Export and role of flavin electron shuttles in Shewanella oneidensis Strain MR-1

University of Minnesota Ph.D. dissertation. September 2014. Major: Microbiology, Immunology and Cancer Biology. Advisor: Jeffrey A. Gralnick. 1 computer file (PDF); ix, 119 pages.Our planet is home to microbes with an impressive diversity of metabolic capabilities. For instance, dissimilatory metal reducing bacteria (DMRB) can respire minerals, heavy metals, and electrodes. Electrode respiration is important both for investigating DMRB and biotechnology applications including biosensors, bioenergy, and wastewater treatment. Placing electrodes in wastewater allows for electricity production and oxidation of carbon sources to carbon dioxide. Shewanella oneidensis MR-1, a model DMRB, transfers electrons generated from metabolism across the inner and outer membranes to insoluble external electron acceptors. This feat is accomplished using an electron conduit consisting of multi-heme cytochromes and flavin electron shuttles. The electron conduit in Shewanella evolved to move electrons out of the cell, but in controlled environments, the conduit is functionally reversible, meaning electrons can flow either out of or into the cell. In a process termed electrosynthesis, electrons are transferred from an electrode into a cell to direct metabolism to produce specific metabolites. A useful way to study electrosynthesis in cells is to use three-electrode bioreactors. The working electrode in a bioreactor is poised at an oxidizing or reducing potential and continuously measures electrons entering or leaving the electrode. When electrodes were used to donate electrons to attached Shewanella cells, intracellular ATP was produced. Mutants defective in generating or using proton motive force were unable to produce wild-type levels of ATP. If significant quantities of ATP can be produced by reverse electron flow, it will be a critical step towards fixing carbon dioxide into metabolic precursors and eventually into biofuels

A Re-evaluation of Electron-Transfer Mechanisms in Microbial Electrochemistry: Shewanella Releases Iron that Mediates Extracellular Electron Transfer

Exoelectrogenic bacteria can couple their metabolism to extracellular electron acceptors, including macroscopic electrodes, and this has applications in energy production, bioremediation and biosensing. Optimisation of these technologies relies on a detailed molecular understanding of extracellular electron-transfer (EET) mechanisms, and Shewanella oneidensis MR-1 (MR-1) has become a model organism for such fundamental studies. Here, cyclic voltammetry was used to determine the relationship between the surface chemistry of electrodes (modified gold, ITO and carbon electrodes) and the EET mechanism. On ultra-smooth gold electrodes modified with self-assembled monolayers containing carboxylic-acid-terminated thiols, an EET pathway dominates with an oxidative catalytic onset at 0.1V versus SHE. Addition of iron(II)chloride enhances the catalytic current, whereas the siderophore deferoxamine abolishes this signal, leading us to conclude that this pathway proceeds via an iron mediated electron transfer mechanism. The same EET pathway is observed at other electrodes, but the onset potential is dependent on the electrolyte composition and electrode surface chemistry. EET pathways with onset potentials above -0.1V versus SHE have previously been ascribed to direct electron-transfer (DET) mechanisms through the surface exposed decaheme cytochromes (MtrC/OmcA) of MR-1. In light of the results reported here, we propose that the previously identified DET mechanism of MR-1 needs to be re-evaluated

Use of magnetic source imaging to assess recovery after severe traumatic brain injury—an MEG pilot study

RationaleSevere TBI (sTBI) is a devastating neurological injury that comprises a significant global trauma burden. Early comprehensive neurocritical care and rehabilitation improve outcomes for such patients, although better diagnostic and prognostic tools are necessary to guide personalized treatment plans.MethodsIn this study, we explored the feasibility of conducting resting state magnetoencephalography (MEG) in a case series of sTBI patients acutely after injury (~7 days), and then about 1.5 and 8 months after injury. Synthetic aperture magnetometry (SAM) was utilized to localize source power in the canonical frequency bands of delta, theta, alpha, beta, and gamma, as well as DC–80 Hz.ResultsAt the first scan, SAM source maps revealed zones of hypofunction, islands of preserved activity, and hemispheric asymmetry across bandwidths, with markedly reduced power on the side of injury for each patient. GCS scores improved at scan 2 and by scan 3 the patients were ambulatory. The SAM maps for scans 2 and 3 varied, with most patients showing increasing power over time, especially in gamma, but a continued reduction in power in damaged areas and hemispheric asymmetry and/or relative diminishment in power at the site of injury. At the group level for scan 1, there was a large excess of neural generators operating within the delta band relative to control participants, while the number of neural generators for beta and gamma were significantly reduced. At scan 2 there was increased beta power relative to controls. At scan 3 there was increased group-wise delta power in comparison to controls.ConclusionIn summary, this pilot study shows that MEG can be safely used to monitor and track the recovery of brain function in patients with severe TBI as well as to identify patient-specific regions of decreased or altered brain function. Such MEG maps of brain function may be used in the future to tailor patient-specific rehabilitation plans to target regions of altered spectral power with neurostimulation and other treatments

The role of riboflavin in decolourisation of Congo red and bioelectricity production using Shewanella oneidensis-MR1 under MFC and non-MFC conditions

Dissimilatory metal reducing bacteria can exchange electrons extracellularly and hold great promise for their use in simultaneous wastewater treatment and electricity production. This study investigated the role of riboflavin, an electron carrier, in the decolourisation of Congo red in microbial fuel cells (MFCs) using Shewanella oneidensis MR-1 as a model organism. The contribution of the membrane-bound protein MtrC to the decolourisation process was also investigated. Within the range of riboflavin concentrations tested, 20 µM was found to be the best with >95% of the dye (initial concentration 200 mg/L) decolourised in MFCs within 50 h compared to 90% in the case where no riboflavin was added. The corresponding maximum power density was 45 mW/m2. There was no significant difference in the overall decolourisation efficiencies of Shewanela oneidensis MR-1 ΔMtrC mutants compared to the wild type. However, in terms of power production the mutant produced more power (Pmax 76 mW/m2) compared to the wild type (Pmax 46 mW/m2) which was attributed to higher levels of riboflavin secreted in solution. Decolourisation efficiencies in non-MFC systems (anaerobic bottles) were similar to those under MFC systems indicating that electricity generation in MFCs does not impair dye decolourisation efficiencies. The results suggest that riboflavin enhances both decolourisation of dyes and simultaneous electricity production in MFCs

Photoreduction of Shewanella oneidensis Extracellular Cytochromes by Organic Chromophores and Dye-Sensitized TiO2.

The transfer of photoenergized electrons from extracellular photosensitizers across a bacterial cell envelope to drive intracellular chemical transformations represents an attractive way to harness nature's catalytic machinery for solar-assisted chemical synthesis. In Shewanella oneidensis MR-1 (MR-1), trans-outer-membrane electron transfer is performed by the extracellular cytochromes MtrC and OmcA acting together with the outer-membrane-spanning porin⋅cytochrome complex (MtrAB). Here we demonstrate photoreduction of solutions of MtrC, OmcA, and the MtrCAB complex by soluble photosensitizers: namely, eosin Y, fluorescein, proflavine, flavin, and adenine dinucleotide, as well as by riboflavin and flavin mononucleotide, two compounds secreted by MR-1. We show photoreduction of MtrC and OmcA adsorbed on RuII -dye-sensitized TiO2 nanoparticles and that these protein-coated particles perform photocatalytic reduction of solutions of MtrC, OmcA, and MtrCAB. These findings provide a framework for informed development of strategies for using the outer-membrane-associated cytochromes of MR-1 for solar-driven microbial synthesis in natural and engineered bacteria.This work was supported by the UK Biotechnology and Biological Sciences Research Council (grants BB/K009753/1, BB/K010220/1, BB/K009885/1, and BB/K00929X/1), the Engineering and Physical Sciences Research Council (EP/M001989/1, PhD studentship 1307196 to E.V.A.), a Royal Society Leverhulme Trust Senior Research Fellowship to J.N.B., the Christian Doppler Research Association, and OMV group

Opportunities for improving animal welfare in rodent models of epilepsy and seizures

Animal models of epilepsy and seizures, mostly involving mice and rats, are used to understand the pathophysiology of the different forms of epilepsy and their comorbidities, to identify biomarkers, and to discover new antiepileptic drugs and treatments for comorbidities. Such models represent an important area for application of the 3Rs (replacement, reduction and refinement of animal use). This report provides background information and recommendations aimed at minimising pain, suffering and distress in rodent models of epilepsy and seizures in order to improve animal welfare and optimise the quality of studies in this area. The report includes practical guidance on principles of choosing a model, induction procedures, in vivo recordings, perioperative care, welfare assessment, humane endpoints, social housing, environmental enrichment, reporting of studies and data sharing. In addition, some model-specific welfare considerations are discussed, and data gaps and areas for further research are identified. The guidance is based upon a systematic review of the scientific literature, survey of the international epilepsy research community, consultation with veterinarians and animal care and welfare officers, and the expert opinion and practical experience of the members of a Working Group convened by the United Kingdom's National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs)

Hippocampal involvement in nonpathological déjà vu: Subfield vulnerability rather than temporal lobe epilepsy equivalent

Introduction Morphological correlates of nonpathological déjà vu (DV) have been identified recently within the human brain. Significantly reduced gray matter volume (GMV) within a set of cortical and subcortical regions reported in subjects experiencing DV seems to mirror the distribution of GMV reduction in mesial temporal lobe epilepsy (MTLE) patients but vary in terms of the hippocampus. Another condition associated with hippocampal GMV reduction and DV alike disturbance in memory processing is schizophrenia (SCH). Here, we tested the hypothesis that hippocampal involvement in nonpathological DV resembles more closely the pattern of GMV decrease observed in MTLE compared with that occurring in SCH. Methods Using automated segmentation of the MRI data we compared the medians of GMV within 12 specific hippocampal subfields in healthy individuals that do (DV+; N = 87) and do not report déjà vu experience (DV−; N = 26), and patients with MTLE (N = 47) and SCH (N = 29). By Pearson correlation, we then evaluated the similarity of MTLE and SCH groups to DV+ group with respect to spatial distribution of GMV deviation from DV− group. Results Significant GMV decrease was found in MTLE group in most of the subfields. There were just trends in the hippocampal GMV decrease found in DV+ or SCH groups. Concerning the spatial distribution of GMV decrease, we revealed statistically significant correlation for the left hippocampus for SCH vs DV+. Otherwise there was no statistically significant correlation. Conclusions Our findings reveal structural features of hippocampal involvement in nonpathological DV, MTLE, and SCH. Despite our expectations, the pattern of GMV reduction in the DV+ relative to the DV− group does not resemble the pattern observed in MTLE any more than that observed in SCH. The highly similar patterns of the three clinical groups rather suggest an increased vulnerability of certain hippocampal subfields; namely, Cornu Ammonis (CA)4, CA3, dentate gyrus granular cell layer (GC‐DG), hippocampal–amygdaloid transition area (HATA) and subiculum

Cellular injury and neuroinflammation in children with chronic intractable epilepsy

<p>Abstract</p> <p>Objective</p> <p>To elucidate the presence and potential involvement of brain inflammation and cell death in neurological morbidity and intractable seizures in childhood epilepsy, we quantified cell death, astrocyte proliferation, microglial activation and cytokine release in brain tissue from patients who underwent epilepsy surgery.</p> <p>Methods</p> <p>Cortical tissue was collected from thirteen patients with intractable epilepsy due to focal cortical dysplasia (6), encephalomalacia (5), Rasmussen's encephalitis (1) or mesial temporal lobe epilepsy (1). Sections were processed for immunohistochemistry using markers for neuron, astrocyte, microglia or cellular injury. Cytokine assay was performed on frozen cortices. Controls were autopsy brains from eight patients without history of neurological diseases.</p> <p>Results</p> <p>Marked activation of microglia and astrocytes and diffuse cell death were observed in epileptogenic tissue. Numerous fibrillary astrocytes and their processes covered the entire cortex and converged on to blood vessels, neurons and microglia. An overwhelming number of neurons and astrocytes showed DNA fragmentation and its magnitude significantly correlated with seizure frequency. Majority of our patients with abundant cell death in the cortex have mental retardation. IL-1beta, IL-8, IL-12p70 and MIP-1beta were significantly increased in the epileptogenic cortex; IL-6 and MCP-1 were significantly higher in patients with family history of epilepsy.</p> <p>Conclusions</p> <p>Our results suggest that active neuroinflammation and marked cellular injury occur in pediatric epilepsy and may play a common pathogenic role or consequences in childhood epilepsy of diverse etiologies. Our findings support the concept that immunomodulation targeting activated microglia and astrocytes may be a novel therapeutic strategy to reduce neurological morbidity and prevent intractable epilepsy.</p

Hippocampal-Dependent Spatial Memory in the Water Maze is Preserved in an Experimental Model of Temporal Lobe Epilepsy in Rats

Cognitive impairment is a major concern in temporal lobe epilepsy (TLE). While different experimental models have been used to characterize TLE-related cognitive deficits, little is known on whether a particular deficit is more associated with the underlying brain injuries than with the epileptic condition per se. Here, we look at the relationship between the pattern of brain damage and spatial memory deficits in two chronic models of TLE (lithium-pilocarpine, LIP and kainic acid, KA) from two different rat strains (Wistar and Sprague-Dawley) using the Morris water maze and the elevated plus maze in combination with MRI imaging and post-morten neuronal immunostaining. We found fundamental differences between LIP- and KA-treated epileptic rats regarding spatial memory deficits and anxiety. LIP-treated animals from both strains showed significant impairment in the acquisition and retention of spatial memory, and were unable to learn a cued version of the task. In contrast, KA-treated rats were differently affected. Sprague-Dawley KA-treated rats learned less efficiently than Wistar KA-treated animals, which performed similar to control rats in the acquisition and in a probe trial testing for spatial memory. Different anxiety levels and the extension of brain lesions affecting the hippocampus and the amydgala concur with spatial memory deficits observed in epileptic rats. Hence, our results suggest that hippocampal-dependent spatial memory is not necessarily affected in TLE and that comorbidity between spatial deficits and anxiety is more related with the underlying brain lesions than with the epileptic condition per se

Th17 cytokines and arthritis

Th17 cells are implicated in human autoimmune diseases, such as rheumatoid arthritis (RA), although it has not been established whether this persistent destructive arthritis is driven by Th1 and/or Th17 cells. Interleukin-17A (IL-17A) contributes to the pathogenesis of arthritis as has been shown in several experimental arthritis models. Importantly, recent data from first clinical trials with anti-IL-17A antibody treatment in psoriatic arthritis patients and RA patients looks promising. This review summarizes the findings about the role of Th17 cells in arthritis and discusses the impact of the different Th17 cytokines in the pathogenesis of this disease. However, further studies are needed to unravel the interplay between IL-17A and other Th17 cytokines such as IL-17F, IL-22, and IL-21 in the pathoimmunological process of this crippling disease, in particular, whether regulating Th17 cell activity or specific combinations of Th17 cytokines will have additional value compared to neutralizing IL-17A activity alone. Moreover, tumor necrosis factor-positive Th17 cells are discussed as potential dangerous cells in driving persistent arthritis in human early RA