Ethane-beta-Sultam Modifies the Activation of the Innate Immune System Induced by Intermittent Ethanol Administration in Female Adolescent Rats

Intermittent ethanol abuse or ‘binge drinking’ during adolescence induces neuronal damage, which may be associated with cognitive dysfunction. To investigate the neurochemical processes involved, rats were administered either 1 g/kg or 2 g/kg ethanol in a ‘binge drinking’ regime. After only 3 weeks, significant activation of phagocytic cells in the peripheral (alveolar macrophages) and the hippocampal brain region (microglia cells) was present,as exemplified by increases in the release of pro-inflammatory cytokines in the macrophages and of iNOS in the microglia. This was associated with neuronal loss in the hippocampus CA1 region. Daily supplementation with a taurine prodrug, ethane-β-sultam, 0.028 g/kg, during the intermittent ethanol loading regime, supressed the release of the pro-inflammatory cytokines and of reactive nitrogen species, as well as neuronal loss, particularly in the rats administered the lower dose of ethanol, 1 g/kg. Plasma, macrophage and hippocampal taurine levels increased marginally after ethane-β-sultam supplementation. The ‘binge drinking’ ethanol rats administered 1 g/kg ethanol showed increased latencies to those of the control rats in their acquisition of spacial navigation in the Morris Water Maze, which was normalised to that of the controls values after ethane-β-sultam administration. Such results confirm that the administration of ethane-β-sultam to binge drinking rats reduces neuroinflammation in both the periphery and the brain, suppresses neuronal loss, and improved working memory of rats in a water maze study

Detection of intrinsic source structure at ~3 Schwarzschild radii with Millimeter-VLBI observations of SAGITTARIUS A*

We report results from very long baseline interferometric (VLBI) observations of the supermassive black hole in the Galactic center, Sgr A*, at 1.3 mm (230 GHz). The observations were performed in 2013 March using six VLBI stations in Hawaii, California, Arizona, and Chile. Compared to earlier observations, the addition of the APEX telescope in Chile almost doubles the longest baseline length in the array, provides additional {\it uv} coverage in the N-S direction, and leads to a spatial resolution of $\sim$30 $\mu$as ($\sim$3 Schwarzschild radii) for Sgr A*. The source is detected even at the longest baselines with visibility amplitudes of $\sim$4-13% of the total flux density. We argue that such flux densities cannot result from interstellar refractive scattering alone, but indicate the presence of compact intrinsic source structure on scales of $\sim$3 Schwarzschild radii. The measured nonzero closure phases rule out point-symmetric emission. We discuss our results in the context of simple geometric models that capture the basic characteristics and brightness distributions of disk- and jet-dominated models and show that both can reproduce the observed data. Common to these models are the brightness asymmetry, the orientation, and characteristic sizes, which are comparable to the expected size of the black hole shadow. Future 1.3 mm VLBI observations with an expanded array and better sensitivity will allow a more detailed imaging of the horizon-scale structure and bear the potential for a deep insight into the physical processes at the black hole boundary.Comment: 11 pages, 5 figures, accepted to Ap

Bypass of mutagenic O 6 -Carboxymethylguanine DNA Adducts by Human Y- and B-Family Polymerases

The generation of chemical alkylating agents from nitrosation of glycine and bile acid conjugates in the gastrointestinal tract is hypothesized to initiate carcinogenesis. O6-carboxymethylguanine (O6-CMG) is a product of DNA alkylation derived from nitrosated glycine. Although the tendency of the structurally related adduct O6-methylguanine to code for the misincoporation of TTP during DNA replication is well-established, the impact of the presence of the O6-CMG adduct in a DNA template on the efficiency and fidelity of translesion DNA synthesis (TLS) by human DNA polymerases (Pols) has hitherto not been described. Herein, we characterize the ability of the four human TLS Pols η, ι, κ, and ζ and the replicative Pol δ to bypass O6-CMG in a prevalent mutational hot-spot for colon cancer. The results indicate that Pol η replicates past O6-CMG, incorporating dCMP or dAMP, whereas Pol κ incorporates dCMP only, and Pol ι incorporates primarily dTMP. Additionally, the subsequent extension step was carried out with high efficiency by TLS Pols η, κ, and ζ, while Pol ι was unable to extend from a terminal mismatch. These results provide a first basis of O6-CMG-promoted base misincorporation by Y- and B-family polymerases potentially leading to mutational signatures associated with colon cancer

High resolution and dynamic imaging of biopersistence and bioreactivity of extra and intracellular MWNTs exposed to microglial cells

Multi-walled carbon nanotubes (MWNTs) are increasingly being developed both as neuro-therapeutic drug delivery systems to the brain and as neural scaffolds to drive tissue regeneration across lesion sites. MWNTs with different degrees of acid oxidation may have different bioreactivities and propensities to aggregate in the extracellular environment, and both individualised and aggregated MWNTs may be expected to be found in the brain. Before practical application, it is vital to understand how both aggregates and individual MWNTs will interact with local phagocytic immune cells, the microglia, and ultimately to determine their biopersistence in the brain. The processing of extra- and intracellular MWNTs (both pristine and when acid oxidised) by microglia was characterised across multiple length scales by correlating a range of dynamic, quantitative and multi-scale techniques, including: UV-vis spectroscopy, light microscopy, focussed ion beam scanning electron microscopy and transmission electron microscopy. Dynamic, live cell imaging revealed the ability of microglia to break apart and internalise micron-sized extracellular agglomerates of acid oxidised MWNTs, but not pristine MWNTs. The total amount of MWNTs internalised by, or strongly bound to, microglia was quantified as a function of time. Neither the significant uptake of oxidised MWNTs, nor the incomplete uptake of pristine MWNTs affected microglial viability, pro-inflammatory cytokine release or nitric oxide production. However, after 24 h exposure to pristine MWNTs, a significant increase in the production of reactive oxygen species was observed. Small aggregates and individualised oxidised MWNTs were present in the cytoplasm and vesicles, including within multilaminar bodies, after 72 h. Some evidence of morphological damage to oxidised MWNT structure was observed including highly disordered graphitic structures, suggesting possible biodegradation. This work demonstrates the utility of dynamic, quantitative and multi-scale techniques in understanding the different cellular processing routes of functionalised nanomaterials. This correlative approach has wide implications for assessing the biopersistence of MWNT aggregates elsewhere in the body, in particular their interaction with macrophages in the lung

Persistent Asymmetric Structure of Sagittarius A* on Event Horizon Scales

The Galactic Center black hole Sagittarius A* (Sgr A*) is a prime observing target for the Event Horizon Telescope (EHT), which can resolve the 1.3 mm emission from this source on angular scales comparable to that of the general relativistic shadow. Previous EHT observations have used visibility amplitudes to infer the morphology of the millimeter-wavelength emission. Potentially much richer source information is contained in the phases. We report on 1.3 mm phase information on Sgr A* obtained with the EHT on a total of 13 observing nights over 4 years. Closure phases, the sum of visibility phases along a closed triangle of interferometer baselines, are used because they are robust against phase corruptions introduced by instrumentation and the rapidly variable atmosphere. The median closure phase on a triangle including telescopes in California, Hawaii, and Arizona is nonzero. This result conclusively demonstrates that the millimeter emission is asymmetric on scales of a few Schwarzschild radii and can be used to break 180-degree rotational ambiguities inherent from amplitude data alone. The stability of the sign of the closure phase over most observing nights indicates persistent asymmetry in the image of Sgr A* that is not obscured by refraction due to interstellar electrons along the line of sight.Comment: 11 pages, accepted to Ap

Neuroprotective role for RORA in Parkinson’s disease revealed by analysis of post-mortem brain and a dopaminergic cell line

Parkinson's disease (PD) is almost twice as prevalent in men, which has largely been attributed to neuroprotective effect of oestradiol in women. RORA (retinoic acid receptor-related orphan receptor alpha) regulates the transcription of central aromatase, the enzyme responsible for local oestradiol synthesis, simultaneously, RORA expression is regulated by sex hormones. Moreover, RORA protects neurones against oxidative stress, a key mechanism contributing to the loss of dopaminergic neurones in PD. Therefore, we hypothesized that there would be sex differences in RORA expression in the substantia nigra pars compacta (SNpc), which could contribute to sex differences observed in PD prevalence and pathogenesis. In a case control study, qPCR and western blot analyses were used to quantify gene and protein expression in the SNpc of post-mortem brains (n = 14 late-stage PD and 11 age and sex matched controls). The neuroprotective properties of a RORA agonist were then investigated directly using a cell culture toxin-based model of PD coupled with measures of viability, mitochondrial function and apoptosis. RORA was expressed at significantly higher levels in the SNpc from control females' brains compared to males. In PD, we found a significant increase in SNpc RORA expression in male PD compared to female PD. Treatment with a RORA agonist showed a significant neuroprotection in our cell culture model of PD and revealed significant effects on intracellular factors involved in neuronal survival and demise. This study is the first to demonstrate a sex specific pattern of RORA protein and gene expression in the SNpc of controls post-mortem human brains, and to show that this is differentially altered in male and female PD subjects, thus supporting a role for RORA in sex-specific aspects of PD. Furthermore, our in vitro PD model indicates mechanisms whereby a RORA agonist exerts its neuroprotective effect, thereby highlighting the translational potential for RORA ligands in PD

Microglial inflammation in the parkinsonian substantia nigra: relationship to alpha-synuclein deposition

BACKGROUND: The role of both microglial activation and alpha-synuclein deposition in Parkinson's disease remain unclear. We have tested the hypothesis that if microglia play a primary role in Parkinson's disease pathogenesis, the microglial "activated" phenotype should be associated with histopathological and/or clinical features of the disease. METHODS: We have examined microglial MHC class II expression, a widely used marker of microglial activation, the occurrence of CD68-positive phagocytes and alpha-synuclein immunoreactivity in post-mortem human substantia nigra affected by idiopathic Parkinson's disease (PD). Using semi-quantitative severity ratings, we have examined the relationship between microglial activation, alpha-synuclein deposition, classical neuropathological criteria for PD, subtype of the disease and clinical course. RESULTS: While we did not observe an association between microglial MHC class II expression and clinical parameters, we did find a correlation between disease duration and the macrophage marker CD68 which is expressed by phagocytic microglia. In addition, we observed a significant correlation between the degree of MHC class II expression and alpha-synuclein deposition in the substantia nigra in PD. CONCLUSION: While microglia appeared to respond to alpha-synuclein deposition, MHC class II antigen expression by microglia in the substantia nigra cannot be used as an indicator of clinical PD severity or disease progression. In addition, a contributory or even causative role for microglia in the neuronal loss associated with PD as suggested by some authors seems unlikely. Our data further suggest that an assessment of microglial activation in the aged brain on the basis of immunohistochemistry for MHC class II antigens alone should be done with caution

Novel 1-hydroxypyridin-2-one metal chelators prevent and res-cue ubiquitin proteasomal-related neuronal injury in an in vitro model of Parkinson’s disease

Ubiquitin proteasome system (UPS) impairment, excessive cellular oxidative stress and iron dyshomeostasis are key to substantia nigra dopaminergic neuronal de-generation in Parkinson's disease (PD); however, a link between these features remains unconfirmed. By using the proteasome inhibitor lactacystin we confirm that nigral injury via UPS impairment disrupts iron homeostasis, in turn increasing oxi-dative stress and promoting protein aggregation. We demonstrate the neuroprotec-tive potential of two novel 1-hydroxy-2(1H)-pyridinone (1,2-HOPO) iron chelators, compounds C6 and C9, against lactacystin-induced cell death. We demonstrate that this cellular preservation relates to the compounds’ iron chelating capabilities and subsequent reduced capacity of iron to form reactive oxygen species (ROS), where we also show that the ligands act as antioxidant agents. Our results also de-monstrate the ability of C6 and C9 to reduce intracellular lactacystin-induced α-synuclein burden. Stability constant measurements confirmed a high affinity of C6 and C9 for Fe3+ and display a 3:1 HOPO:Fe3+ complex formation at physiological pH. Reducing iron reactivity could prevent the demise of nigral dopaminergic neurons. We provide evidence that the lactacystin model presents with several neuropatho-logical hallmarks of PD related to iron dyshomeostasis and that the novel chelating compounds C6 and C9 can protect against lactacystin-related neurotoxicity