Energetic Limitations Of Thermophilic Methanogens And Thiosulfate Reducers In The Subsurface Biosphere At Deep-Sea Hydrothermal Vents

This dissertation examined the substrate and energetic limitations of hydrogenotrophic thermophiles from deep-sea hydrothermal vents. Thermophilic and hyperthermophilic organisms in diffuse hydrothermal venting are thought to represent a hot subsurface biosphere associated with deep-sea hydrothermal vents, where primary production is dominated by hydrogenotrophy rather than sulfide oxidation as at the vent/seawater interface of hydrothermal sulfide chimneys. Methanogens and sulfur-reducers are known to compete for hydrogen in mesophilic, freshwater systems, and likely do so in deep-sea hydrothermal vent environments as well. However, the exact size and biomass of the subsurface biosphere is difficult to determine through direct sampling. Firstly, the distribution of thermophilic and hyperthermophilic methanogens, sulfur-reducers, and heterotrophs in diffuse venting fluids at our field site, Axial Volcano (on the Juan de Fuca Ridge), was examined using culture-dependent (Most-Probable-Number) and independent (omics) techniques. It was confirmed that methane production in diffuse venting fluids could be stimulated by the sole addition of hydrogen and incubation at thermophilic and/or hyperthermophilic temperatures, indicating that methanogens in this system are not limited significantly by nutrient or trace element requirements. To determine why one novel hyperthermophilic methanogen from our field site (Methanocaldococcus bathoardescens) appeared to prefer high levels of nitrogen when grown in the lab, its genome was examined for nitrogen assimilation-related genes. In the laboratory, the growth energies of Methanocaldococcus and Methanothermococcus spp. over their full temperature ranges were measured in order to determine Arrhenius constants for their production of methane. They were also grown in continuous flow chemostat culture to determine their hydrogen limitations at both optimal and sub-optimal temperatures for growth, and the Monod kineticsfor their hydrogen use and methane production were measured. Additionally, the minimum hydrogen and thiosulfate requirements, as well as Monod kinetics, were measured in batch bioreactor culture for a thiosulfate-reducing, hydrogenotrophic, thermophilic Desulfurobacterium sp. isolated from another site on the Juan de Fuca Ridge, the Endeavour Segment, to determine where it might compete with methanogens for hydrogen. Finally, the geochemical and distribution data from Axial Volcano and laboratory-derived kinetic data for thermophilic and hyperthermophilic methanogens were used to create a one-dimensional reactive transport model (RTM) of hydrogenotrophic methanogenesis in the subsurface at Axial Volcano. In this way, the relative dimensions and biomass of methanogens in the subsurface can be predicted without direct sampling. In future, this type of model could be used make predictions about the thermophilic subsurface at other vent locations, as well as expanded to include competition between different types of hydrogenotrophs (rather than just hydrogenotrophic methanogens with different optimum temperatures) and interactions with other organisms, such as hydrogen-producing hyperthermophilic heterotrophs

A primary fish gill cell culture model to assess pharmaceutical uptake and efflux:evidence for passive and facilitated transport

AbstractThe gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ngL−1), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport

Biomarkers of Rehabilitation Therapy Vary According To Stroke Severity

Biomarkers that capture treatment effects could improve the precision of clinical decision making for restorative therapies. We examined the performance of candidate structural, functional,and angiogenesis-related MRI biomarkers before and after a 3-week course of standardized robotic therapy in 18 patients with chronic stroke and hypothesized that results vary significantly according to stroke severity. Patients were 4.1 ± 1 months poststroke, with baseline arm Fugl-Meyer scores of 20–60. When all patients were examined together, no imaging measure changed over time in a manner that correlated with treatment-induced motor gains. However, when also considering the interaction with baseline motor status, treatment-induced motor gains were significantly related to change in three functional connectivity measures: ipsilesional motor cortex connectivity with (1) contralesional motor cortex (p = 0 003), (2) contralesional dorsal premotor cortex (p = 0 005), and (3) ipsilesional dorsal premotor cortex (p = 0 004). In more impaired patients, larger treatment gains were associated with greater increases in functional connectivity, whereas in less impaired patients larger treatment gains were associated with greater decreases in functional connectivity. Functional connectivity measures performed best as biomarkers of treatment effects after stroke. The relationship between changes in functional connectivity and treatment gains varied according to baseline stroke severity. Biomarkers of restorative therapy effects are not one-size-fits-all after stroke

Assessing the reliability of uptake and elimination kinetics modelling approaches for estimating bioconcentration factors in the freshwater invertebrate, Gammarus pulex

This study considers whether the current standard toxicokinetic methods are an accurate and applicable assessment of xenobiotic exposure in an aquatic freshwater invertebrate. An in vivo exposure examined the uptake and elimination kinetics for eight pharmaceutical compounds in the amphipod crustacean, Gammarus pulex by measuring their concentrations in both biological material and in the exposure medium over a 96 h period. Selected pharmaceuticals included two anti-inflammatories (diclofenac and ibuprofen), two beta-blockers (propranolol and metoprolol), an anti-depressant (imipramine), an anti-histamine (ranitidine) and two beta-agonists (formoterol and terbutaline). Kinetic bioconcentration factors (BCFs) for the selected pharmaceuticals were derived from a first-order one-compartment model using either the simultaneous or sequential modelling methods. Using the simultaneous method for parameter estimation, BCF values ranged from 12 to 212. In contrast, the sequential method for parameter estimation resulted in bioconcentration factors ranging from 19 to 4533. Observed toxicokinetic plots showed statistically significant lack-of-fits and further interrogation of the models revealed a decreasing trend in the uptake rate constant over time for rantidine, diclofenac, imipramine, metoprolol, formoterol and terbutaline. Previous published toxicokinetic data for 14 organic micro-pollutants were also assessed and similar trends were identified to those observed in this study. The decreasing trend of the uptake rate constant over time highlights the need to interpret modelled data more comprehensively to ensure uncertainties associated with uptake and elimination parameters for determining bioconcentration factors are minimised

Role of Corpus Callosum Integrity in Arm Function Differs Based on Motor Severity After Stroke

While the corpus callosum (CC) is important to normal sensorimotor function, its role in motor function after stroke is less well understood. This study examined the relationship between structural integrity of the motor and sensory sections of the CC, as reflected by fractional anisotropy (FA), and motor function in individuals with a range of motor impairment level due to stroke. Fifty-five individuals with chronic stroke (Fugl-Meyer motor score range 14 to 61) and 18 healthy controls underwent diffusion tensor imaging and a set of motor behavior tests. Mean FA from the motor and sensory regions of the CC and from corticospinal tract (CST) were extracted and relationships with behavioral measures evaluated. Across all participants, FA in both CC regions was significantly decreased after stroke (p \u3c 0.001) and showed a significant, positive correlation with level of motor function. However, these relationships varied based on degree of motor impairment: in individuals with relatively less motor impairment (Fugl-Meyer motor score \u3e 39), motor status correlated with FA in the CC but not the CST, while in individuals with relatively greater motor impairment (Fugl-Meyer motor score ≤ 39), motor status correlated with FA in the CST but not the CC. The role interhemispheric motor connections play in motor function after stroke may differ based on level of motor impairment. These findings emphasize the heterogeneity of stroke, and suggest that biomarkers and treatment approaches targeting separate subgroups may be warranted

Hydrogen limitation and syntrophic growth among natural assemblages of thermophilic methanogens at deep-sea hydrothermal vents

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 1240, doi:10.3389/fmicb.2016.01240.Thermophilic methanogens are common autotrophs at hydrothermal vents, but their growth constraints and dependence on H2 syntrophy in situ are poorly understood. Between 2012 and 2015, methanogens and H2-producing heterotrophs were detected by growth at 80∘C and 55∘C at most diffuse (7–40∘C) hydrothermal vent sites at Axial Seamount. Microcosm incubations of diffuse hydrothermal fluids at 80∘C and 55∘C demonstrated that growth of thermophilic and hyperthermophilic methanogens is primarily limited by H2 availability. Amendment of microcosms with NH4+ generally had no effect on CH4 production. However, annual variations in abundance and CH4 production were observed in relation to the eruption cycle of the seamount. Microcosm incubations of hydrothermal fluids at 80∘C and 55∘C supplemented with tryptone and no added H2 showed CH4 production indicating the capacity in situ for methanogenic H2 syntrophy. 16S rRNA genes were found in 80∘C microcosms from H2-producing archaea and H2-consuming methanogens, but not for any bacteria. In 55∘C microcosms, sequences were found from H2-producing bacteria and H2-consuming methanogens and sulfate-reducing bacteria. A co-culture of representative organisms showed that Thermococcus paralvinellae supported the syntrophic growth of Methanocaldococcus bathoardescens at 82∘C and Methanothermococcus sp. strain BW11 at 60∘C. The results demonstrate that modeling of subseafloor methanogenesis should focus primarily on H2 availability and temperature, and that thermophilic H2 syntrophy can support methanogenesis within natural microbial assemblages and may be an important energy source for thermophilic autotrophs in marine geothermal environments.This work was funded by the Gordon and Betty Moore Foundation grant GBMF 3297, the NASA Earth and Space Science Fellowship Program grant NNX11AP78H, the National Science Foundation grant OCE-1547004, with funding from NOAA/PMEL, contribution #4493, and JISAO under NOAA Cooperative Agreement NA15OAR4320063, contribution #2706

Experimental investigation on the controls of clumped isotopologue and hydrogen isotope ratios in microbial methane

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Geochimica et Cosmochimica Acta 237 (2018): 339-356, doi:10.1016/j.gca.2018.06.029.The abundance of methane isotopologues with two rare isotopes (e.g., 13CH3D) has been proposed as a tool to estimate the temperature at which methane is formed or thermally equilibrated. It has been shown, however, that microbial methane from surface environments and from laboratory cultures is characterized by low 13CH3D abundance, corresponding to anomalously high apparent 13CH3D equilibrium temperatures. We carried out a series of batch culture experiments to investigate the origin of the non-equilibrium signals in microbial methane by exploring a range of metabolic pathways, growth temperatures, and hydrogen isotope compositions of the media. We found that thermophilic methanogens (Methanocaldococcus jannaschii, Methanothermococcus thermolithotrophicus, and Methanocaldococcus bathoardescens) grown on H2+CO2 at temperatures between 60 and 80°C produced methane with Δ13CH3D values (defined as the deviation from stochastic abundance) of 0.5 to 2.5‰, corresponding to apparent 13CH3D equilibrium temperatures of 200 to 600°C. Mesophilic methanogens (Methanosarcina barkeri and Methanosarcina mazei) grown on H2+CO2, acetate, or methanol produced methane with consistently low Δ13CH3D values, down to -5.2‰. Closed system effects can explain part of the non-equilibrium signals for methane from thermophilic methanogens. Experiments with M. barkeri using D-spiked water or D-labeled acetate (CD3COO-) indicate that 1.6 to 1.9 out of four H atoms in methane originate from water, but Δ13CH3D values of product methane only weakly correlate with the D/H ratio of medium water. Our experimental results demonstrate that low Δ13CH3D values are not specific to the metabolic pathways of methanogenesis, suggesting that they could be produced during enzymatic reactions common in the three methanogenic pathways, such as the reduction of methyl-coenzyme M. Nonetheless C-H bonds inherited from precursor methyl groups may also carry part of non-equilibrium signals.Grants from the National Science Foundation (EAR-1250394 to S.O.), N. Braunsdorf and D. Smit of Shell PTI/EG (to S.O.), the Deep Carbon Observatory (to S.O., M.K., K.-U.H., D.S.G.), the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft (HI 616-14-1 to K.- U.H.), and the Heisenberg Program (KO3651-3-1 to M.K.) of the Deutsche Forschungsgemeinschaft supported this study. D.S.G. was also supported by a National Science Foundation Graduate Research Fellowship, the Neil and Anna Rasmussen Foundation Fund, the Grayce B. Kerr Fellowship, and a Shell-MIT Energy Initiative Graduate Fellowship. D.T.W. was supported by a National Defense Science and Engineering Graduate Fellowship. L.C.S. was supported by a NASA Earth and Space Science Fellowship (grant NNX11AP78H)

Fluid geochemistry, local hydrology, and metabolic activity define methanogen community size and composition in deep-sea hydrothermal vents

The size and biogeochemical impact of the subseafloor biosphere in oceanic crust remain largely unknown due to sampling limitations. We used reactive transport modeling to estimate the size of the subseafloor methanogen population, volume of crust occupied, fluid residence time, and nature of the subsurface mixing zone for two low-temperature hydrothermal vents at Axial Seamount. Monod CH4 production kinetics based on chemostat H2 availability and batch-culture Arrhenius growth kinetics for the hyperthermophile Methanocaldococcus jannaschii and thermophile Methanothermococcus thermolithotrophicus were used to develop and parameterize a reactive transport model, which was constrained by field measurements of H2, CH4, and metagenome methanogen concentration estimates in 20–40 °C hydrothermal fluids. Model results showed that hyperthermophilic methanogens dominate in systems where a narrow flow path geometry is maintained, while thermophilic methanogens dominate in systems where the flow geometry expands. At Axial Seamount, the residence time of fluid below the surface was 29–33 h. Only 1011 methanogenic cells occupying 1.8–18 m3 of ocean crust per m2 of vent seafloor area were needed to produce the observed CH4 anomalies. We show that variations in local geology at diffuse vents can create fluid flow paths that are stable over space and time, harboring persistent and distinct microbial communities

Collisions between equal sized ice grain agglomerates

Following the recent insight in the material structure of comets, protoplanetesimals are assumed to have low densities and to be highly porous agglomerates. It is still unclear if planetesimals can be formed from these objects by collisional growth. Therefore, it is important to study numerically the collisional outcome from low velocity impacts of equal sized porous agglomerates which are too large to be examined in a laboratory experiment. We use the Lagrangian particle method Smooth Particle Hydrodynamics to solve the equations that describe the dynamics of elastic and plastic bodies. Additionally, to account for the influence of porosity, we follow a previous developed equation of state and certain relations between the material strength and the relative density. Collisional growth seems possible for rather low collision velocities and particular material strengths. The remnants of collisions with impact parameters that are larger than 50% of the radius of the colliding objects tend to rotate. For small impact parameters, the colliding objects are effectively slowed down without a prominent compaction of the porous structure, which probably increases the possibility for growth. The protoplanetesimals, however, do not stick together for the most part of the employed material strengths. An important issue in subsequent studies has to be the influence of rotation to collisional growth. Moreover, for realistic simulations of protoplanetesimals it is crucial to know the correct material parameters in more detail.Comment: 7 pages, 11 figures, accepted by A&

T Cell Cross-Reactivity and Conformational Changes during TCR Engagement

All thymically selected T cells are inherently cross-reactive, yet many data indicate a fine specificity in antigen recognition, which enables virus escape from immune control by mutation in infections such as the human immunodeficiency virus (HIV). To address this paradox, we analyzed the fine specificity of T cells recognizing a human histocompatibility leukocyte antigen (HLA)-A2–restricted, strongly immunodominant, HIV gag epitope (SLFNTVATL). The majority of 171 variant peptides tested bound HLA-A2, but only one third were recognized. Surprisingly, one recognized variant (SLYNTVATL) showed marked differences in structure when bound to HLA-A2. T cell receptor (TCR) recognition of variants of these two peptides implied that they adopted the same conformation in the TCR–peptide–major histocompatibility complex (MHC) complex. However, the on-rate kinetics of TCR binding were identical, implying that conformational changes at the TCR–peptide–MHC binding interface occur after an initial permissive antigen contact. These findings have implications for the rational design of vaccines targeting viruses with unstable genomes