asymmetric stress. Furthermore, we demonstrated that auxin signaling and cell wall remodeling act downstream of the systemic ROS signaling to promote LR development. Collectively, our study reveals an RBOH-ROS-auxin signaling cascade that enables rice roots to avoid localized stress of heavy metals and salt and provides new insight into root system plasticity in heterogenous soil.

Electrospun polyvinyl alcohol-chitosan dressing stimulates infected diabetic wound healing with combined reactive oxygen species scavenging and antibacterial abilities

Diabetic wounds (DW) are constantly challenged by excessive reactive oxygen species (ROS) accumulation and susceptibility to bacterial contamination. Therefore, the elimination of ROS in the immediate vicinity and the eradication of local bacteria are critical to stimulating the efficient healing of diabetic wounds. In the current study, we encapsulated mupirocin (MP) and cerium oxide nanoparticles (CeNPs) into a polyvinyl alcohol/chitosan (PVA/CS) polymer, and then a PVA/chitosan nanofiber membrane wound dressing was fabricated using electrostatic spinning, which is a simple and efficient method for fabricating membrane materials. The PVA/chitosan nanofiber dressing provided a controlled release of MP, which produced rapid and long-lasting bactericidal activity against both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) strains. Simultaneously, the CeNPs embedded in the membrane exhibited the desired ROS scavenging capacity to maintain the local ROS at a normal physiological level. Moreover, the biocompatibility of the multifunctional dressing was evaluated both in vitro and in vivo. Taken together, PVA-CS-CeNPs-MP integrated the desirable features of a wound dressing, including rapid and broad-spectrum antimicrobial and ROS scavenging activities, easy application, and good biocompatibility. The results validated the effectiveness of our PVA/chitosan nanofiber dressing, highlighting its promising translational potential in the treatment of diabetic wounds.

Quantitatively Assessing the Respiratory Burst in Innate Immune Cells.

The respiratory burst is a rapid cellular consumption of oxygen resulting in abundant production of reactive oxygen species (ROS), most often associated with primary mediators of innate immunity, neutrophils and macrophages. These myeloid cells convert ROS into potent antimicrobial oxidants that efficiently kill pathogens. The respiratory burst also can have destructive consequences, as ROS are well known to support chronic inflammation and aberrant autoimmune responses. Interestingly, ROS perform conflicting roles in the tumor microenvironment; ROS and derived cytotoxic products can destroy cancer cells but also suppress important tumor-fighting functions of T cells or natural killer cells, or yield mutagenized proteins that can promote tumorigenesis or support tumor cell growth. Moreover, high numbers of neutrophils or macrophages in tumors are associated with poor prognosis. Therefore, accurate and quantitative assays to assess the respiratory burst are an important tool for measuring ROS production by neutrophils or cells of the monocyte/macrophage system, each recently identified in the tumor microenvironment. Described are methods to derive mouse or human models of neutrophils or macrophages, which are then used in a detailed assay to quantitatively measure ROS produced by either cell type using luminescence-enhanced reagents and a multi-well platform along with different stimulants that cause rapid ROS production.

Recent progress in metal-organic framework-based sonosensitizers for sonodynamic tumor therapy.

In recent years, sonodynamic therapy (SDT) has emerged as a promising approach in biomedicine, due to its low toxicity, non-invasiveness, and deep tissue penetration for effective treatment of deep tumors. SDT utilizes ultrasound to irradiate sonosensitizers accumulated in tumors to generate reactive oxygen species (ROS), which can kill tumors by inducing apoptosis or necrosis in tumor cells. Developing safe and efficient sonosensitizers is a top priority in SDT. Recently reported sonosensitizers can be divided into three basic categories: organic, inorganic, and organic-inorganic hybrid sonosensitizers. Among these, metal-organic frameworks (MOFs) are a promising class of hybrid sonosensitizers due to their advantages of the linker-to-metal charge transfer mechanism for rapid ROS generation and the porous structure to eliminate self-quenching to increase the ROS generation efficiency. Moreover, MOF-based sonosensitizers can be combined with other therapies due to their large specific surface area, high porosity, and easy modification, which can increase the therapeutic efficacy through various synergistic effects. This review focuses on the latest progress in MOF-based sonosensitizers, strategies to improve the therapeutic effect, and the use of MOF-based sonosensitizers as multifunctional platforms for combination therapies emphasizing on increased therapeutic efficacy. Additionally, the challenges of MOF-based sonosensitizers from a clinical perspective are discussed.

Circulating Permeability Factors in Focal Segmental Glomerulosclerosis: In Vitro Detection

The recurrence of proteinuria after kidney transplantation in patients with focal segmental glomerulosclerosis (FSGS) is considered proof of the presence of circulating permeability factors (CPFs). The aim of this study is to demonstrate the presence of plasma CPFs using series of invitro assays. Podocytes and endothelial cells (glomerular microvascular endothelial cells [GMVECs]) were incubated with plasma from FSGS patients with presumed CPFs in relapse and remission and from steroid-resistant nephrotic syndrome (SRNS), steroid-sensitive nephrotic syndrome (SSNS), membranous nephropathy (MN), and healthy controls (hCtrls). Cell viability, podocyte actin cytoskeleton architecture, and reactive oxygen species (ROS) formation with or without ROS scavenger were investigated by Cell Counting Kit-8 assay, immunofluorescence staining, and CM-H2DCFDA probing, respectively. Presumed CPF-containing plasma causes a series of events in podocytes but not in GMVECs. These events include actin cytoskeleton rearrangement and excessive formation of ROS, which results in podocyte loss. These effects were solely observed in response to CPF plasma collected during relapse, but not in response to plasma of hCtrls, or patients with SRNS, SSNS, and MN. The copresence of dimethylthiourea, a scavenger of ROS, abolished the aforementioned effects of CPF plasma. We provide a panel of invitro bioassays to measure podocyte injury and predict the presence of CPFs in plasma of patients with nephrotic syndrome (NS), providing a new framework for monitoring CPF activity that may contribute to future NS diagnostics or used for disease monitoring purposes. Moreover, our findings suggest that the inhibition of ROS formation or facilitating rapid ROS scavenging may exert beneficial effects in patients with CPFs.

An engineered, self-propelled nanozyme as reactive oxygen species scavenger

The development of enzyme-mimicking nanomaterials (nanozymes) with excellent reactive oxygen species (ROS)-scavenging abilities and low biotoxicity is desired for clinical translation. The use of nanomotors as nanozymes for active ROS elimination is a novel strategy that involves rapid ROS diffusion through autonomous movement. Here, we report an engineered, self-propelled nanozyme based on a Janus Pt/SiO2 nanomotor with Pt nanoparticle active centers, which possess GPx-like, CAT-like and SOD-like enzyme activities as well as ∙ OH scavenging ability. Nanozymes modified with black phosphorous quantum dots (BPQDs) achieved efficient self-diffusiophoretic motion under physiological concentrations of H2O2, independent of a fuel source. Moreover, the OH and O2- removal ability of BPQDs further improved antioxidant ability of this nanozyme. We hope that such strategy involving self-propulsion will facilitate the development of nanozymes and expand biomedical applications of nanomotors.

Copper doped Fe-N-C as an excellent Fenton-like catalyst for membrane fouling mitigation against natural organic matters at neutral pH

The presence of humic substances as a component of natural organic matter (NOM) may cause inefficiency towards drinking water treatment processes, especially the membrane fouling during filtration process. Therefore, to enhance the efficiency of the conventional membrane filtration process, several copper doped Fe-N-C bimetallic heterogeneous Fenton-like catalysts (Cux@FeNC) were prepared and evaluated by executing their catalytic degradation performance against NOM. Results indicated that humic acid (HA, 10 mg/L) could be significantly degraded (95%) within 30 min application of Cu5p@FeNC-Fenton under neutral pH, which was relatively higher than of FeNC-Fenton (70%) under identical conditions. The macromolecular organic matters in NOM could be destroyed more effectively while the overall mineralization degree also became relatively higher. Meanwhile, a lower membrane fouling of UF (PES 20 kDa)/NF (DOW 270) and highest flux recovery after backwash were observed with Cu5p@FeNC-Fenton treatments. Characterization and chemical analysis further explored the introduction of Cux in FeNC was responsible for the rapid reactive oxygen species (ROS) generation and efficient degradation of NOM. Additionally, a possible copper-iron synergetic mechanism in Fenton-like system was proposed. This work gives a deep insight into the potential NOM degradation using Fenton-like Cux@FeNC catalysts and ensures the membrane fouling mitigation for effective water treatment processes.

A negative feedback loop controls ROS production in plant immunity

A negative feedback loop controls ROS production in plant immunity

Bacillus amyloliquefaciens strains control strawberry anthracnose through antagonistic activity and plant immune response intensification

Anthracnose disease caused by Colletotrichum gloeoesporioides can have devastating effects on strawberry production. In addition to the use of fungicides, feasible strategies for controlling this disease include the application of beneficial microorganisms. In this study, we assayed the effects of two Bacillus amyloliquefaciens strains, PMB04 and PMB05, on the control of strawberry anthracnose. First, an in vitro inhibitory assay revealed that the hyphal growth of C. gloeosporioide SC01 was inhibited by PMB04 more strongly than by PMB05. We further found that the culture filtrate from PMB04 was lethal to the conidia of SC01. Meanwhile, PMB05 was previously demonstrated to be a rhizobacterium that intensifies plant immunity in response to bacterial elicitors. Thus, its effect on plant immune signals, such as the rapid reactive oxygen species (ROS) generation and callose deposition triggered by the conidia of SC01 on strawberry leaves, were evaluated in this study. The results showed that only the PMB05 strain could intensify the plant immune signals activated by SC01. In an inoculation assay, meanwhile, both strains exhibited superior efficacy in reducing the occurrence of anthracnose on strawberry seedlings and fruits. More specifically, PMB04 and PMB05 showed better ability to control anthracnose on fruits and seedlings, respectively. We concluded that both the lethal effect on spores caused by PMB04 and the intensification of plant immunity caused by PMB05 could be key factors in controlling strawberry anthracnose. More interestingly, we present a case in which a plant immune response-intensifying strain was applied to control strawberry anthracnose.

54P The combination of copper oxide nanoparticles and N-acetyl-L-cysteine triggers rapid oxidative burst and death of wild-type and multidrug-resistant tumour cells

Overcoming drug resistance is a major problem in cancer therapy. Tumor cells are vulnerable to reactive oxygen species (ROS). The oxygen burst can be evoked by metal oxide nanoparticles (NPs) alone and/or together with conventional or novel compounds. We investigated the antitumor potential of the combination of copper oxide NPs and N-acetyl-L-cysteine (NAC) against various human tumor cell lines. CuO NPs (80±20 nm as determined by dynamic light scattering) were synthesized by the precipitation method. Intracellular accumulation of CuO NPs was measured by atomic absorption spectroscopy. The panel of cell lines included HCT116 colon and MDA-MB-231 triple-negative breast carcinomas as well as K562 (CML) cell line and its multidrug-resistant K562/4 subline. The cytotoxicity of CuO NPs, NAC and their combination were determined in MTT assays. Flow cytometry was used to analyze the mechanisms of cell response. Caspase-3 and poly(ADPribose) polymerase (PARP) were detected by immunoblotting. CuO NPs showed a slight toxicity for all tested cell lines after 72 h, while in combination with the non-toxic NAC a rapid (within 2 h) ROS generation and cell death by 8 h were detected. CuO NPs weakly accumulated in the cells, so ROS generation is likely to occur mainly outside the cells. The IC50 of the combination was 2-3 orders of magnitude smaller compared to NPs alone. Death associated events differed between the cell lines but generally caspase-independent apoptosis was followed by irreversible damage of the plasma membrane. Importantly, the combination of CuO NPs with NAC was equally potent for K562 cells and the multidrug-resistant K562/4 subline. Reduction of Cu2+ to Cu+ upon interaction with the thiol group in NAC can trigger ROS formation. The combination of CuO NPs and NAC killed human tumor cell lines including the multidrug-resistant variant. Rapid ROS generation can be promising in situations where tumor cells acquire multidrug resistance during treatment. Also, engineering of supramolecular systems will allow to guide copper to critical cysteine residues in specific proteins for their oxidative destruction.

Vascular Bundles Mediate Systemic Reactive Oxygen Signaling during Light Stress.

Systemic signaling and systemic acquired acclimation (SAA) are essential for plant survival during episodes of environmental stress. Recent studies highlighted a key role for reactive oxygen species (ROS) signaling in mediating systemic responses and SAA during light stress in Arabidopsis (Arabidopsis thaliana). These studies further identified the RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD) protein as a key player in mediating rapid systemic ROS responses. Here, we report that tissue-specific expression of RBOHD in phloem or xylem parenchyma cells of the rbohD mutant restores systemic ROS signaling, systemic stress-response transcript expression, and SAA to a local treatment of light stress. We further demonstrate that RBOHD and RBOHF are both required for local and systemic ROS signaling at the vascular bundles of Arabidopsis. Taken together, our findings highlight a key role for RBOHD-driven ROS production at the vascular bundles of Arabidopsis in mediating light stress-induced systemic signaling and SAA. In addition, they suggest that the integration of ROS, calcium, electric, and hydraulic signals, during systemic signaling, occurs at the vascular bundles.

Cannabidiol Protects Dopaminergic Neuronal Cells from Cadmium

The protective effect of cannabidiol (CBD), the non-psychoactive component of Cannabis sativa, against neuronal toxicity induced by cadmium chloride (CdCl2 10 μM) was investigated in a retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cell line. CBD (1 μM) was applied 24 h before and removed during cadmium (Cd) treatment. In differentiated neuronal cells, CBD significantly reduced the Cd-dependent decrease of cell viability, and the rapid reactive oxygen species (ROS) increase. CBD significantly prevented the endoplasmic reticulum (ER) stress (GRP78 increase) and the subcellular distribution of the cytochrome C, as well as the overexpression of the pro-apoptotic protein BAX. Immunocytochemical analysis as well as quantitative protein evaluation by western blotting revealed that CBD partially counteracted the depletion of the growth associated protein 43 (GAP43) and of the neuronal specific class III β-tubulin (β3 tubulin) induced by Cd treatment. These data showed that Cd-induced neuronal injury was ameliorated by CBD treatment and it was concluded that CBD may represent a potential option to protect neuronal cells from the detrimental effects of Cd toxicity.

Nitric Oxide Enhances Cytotoxicity of Lead by Modulating the Generation of Reactive Oxygen Species and Is Involved in the Regulation of Pb2+ and Ca2+ Fluxes in Tobacco BY-2 Cells.

Lead is a heavy metal known to be toxic to both animals and plants. Nitric oxide (NO) was reported to participate in plant responses to different heavy metal stresses. In this study, we analyzed the function of exogenous and endogenous NO in Pb-induced toxicity in tobacco BY-2 cells, focusing on the role of NO in the generation of reactive oxygen species (ROS) as well as Pb2+ and Ca2+ fluxes using non-invasive micro-test technology (NMT). Pb treatment induced BY-2 cell death and rapid NO and ROS generation, while NO burst occurred earlier than ROS accumulation. The elimination of NO by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) resulted in a decrease of ROS, and the supplementation of NO by sodium nitroprusside (SNP) caused an increased accumulation of ROS. Furthermore, the addition of exogenous NO stimulated Pb2+ influx, thus promoting Pb uptake in cells and aggravating Pb-induced toxicity in cells, whereas the removal of endogenous NO produced the opposite effect. Moreover, we also found that both exogenous and endogenous NO enhanced Pb-induced Ca2+ effluxes and calcium homeostasis disorder. These results suggest that exogenous and endogenous NO played a critical regulatory role in BY-2 cell death induced by Pb stress by promoting Pb2+ influx and accumulation and disturbing calcium homeostasis.

Flexible, mesoporous, and monodispersed metallic cobalt-embedded inorganic nanofibrous membranes enable ultra-fast and high-efficiency killing of bacteria

Severe emerging infectious diseases, deriving from the contamination of pathogenic bacteria, are threatening human’s survival and development. Metal-embedded inorganic porous material with large mesopores and high flexibility, has proven to be one of the most promising activators of peroxymonosulfate (PMS) to generate reactive oxygen species (ROS) for the high-efficiency pathogenic bacteria elimination. However, there still exists huge challenges to develop such materials. Herein, a robust strategy was reported to create flexible metallic cobalt (Co)-embedded inorganic nanofibrous membranes (CINMs) with interconnected mesoporous structures and monodispersed Co nanoparticles, for the first time, by double-template electrospinning approach and in situ carbonization reduction method. The monodispersed Co particles throughout fibers could activate PMS to effectively produce ROS, enabling the complete bacterial inactivation with a 7 log reduction within only 3 min. Benefitting from the integrated features of high porosity, robust mechanical property, and rapid ROS production, the obtained CINMs exhibit an ultra-high bactericidal efficiency of 99.99999% and a high permeate flux of 3.4 × 104 L m−2 h−1 merely driven by gravity (≈1.2 kPa). Moreover, a novel CINMs/PMS-based spray type sterilizer was successfully created for the convenient and high-efficiency sterilization of solid surfaces, which could completely inactivate bacteria on the surfaces after being sprayed for 10 s. The successful synthesis of such fascinating materials may shed light on designing new types of inorganic nanofiber-based antimicrobials for various public health and environment protection.

Increasing Pyruvate Concentration Enhances Conidial Thermotolerance in the Entomopathogenic Fungus Metarhizium robertsii.

The fungal entomopathogens Metarhizium spp. have been developed as environmentally friendly mycoinsecticides. However, heat stress severely reduces the viability of Metarhizium conidia in the field, which is an important obstacle to the successful use of these mycoinsecticides. Heat treatment induces rapid accumulation of pyruvate, which timely scavenges heat-induced ROS (reactive oxygen species) in hyphal cells of M. robertsii. However, in heat-treated conidia, pyruvate accumulation occurs later than the rapid production of ROSs, which could harm the conidial cells. In the present study, a transgenic M. robertsii strain was constructed with the pyruvate kinases gene overexpressed during conidiation. Two independent transformants of the transgenic strain produced conidia under optimal conditions with elevated pyruvate concentration. This inhibits the rapid heat-induced ROS production and prevents the collapse of mitochondrial membrane potential, thereby increasing conidial tolerance to heat stress. In conclusion, the tolerance of M. robertsii conidia to heat stress was improved by increasing the conidial pyruvate concentration, which could be translated into a more effective pest control.

Respiratory Burst Oxidase Homolog Gene A Is Crucial for Rhizobium Infection and Nodule Maturation and Function in Common Bean.

Reactive oxygen species (ROS) produced by respiratory burst oxidase homologs (RBOHs) regulate numerous plant cell processes, including the symbiosis between legumes and nitrogen-fixing bacteria. Rapid and transient ROS production was reported after Phaseolus vulgaris root hairs were treated with Nod factors, indicating the presence of a ROS-associated molecular signature in the symbiosis signaling pathway. Rboh is a multigene family containing nine members (RbohA–I) in P. vulgaris. RNA interference of RbohB suppresses ROS production and attenuates rhizobial infection thread (IT) progression in P. vulgaris root hairs. However, the roles of other Rboh members in symbiotic interactions are largely unknown. In this study, we characterized the role of the NADPH oxidase-encoding gene RbohA (Phvulv091020621) in the P. vulgaris–Rhizobium tropici symbiosis. The spatiotemporal activity of the RbohA promoter colocalized with growing ITs and was associated with vascular bundles in developing nodules. Subcellular localization studies indicated that RBOHA was localized in the plasma membrane of P. vulgaris root hairs. After rhizobial inoculation, PvRBOHA was mainly distributed in the infection pocket and, to a lesser extent, throughout the IT. In PvRbohA RNAi lines, the rhizobial infection events were significantly reduced and, in successful infections, IT progression was arrested within the root hair, but did not impede cortical cell division. PvRbohA-RNAi nodules failed to fix nitrogen, since the infected cells in the few nodules formed were empty. RbohA-dependent ROS production and upregulation of several antioxidant enzymes was attenuated in rhizobia-inoculated PvRbohA-RNAi roots. These combined results indicate that PvRbohA is crucial for effective Rhizobium infection and its release into the nodule cells. This oxidase is partially or indirectly required to promote nodule organogenesis, altering the expression of auxin- and cyclin-related genes and genes involved in cell growth and division.

Root phonotropism: Early signalling events following sound perception in Arabidopsis roots

Sound is a fundamental form of energy and it has been suggested that plants can make use of acoustic cues to obtain information regarding their environments and alter and fine-tune their growth and development. Despite an increasing body of evidence indicating that it can influence plant growth and physiology, many questions concerning the effect of sound waves on plant growth and the underlying signalling mechanisms remains unknown. Here we show that in Arabidopsis thaliana, exposure to sound waves (200Hz) for 2 weeks induced positive phonotropism in roots, which grew towards to sound source. We found that sound waves triggered very quickly (within minutes) an increase in cytosolic Ca2+, possibly mediated by an influx through plasma membrane and a release from internal stock. Sound waves likewise elicited rapid reactive oxygen species (ROS) production and K+ efflux. Taken together these results suggest that changes in ion fluxes (Ca2+ and K+) and an increase in superoxide production are involved in sound perception in plants, as previously established in animals.

The IDA-LIKE peptides IDL6 and IDL7 are negative modulators of stress responses in Arabidopsis thaliana.

Small signalling peptides have emerged as important cell to cell messengers in plant development and stress responses. However, only a few of the predicted peptides have been functionally characterized. Here, we present functional characterization of two members of the IDA-LIKE (IDL) peptide family in Arabidopsis thaliana, IDL6 and IDL7. Localization studies suggest that the peptides require a signal peptide and C-terminal processing to be correctly transported out of the cell. Both IDL6 and IDL7 appear to be unstable transcripts under post-transcriptional regulation. Treatment of plants with synthetic IDL6 and IDL7 peptides resulted in down-regulation of a broad range of stress-responsive genes, including early stress-responsive transcripts, dominated by a large group of ZINC FINGER PROTEIN (ZFP) genes, WRKY genes, and genes encoding calcium-dependent proteins. IDL7 expression was rapidly induced by hydrogen peroxide, and idl7 and idl6 idl7 double mutants displayed reduced cell death upon exposure to extracellular reactive oxygen species (ROS). Co-treatment of the bacterial elicitor flg22 with IDL7 peptide attenuated the rapid ROS burst induced by treatment with flg22 alone. Taken together, our results suggest that IDL7, and possibly IDL6, act as negative modulators of stress-induced ROS signalling in Arabidopsis.

Time courses of post-injury mitochondrial oxidative damage and respiratory dysfunction and neuronal cytoskeletal degradation in a rat model of focal traumatic brain injury

Traumatic brain injury (TBI) results in rapid reactive oxygen species (ROS) production and oxidative damage to essential brain cellular components leading to neuronal dysfunction and cell death. It is increasingly appreciated that a major player in TBI-induced oxidative damage is the reactive nitrogen species (RNS) peroxynitrite (PN) which is produced in large part in injured brain mitochondria. Once formed, PN decomposes into highly reactive free radicals that trigger membrane lipid peroxidation (LP) of polyunsaturated fatty acids (e.g. arachidonic acid) and protein nitration (3-nitrotyrosine, 3-NT) in mitochondria and other cellular membranes causing various functional impairments to mitochondrial oxidative phosphorylation and calcium (Ca2+) buffering capacity. The LP also results in the formation of neurotoxic reactive aldehyde byproducts including 4-hydroxynonenal (4-HNE) and propenal (acrolein) which exacerbates ROS/RNS production and oxidative protein damage in the injured brain. Ultimately, this results in intracellular Ca2+ overload that activates proteolytic degradation of α-spectrin, a neuronal cytoskeletal protein. Therefore, the aim of this study was to establish the temporal evolution of mitochondrial dysfunction, oxidative damage and cytoskeletal degradation in the brain following a severe controlled cortical impact (CCI) TBI in young male adult rats. In mitochondria isolated from an 8 mm diameter cortical punch including the 5 mm wide impact site and their respiratory function studied ex vivo, we observed an initial decrease in complex I and II mitochondrial bioenergetics within 3 h (h). For complex I bioenergetics, this partially recovered by 12–16 h, whereas for complex II respiration the recovery was complete by 12 h. During the first 24 h, there was no evidence of an injury-induced increase in LP or protein nitration in mitochondrial or cellular homogenates. However, beginning at 24 h, there was a gradual secondary decline in complex I and II respiration that peaked at 72 h. post-TBI that coincided with progressive peroxidation of mitochondrial and cellular lipids, protein nitration and protein modification by 4-HNE and acrolein. The oxidative damage and respiratory failure paralleled an increase in Ca2+-induced proteolytic degradation of the neuronal cytoskeletal protein α-spectrin indicating a failure of intracellular Ca2+ homeostasis. These findings of a surprisingly delayed peak in secondary injury, suggest that the therapeutic window and needed treatment duration for certain antioxidant treatment strategies following CCI-TBI in rodents may be longer than previously believed.

Clostridium difficile Toxin A Induces Reactive Oxygen Species Production and p38 MAPK Activation to Exert Cellular Toxicity in Neuronal Cells.

Clostridium difficile releases two exotoxins, toxin A and toxin B, which disrupt the epithelial cell barrier in the gut to increase mucosal permeability and trigger inflammation with severe diarrhea. Many studies have suggested that enteric nerves are also directly involved in the progression of this toxin-mediated inflammation and diarrhea. C. difficile toxin A is known to enhance neurotransmitter secretion, increase gut motility, and suppress sympathetic neurotransmission in the guinea pig colitis model. Although previous studies have examined the pathophysiological role of enteric nerves in gut inflammation, the direct effect of toxins on neuronal cells and the molecular mechanisms underlying toxin-induced neuronal stress remained to be unveiled. Here, we examined the toxicity of C. difficile toxin A against neuronal cells (SH-SY5Y). We found that toxin A treatment time- and dose-dependently decreased cell viability and triggered apoptosis accompanied by caspase-3 activation in this cell line. These effects were found to depend on the up-regulation of reactive oxygen species (ROS) and the subsequent activation of p38 MAPK and induction of p21Cip1/Waf1. Moreover, the N-acetyl-L-cysteine (NAC)-induced down-regulation of ROS could recover the viability loss and apoptosis of toxin A-treated neuronal cells. These results collectively suggest that C. difficile toxin A is toxic for neuronal cells, and that this is associated with rapid ROS generation and subsequent p38 MAPK activation and p21Cip1/Waf1 up-regulation. Moreover, our data suggest that NAC could inhibit the toxicity of C. difficile toxin A toward enteric neurons.