Antioxidant Carbon Nanoparticles Inhibit Fibroblast-Like Synoviocyte Invasiveness and Reduce Disease Severity in a Rat Model of Rheumatoid Arthritis.

Reactive oxygen species have been involved in the pathogenesis of rheumatoid arthritis (RA). Our goal was to determine the effects of selectively scavenging superoxide (O2•-) and hydroxyl radicals with antioxidant nanoparticles, called poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), on the pathogenic functions of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and on the progression of an animal model of RA. We used human FLS from patients with RA to determine PEG-HCC internalization and effects on FLS cytotoxicity, invasiveness, proliferation, and production of proteases. We used the pristane-induced arthritis (PIA) rat model of RA to assess the benefits of PEG-HCCs on reducing disease severity. PEG-HCCs were internalized by RA-FLS, reduced their intracellular O2•-, and reduced multiple measures of their pathogenicity in vitro, including proliferation and invasion. In PIA, PEG-HCCs caused a 65% reduction in disease severity, as measured by a standardized scoring system of paw inflammation and caused a significant reduction in bone and tissue damage, and circulating rheumatoid factor. PEG-HCCs did not induce lymphopenia during PIA. Our study demonstrated a role for O2•- and hydroxyl radicals in the pathogenesis of a rat model of RA and showed efficacy of PEG-HCCs in treating a rat model of RA.

Catalytic oxidation and reduction reactions of hydrophilic carbon clusters with NADH and cytochrome C: features of an electron transport nanozyme.

Previously, our group reported on the promising efficacy of poly(ethylene glycol)-hydrophilic carbon clusters (PEG-HCCs) to work as broadly active and high capacity antioxidants in brain ischemia and injury models including stroke and traumatic brain injury coupled with hemorrhagic shock. PEG-HCCs are a carbon nanomaterial derived from harsh oxidation of single wall carbon nanotubes and covalently modified with poly(ethylene glycol). They retain no tubular remnants and are composed of a highly oxidized carbon core functionalized with epoxy, peroxyl, quinone, ketone, carboxylate, and hydroxyl groups. HCCs are the redox active carbon core of PEG-HCCs, which have a broad reduction potential range starting at +200 mV and extending to -2 V. Here we describe a new property of these materials: the ability to catalytically transfer electrons between key surrogates and proteins of the mitochondrial electron transport complex in a catalytic fashion consistent with the concept of a nanozyme. The estimated reduction potential of PEG-HCCs is similar to that of ubiquinone and they enabled the catalytic transfer of electrons from low reduction potential species to higher reduction electron transport complex constituents. PEG-HCCs accelerated the reduction of resazurin (a test indicator of mitochondrial viability) and cytochrome c by NADH and ascorbic acid in solution. Kinetic experiments suggested a transient tertiary complex. Electron paramagnetic resonance demonstrated NADH increased the magnitude of PEG-HCCs' intrinsic radical, which then reduced upon subsequent addition of cytochrome c or resazurin. Deconvolution microscopy identified PEG-HCCs in close proximity to mitochondria after brief incubation with cultured SHSY-5Y human neuroblastoma cells. Compared to methylene blue (MB), considered a prototypical small molecule electron transport shuttle, PEG-HCCs were more protective against toxic effects of hydrogen peroxide in vitro and did not demonstrate impaired cell viability as did MB. PEG-HCCs were protective in vitro when cells were exposed to sodium cyanide, a mitochondrial complex IV poison. Because mitochondria are a major source of free radicals in pathology, we suggest that this newly described nanozyme action helps explain their in vivo efficacy in a range of injury models. These findings may also extend their use to mitochondrial disorders.

Highly Oxidized Graphene Quantum Dots from Coal as Efficient Antioxidants.

Graphene quantum dots (GQDs) have recently been employed in various fields including medicine as antioxidants, primarily because of favorable biocompatibility in comparison to common inorganic quantum dots, although the structural features that lead to the biological activities of GQDs are poorly understood. Here, we report that coal-derived GQDs and their poly(ethylene glycol)-functionalized derivatives serve as efficient antioxidants, and we evaluate their electrochemical, chemical, and in vitro biological activities.

Inducible lung epithelial resistance requires multisource reactive oxygen species generation to protect against bacterial infections.

Pneumonia remains a global health threat, in part due to expanding categories of susceptible individuals and increasing prevalence of antibiotic resistant pathogens. However, therapeutic stimulation of the lungs' mucosal defenses by inhaled exposure to a synergistic combination of Toll-like receptor (TLR) agonists known as Pam2-ODN promotes mouse survival of pneumonia caused by a wide array of pathogens. This inducible resistance to pneumonia relies on intact lung epithelial TLR signaling, and inducible protection against viral pathogens has recently been shown to require increased production of epithelial reactive oxygen species (ROS) from multiple epithelial ROS generators. To determine whether similar mechanisms contribute to inducible antibacterial responses, the current work investigates the role of ROS in therapeutically-stimulated protection against Pseudomonas aerugnosa challenges. Inhaled Pam2-ODN treatment one day before infection prevented hemorrhagic lung cytotoxicity and mouse death in a manner that correlated with reduction in bacterial burden. The bacterial killing effect of Pam2-ODN was recapitulated in isolated mouse and human lung epithelial cells, and the protection correlated with inducible epithelial generation of ROS. Scavenging or targeted blockade of ROS production from either dual oxidase or mitochondrial sources resulted in near complete loss of Pam2-ODN-induced bacterial killing, whereas deficiency of induced antimicrobial peptides had little effect. These findings support a central role for multisource epithelial ROS in inducible resistance against a bacterial pathogen and provide mechanistic insights into means to protect vulnerable patients against lethal infections.

Effect of Graphene Nanoribbons (TexasPEG) on locomotor function recovery in a rat model of lumbar spinal cord transection.

A sharply transected spinal cord has been shown to be fused under the accelerating influence of membrane fusogens such as polyethylene glycol (PEG) (GEMINI protocol). Previous work provided evidence that this is in fact possible. Other fusogens might improve current results. In this study, we aimed to assess the effects of PEGylated graphene nanoribons (PEG-GNR, and called "TexasPEG" when prepared as 1wt% dispersion in PEG600) versus placebo (saline) on locomotor function recovery and cellular level in a rat model of spinal cord transection at lumbar segment 1 (L1) level. In vivo and in vitro experiments (n = 10 per experiment) were designed. In the in vivo experiment, all rats were submitted to full spinal cord transection at L1 level. Five weeks later, behavioral assessment was performed using the Basso Beattie Bresnahan (BBB) locomotor rating scale. Immunohistochemical staining with neuron marker neurofilament 200 (NF200) antibody and astrocytic scar marker glial fibrillary acidic protein (GFAP) was also performed in the injured spinal cord. In the in vitro experiment, the effects of TexasPEG application for 72 hours on the neurite outgrowth of SH-SY5Y cells were observed under the inverted microscope. Results of both in vivo and in vitro experiments suggest that TexasPEG reduces the formation of glial scars, promotes the regeneration of neurites, and thereby contributes to the recovery of locomotor function of a rat model of spinal cord transfection.

Inducible Lung Epithelial Resistance Requires Multisource Reactive Oxygen Species Generation To Protect against Viral Infections.

Viral pneumonias cause profound worldwide morbidity, necessitating novel strategies to prevent and treat these potentially lethal infections. Stimulation of intrinsic lung defenses via inhalation of synergistically acting Toll-like receptor (TLR) agonists protects mice broadly against pneumonia, including otherwise-lethal viral infections, providing a potential opportunity to mitigate infectious threats. As intact lung epithelial TLR signaling is required for the inducible resistance and as these cells are the principal targets of many respiratory viruses, the capacity of lung epithelial cells to be therapeutically manipulated to function as autonomous antiviral effectors was investigated. Our work revealed that mouse and human lung epithelial cells could be stimulated to generate robust antiviral responses that both reduce viral burden and enhance survival of isolated cells and intact animals. The antiviral protection required concurrent induction of epithelial reactive oxygen species (ROS) from both mitochondrial and dual oxidase sources, although neither type I interferon enrichment nor type I interferon signaling was required for the inducible protection. Taken together, these findings establish the sufficiency of lung epithelial cells to generate therapeutically inducible antiviral responses, reveal novel antiviral roles for ROS, provide mechanistic insights into inducible resistance, and may provide an opportunity to protect patients from viral pneumonia during periods of peak vulnerability.IMPORTANCE Viruses are the most commonly identified causes of pneumonia and inflict unacceptable morbidity, despite currently available therapies. While lung epithelial cells are principal targets of respiratory viruses, they have also been recently shown to contribute importantly to therapeutically inducible antimicrobial responses. This work finds that lung cells can be stimulated to protect themselves against viral challenges, even in the absence of leukocytes, both reducing viral burden and improving survival. Further, it was found that the protection occurs via unexpected induction of reactive oxygen species (ROS) from spatially segregated sources without reliance on type I interferon signaling. Coordinated multisource ROS generation has not previously been described against viruses, nor has ROS generation been reported for epithelial cells against any pathogen. Thus, these findings extend the potential clinical applications for the strategy of inducible resistance to protect vulnerable people against viral infections and also provide new insights into the capacity of lung cells to protect against infections via novel ROS-dependent mechanisms.

Efficacy of Novel Carbon Nanoparticle Antioxidant Therapy in a Severe Model of Reversible Middle Cerebral Artery Stroke in Acutely Hyperglycemic Rats.

INTRODUCTION: While oxidative stress can be measured during transient cerebral ischemia, antioxidant therapies for ischemic stroke have been clinically unsuccessful. Many antioxidants are limited in their range and/or capacity for quenching radicals and can generate toxic intermediates overwhelming depleted endogenous protection. We developed a new antioxidant class, 40 nm × 2 nm carbon nanoparticles, hydrophilic carbon clusters, conjugated to poly(ethylene glycol) termed PEG-HCCs. These particles are high-capacity superoxide dismutase mimics, are effective against hydroxyl radical, and restore the balance between nitric oxide and superoxide in the vasculature. Here, we report the effects of PEG-HCCs administered during reperfusion after transient middle cerebral artery occlusion (tMCAO) by suture in the rat under hyperglycemic conditions. Hyperglycemia occurs in one-third of stroke patients and worsens clinical outcome. In animal models, this worsening occurs largely by accelerating elaboration of reactive oxygen species (ROS) during reperfusion. METHODS: PEG-HCCs were studied for their protective ability against hydrogen peroxide in b.End3 brain endothelial cell line and E17 primary cortical neuron cultures. In vivo, hyperglycemia was induced by streptozotocin injection 2 days before tMCAO. 58 Male Sprague-Dawley rats were analyzed. They were injected IV with PBS or PEG-HCCs (4 mg/kg 2×) at the time of recanalization after either 90- or 120-min occlusion. Rats were survived for up to 3 days, and infarct volume characteristics and neurological functional outcome (modified Bederson Score) were assessed. RESULTS: PEG-HCCs were protective against hydrogen peroxide in both culture models. In vivo improvement was found after PEG-HCCs with 90-min ischemia with reduction in infarct size (42%), hemisphere swelling (46%), hemorrhage score (53%), and improvement in Bederson score (70%) (p = 0.068-0.001). Early high mortality in the 2-h in the PBS control group precluded detailed analysis, but a trend was found in improvement in all factors, e.g., reduction in infarct volume (48%; p = 0.034) and a 56% improvement in Bederson score (p = 0.055) with PEG-HCCs. CONCLUSION: This nano-antioxidant showed some improvement in several outcome measures in a severe model of tMCAO when administered at a clinically relevant time point. Long-term studies and additional models are required to assess potential for clinical use, especially for patients hyperglycemic at the time of their stroke, as these patients have the worst outcomes.

Physical and electrical characterization of TexasPEG: An electrically conductive neuronal scaffold.

BACKGROUND: Graphene and its derivatives have been shown to be biocompatible and electrically active materials upon which neurons readily grow. The fusogen poly(ethylene glycol) (PEG) has been shown to improve outcomes after cervical and dorsal spinal cord transection. The long and narrow PEGylated graphene nanoribbon stacks (PEG-GNRs) with their 5 µm × 200 nm × 10 nm dimensions can provide a scaffold upon which neurons can grow and fuse. We disclose here the extensive characterization data for the PEG-GNRs. METHODS: PEG-GNRs were chemically synthesized and chemically and electrically characterized. RESULTS: The average aspect ratio of the PEG-GNRs was determined to be ~85, which corresponds to a critical percolation value (the point where insulating material becomes conductive by addition of conductive particles) of 1%. However, there was not a sharp increase in AC conductivity at frequencies relevant to action potentials. CONCLUSION: A robust characterization of PEG-GNRs is discussed, though the precise origin of efficacy in improving outcomes following spinal cord transection is not known.

Conformational dynamics of the frameshift stimulatory structure in HIV-1.

Programmed ribosomal frameshifting (PRF) in HIV-1 is thought to be stimulated by a hairpin in the mRNA, although a pseudoknot-like triplex has also been proposed. Because the conformational dynamics of the stimulatory structure under tension applied by the ribosomal helicase during translation may play an important role in PRF, we used optical tweezers to apply tension to the HIV stimulatory structure and monitor its unfolding and refolding dynamics. The folding and unfolding kinetics and energy landscape of the hairpin were measured by ramping the force on the hairpin up and down, providing a detailed biophysical characterization. Unexpectedly, whereas unfolding reflected the simple two-state behavior typical of many hairpins, refolding was more complex, displaying significant heterogeneity. Evidence was found for multiple refolding pathways as well as previously unsuspected, partially folded intermediates. Measuring a variant mRNA containing only the sequence required to form the proposed triplex, it behaved largely in the same way. Nonetheless, very rarely, high-force unfolding events characteristic of pseudoknot-like structures were observed. The rare occurrence of the triplex suggests that the hairpin is the functional stimulatory structure. The unusual heterogeneity of the hairpin dynamics under tension suggests a possible functional role in PRF similar to the dynamics of other stimulatory structures.

Preferential uptake of antioxidant carbon nanoparticles by T lymphocytes for immunomodulation.

Autoimmune diseases mediated by a type of white blood cell-T lymphocytes-are currently treated using mainly broad-spectrum immunosuppressants that can lead to adverse side effects. Antioxidants represent an alternative approach for therapy of autoimmune disorders; however, dietary antioxidants are insufficient to play this role. Antioxidant carbon nanoparticles scavenge reactive oxygen species (ROS) with higher efficacy than dietary and endogenous antioxidants. Furthermore, the affinity of carbon nanoparticles for specific cell types represents an emerging tactic for cell-targeted therapy. Here, we report that nontoxic poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), known scavengers of the ROS superoxide (O2•-) and hydroxyl radical, are preferentially internalized by T lymphocytes over other splenic immune cells. We use this selectivity to inhibit T cell activation without affecting major functions of macrophages, antigen-presenting cells that are crucial for T cell activation. We also demonstrate the in vivo effectiveness of PEG-HCCs in reducing T lymphocyte-mediated inflammation in delayed-type hypersensitivity and in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Our results suggest the preferential targeting of PEG-HCCs to T lymphocytes as a novel approach for T lymphocyte immunomodulation in autoimmune diseases without affecting other immune cells.

Spinal cord fusion with PEG-GNRs (TexasPEG): Neurophysiological recovery in 24 hours in rats.

BACKGROUND: The GEMINI spinal cord fusion protocol has been developed to achieve a successful cephalosomatic anastomosis. Here, for the first time, we report the effects of locally applied water-soluble, conductive PEG(polyethylene glycol)ylated graphene nanoribbons (PEG-GNRs) on neurophysiologic conduction after sharp cervical cord transection in rats. PEG-GNRs were produced by the polymerization of ethylene oxide from anion-edged graphene nanoribbons. These combine the fusogenic potential of PEG with the electrical conducting properties of the graphene nanoribbons. METHODS: Laminectomy and transection of cervical spinal cord (C5) was performed on Female Sprague-Dawley (SD) rats. After applying PEG-GNR on the severed part, electrophysiological recovery of the reconstructed cervical spinal cord was confirmed by somatosensory evoked potentials (SSEPs) at 24 h after surgery. RESULTS: While no SSEPs were detected in the control group, PEG-GNR treated group showed fast recovery of SSEPs at 24 h after the surgery. CONCLUSION: In this preliminary dataset, for the first time, we report the effect of a novel form of PEG with the goal of rapid reconstruction of a sharply severed spinal cord.

Mechanistic Study of the Conversion of Superoxide to Oxygen and Hydrogen Peroxide in Carbon Nanoparticles.

Hydrophilic carbon clusters (HCCs) are oxidized carbon nanoparticles with a high affinity for electrons. The electron accepting strength of HCCs, employing the efficient conversion of superoxide (O2(•-)) to molecular oxygen (O2) via single-electron oxidation, was monitored using cyclic voltammetry and electron paramagnetic resonance spectroscopy. We found that HCCs possess O2 reduction reaction (ORR) capabilities through a two-electron process with the formation of H2O2. By comparing results from aprotic solvents to those obtained from ORR activity in aqueous media, we propose a mechanism for the origin of the antioxidant and superoxide dismutase mimetic properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs).

Passive Anti-Icing and Active Deicing Films.

Anti-icing and deicing are the two major pathways for suppressing adhesion of ice on surfaces, yet materials with dual capabilities are rare. In this work, we have designed a perfluorododecylated graphene nanoribbon (FDO-GNR) film that takes advantage of both the low polarizability of perfluorinated carbons and the intrinsic conductive nature of graphene nanoribbons. The FDO-GNR films are superhydrophobic with a sheet resistance below 8 kΩ·sq(-1) and then exhibit an anti-icing property that prevents freezing of incoming ice-cold water down to -14 °C. After that point, voltage can be applied to the films to resistively heat and deice the surface. Further a lubricating liquid can be employed to create a slippery surface to improve the film's deicing performance. The FDO-GNR films can be easily switched between the superhydrophobic anti-icing mode and the slippery deicing mode by applying the lubricant. A spray-coating method makes it suitable for large-scale applications. The anti-icing and deicing properties render the FDO-GNR films with promise for use in extreme environments.

The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T(H)17 cell-dependent emphysema.

Smoking-related emphysema is a chronic inflammatory disease driven by the T(H)17 subset of helper T cells through molecular mechanisms that remain obscure. Here we explored the role of the microRNA miR-22 in emphysema. We found that miR-22 was upregulated in lung myeloid dendritic cells (mDCs) of smokers with emphysema and antigen-presenting cells (APCs) of mice exposed to smoke or nanoparticulate carbon black (nCB) through a mechanism that involved the transcription factor NF-κB. Mice deficient in miR-22, but not wild-type mice, showed attenuated T(H)17 responses and failed to develop emphysema after exposure to smoke or nCB. We further found that miR-22 controlled the activation of APCs and T(H)17 responses through the activation of AP-1 transcription factor complexes and the histone deacetylase HDAC4. Thus, miR-22 is a critical regulator of both emphysema and T(H)17 responses.

Regulation of mitotic cytoskeleton dynamics and cytokinesis by integrin-linked kinase in retinoblastoma cells.

During cell division integrin-linked kinase (ILK) has been shown to regulate microtubule dynamics and centrosome clustering, processes involved in cell cycle progression, and malignant transformation. In this study, we examine the effects of downregulating ILK on mitotic function in human retinoblastoma cell lines. These retinal cancer cells, caused by the loss of function of two gene alleles (Rb1) that encode the retinoblastoma tumour suppressor, have elevated expression of ILK. Here we show that inhibition of ILK activity results in a concentration-dependent increase in nuclear area and multinucleated cells. Moreover, inhibition of ILK activity and expression increased the accumulation of multinucleated cells over time. In these cells, aberrant cytokinesis and karyokinesis correlate with altered mitotic spindle organization, decreased levels of cortical F-actin and centrosome de-clustering. Centrosome de-clustering, induced by ILK siRNA, was rescued in FLAG-ILK expressing Y79 cells as compared to those expressing FLAG-tag alone. Inhibition of ILK increased the proportion of cells exhibiting mitotic spindles and caused a significant G2/M arrest as early as 24 hours after exposure to QLT-0267. Live cell analysis indicate ILK downregulation causes an increase in multipolar anaphases and failed cytokinesis (bipolar and multipolar) of viable cells. These studies extend those indicating a critical function for ILK in mitotic cytoskeletal organization and describe a novel role for ILK in cytokinesis of Rb deficient cells.

Anti-frameshifting ligand reduces the conformational plasticity of the SARS virus pseudoknot.

Programmed -1 ribosomal frameshifting (-1 PRF) stimulated by mRNA pseudoknots regulates gene expression in many viruses, making pseudoknots potential targets for anti-viral drugs. The mechanism by which pseudoknots trigger -1 PRF, however, remains controversial, with several competing models. Recent work showed that high -1 PRF efficiency was linked to high pseudoknot conformational plasticity via the formation of alternate conformers. We tested whether pseudoknots bound with an anti-frameshifting ligand exhibited a similar correlation between conformational plasticity and -1 PRF efficiency by measuring the effects of a ligand that was found to inhibit -1 PRF in the SARS coronavirus on the conformational dynamics of the SARS pseudoknot. Using single-molecule force spectroscopy to unfold pseudoknots mechanically, we found that the ligand binding effectively abolished the formation of alternate conformers. This result extends the connection between -1 PRF and conformational dynamics and, moreover, suggests that targeting the conformational dynamics of pseudoknots may be an effective strategy for anti-viral drug design.