Deep-branching acetogens in serpentinized subsurface fluids of Oman.

Little is known of acetogens in contemporary serpentinizing systems, despite widely supported theories that serpentinite-hosted environments supported the first life on Earth via acetogenesis. To address this knowledge gap, genome-resolved metagenomics was applied to subsurface fracture water communities from an area of active serpentinization in the Samail Ophiolite, Sultanate of Oman. Two deeply branching putative bacterial acetogen types were identified in the communities belonging to the Acetothermia (hereafter, types I and II) that exhibited distinct distributions among waters with lower and higher water-rock reaction (i.e., serpentinization influence), respectively. Metabolic reconstructions revealed contrasting core metabolic pathways of type I and II Acetothermia, including in acetogenic pathway components (e.g., bacterial- vs. archaeal-like carbon monoxide dehydrogenases [CODH], respectively), hydrogen use to drive acetogenesis, and chemiosmotic potential generation via respiratory (type I) or canonical acetogen ferredoxin-based complexes (type II). Notably, type II Acetothermia metabolic pathways allow for use of serpentinization-derived substrates and implicate them as key primary producers in contemporary hyperalkaline serpentinite environments. Phylogenomic analyses indicate that 1) archaeal-like CODH of the type II genomes and those of other serpentinite-associated Bacteria derive from a deeply rooted horizontal transfer or origin among archaeal methanogens and 2) Acetothermia are among the earliest evolving bacterial lineages. The discovery of dominant and early-branching acetogens in subsurface waters of the largest near-surface serpentinite formation provides insight into the physiological traits that likely facilitated rock-supported life to flourish on a primitive Earth and possibly on other rocky planets undergoing serpentinization.

Metal-oxide precipitation influences microbiome structure in hyporheic zones receiving acid rock drainage.

Streams impacted by historic mining activity are characterized by acidic pH, unique microbial communities, and abundant metal-oxide precipitation, all of which can influence groundwater-surface water exchange. We investigate how metal-oxide precipitates and hyporheic mixing mediate the composition of microbial communities in two streams receiving acid-rock and mine drainage near Silverton, Colorado, USA. A large, neutral pH hyporheic zone facilitated the precipitation of metal particles/colloids in hyporheic porewaters. A small, low pH hyporheic zone, limited by the presence of a low-permeability, iron-oxyhydroxide layer known as ferricrete, led to the formation of steep geochemical gradients and high dissolved-metal concentrations. To determine how these two hyporheic systems influence microbiome composition, we installed well clusters and deployed in situ microcosms in each stream to sample porewaters and sediments for 16S rRNA gene sequencing. Results indicated that distinct hydrogeochemical conditions were present above and below the ferricrete in the low pH system. A positive feedback loop may be present in the low pH stream where microbially mediated precipitation of iron-oxides contributes to additional clogging of hyporheic pore spaces, separating abundant, iron-oxidizing bacteria (Gallionella spp.) above the ferricrete from rare, low-abundance bacteria below the ferricrete. Metal precipitates and colloids that formed in the neutral pH hyporheic zone were associated with a more diverse phylogenetic community of nonmotile, nutrient-cycling bacteria that may be transported through hyporheic pore spaces. In summary, biogeochemical conditions influence, and are influenced by, hyporheic mixing, which mediates the distribution of micro-organisms and, thus, the cycling of metals in streams receiving acid-rock and mine drainage. IMPORTANCE: In streams receiving acid-rock and mine drainage, the abundant precipitation of iron minerals can alter how groundwater and surface water mix along streams (in what is known as the "hyporheic zone") and may shape the distribution of microbial communities. The findings presented here suggest that neutral pH streams with large, well-mixed hyporheic zones may harbor and transport diverse microorganisms attached to particles/colloids through hyporheic pore spaces. In acidic streams where metal oxides clog pore spaces and limit hyporheic exchange, iron-oxidizing bacteria may dominate and phylogenetic diversity becomes low. The abundance of iron-oxidizing bacteria in acid mine drainage streams has the potential to contribute to additional clogging of hyporheic pore spaces and the accumulation of toxic metals in the hyporheic zone. This research highlights the dynamic interplay between hydrology, geochemistry, and microbiology at the groundwater-surface water interface of acid mine drainage streams.

Seasonal variation in the biological succession of marine diatoms over 316L stainless steel in a coastal environment of Chile.

Characterizing seasonal changes in diatom community profiles in coastal environments is scarce worldwide. Despite diatoms being prevalent in microfouling, their role in microbially influenced corrosion of metallic materials remains poorly understood. This study reports the effect of seasonal variations on the settlement of marine diatoms and corrosion of 316 L stainless steel surfaces exposed to Chilean coastal seawater. Electron microscopy imaging revealed a diverse assembly of diatoms, exhibiting pronounced differences at genus level between summer and winter seasons, with a significant delay in diatom settlement during winter. Electrochemical measurements indicated an active role of diatoms in increasing corrosion current during biofilm development. While the final diatom composition was similar irrespective of the season, the analyses of diatom assemblages over time differed, showing faster colonization when silicate and nitrate were available. This study lays the foundation for future research on the dominant season-specific genera of diatoms to unveil the microbial interactions that could contribute to corrosion and to evaluate their potential as bioindicators for alternative surveillance strategies.

Optode-based chemical imaging of laboratory burned soil reveals millimeter-scale heterogeneous biogeochemical responses.

Soil spatial responses to fire are unclear. Using optical chemical sensing with planar 'optodes', pH and dissolved O2 concentration were tracked spatially with a resolution of 360 µm per pixel for 72 h after burning soil in the laboratory with a butane torch (∼1300 °C) and then sprinkling water to simulate a postfire moisture event. Imaging data from planar optodes correlated with microbial activity (quantified via RNA transcripts). Post-fire and post-wetting, soil pH increased throughout the entire ∼13 cm × 17 cm × 20 cm rectangular cuboid of sandy loam soil. Dissolved O2 concentrations were not impacted until the application of water postfire. pH and dissolved O2 both negatively correlated (p < 0.05) with relative transcript expression for galactose metabolism, the degradation of aromatic compounds, sulfur metabolism, and narH. Additionally, dissolved O2 negatively correlated (p < 0.05) with the relative activity of carbon fixation pathways in Bacteria and Archaea, amoA/amoB, narG, nirK, and nosZ. nifH was not detected in any samples. Only amoB and amoC correlated with depth in soil (p < 0.05). Results demonstrate that postfire soils are spatially complex on a mm scale and that using optode-based chemical imaging as a chemical navigator for RNA transcript sampling is effective.

pH-Induced Conformational Changes of Human Bocavirus Capsids.

Human bocavirus 1 (HBoV1) and HBoV2-4 infect children and immunocompromised individuals, resulting in respiratory and gastrointestinal infections, respectively. Using cryo-electron microscopy and image reconstruction, the HBoV2 capsid structure was determined to 2.7 Å resolution at pH 7.4 and compared to the previously determined HBoV1, HBoV3, and HBoV4 structures. Consistent with previous findings, surface variable region (VR) III of the capsid protein VP3, proposed as a host tissue-tropism determinant, was structurally similar among the gastrointestinal strains HBoV2-4, but differed from HBoV1 with its tropism for the respiratory tract. Towards understanding the entry and trafficking properties of these viruses, HBoV1 and HBoV2 were further analyzed as species representatives of the two HBoV tropisms. Their cell surface glycan-binding characteristics were analyzed, and capsid structures determined to 2.5-2.7 Å resolution at pH 5.5 and 2.6, conditions normally encountered during infection. The data showed that glycans with terminal sialic acid, galactose, GlcNAc or heparan sulfate moieties do not facilitate HBoV1 or HBoV2 cellular attachment. With respect to trafficking, conformational changes common to both viruses were observed at low pH conditions localized to the VP N-terminus under the 5-fold channel, in the surface loops VR-I and VR-V and specific side-chain residues such as cysteines and histidines. The 5-fold conformational movements provide insight into the potential mechanism of VP N-terminal dynamics during HBoV infection and side-chain modifications highlight pH-sensitive regions of the capsid.IMPORTANCE Human bocaviruses (HBoVs) are associated with disease in humans. However, the lack of an animal model and a versatile cell culture system to study their life cycle limits the ability to develop specific treatments or vaccines. This study presents the structure of HBoV2, at 2.7 Å resolution, determined for comparison to the existing HBoV1, HBoV3, and HBoV4 structures, to enable the molecular characterization of strain and genus-specific capsid features contributing to tissue tropism and antigenicity. Furthermore, HBoV1 and HBoV2 structures determined under acidic conditions provide insight into capsid changes associated with endosomal and gastrointestinal acidification. Structural rearrangements of the capsid VP N-terminus, at the base of the 5-fold channel, demonstrate a disordering of a "basket" motif as pH decreases. These observations begin to unravel the molecular mechanism of HBoV infection and provide information for control strategies.

Statistical Learning and Uncommon Soil Microbiota Explain Biogeochemical Responses after Wildfire.

Wildfires are a perennial event globally, and the biogeochemical underpinnings of soil responses at relevant spatial and temporal scales are unclear. Soil biogeochemical processes regulate plant growth and nutrient losses that affect water quality, yet the response of soil after variable intensity fire is difficult to explain and predict. To address this issue, we examined two wildfires in Colorado, United States, across the first and second postfire years and leveraged statistical learning (SL) to predict and explain biogeochemical responses. We found that SL predicts biogeochemical responses in soil after wildfire with surprising accuracy. Of the 13 biogeochemical analytes analyzed in this study, 9 are best explained with a hybrid microbiome + biogeochemical SL model. Biogeochemical-only models best explain 3 features, and 1 feature is explained equally well with the hybrid and biogeochemical-only models. In some cases, microbiome-only SL models are also effective (such as predicting NH4+). Whenever a microbiome component is employed, selected features always involve uncommon soil microbiota (i.e., the "rare biosphere" [existing at <1% mean relative abundance]). Here, we demonstrate that SL paired with DNA sequence and biogeochemical data predicts environmental features in postfire soils, although this approach could likely be applied to any biogeochemical system. IMPORTANCE Soil biogeochemical processes are critical to plant growth and water quality and are substantially disturbed by wildfire. However, soil responses to fire are difficult to predict. To address this issue, we developed a large environmental data set that tracks postfire changes in soil and used statistical learning (SL) to build models that exploit complex data to make predictions about biogeochemical responses. Here, we show that SL depends upon uncommon microbiota in soil (the "rare biosphere") to make surprisingly accurate predictions about soil biogeochemical responses to wildfire. Using SL to explain variation in a natively chaotic environmental system is mechanism independent. Likely, the approach that we describe for combining SL with microbiome and biogeochemical parameters has practical applications across a range of issues in the environmental sciences where predicting responses would be useful.

Olfactory bulb-targeted quantum dot (QD) bioconjugate and Kv1.3 blocking peptide improve metabolic health in obese male mice.

The olfactory system is a driver of feeding behavior, whereby olfactory acuity is modulated by the metabolic state of the individual. The excitability of the major output neurons of the olfactory bulb (OB) can be modulated through targeting a voltage-dependent potassium channel, Kv1.3, which responds to changes in metabolic factors such as insulin, glucose, and glucagon-like peptide-1. Because gene-targeted deletion or inhibition of Kv1.3 in the periphery has been found to increase energy metabolism and decrease body weight, we hypothesized that inhibition of Kv1.3 selectively in the OB could enhance excitability of the output neurons to evoke changes in energy homeostasis. We thereby employed metal-histidine coordination to self-assemble the Kv1.3 inhibitor margatoxin (MgTx) to fluorescent quantum dots (QDMgTx) as a means to label cells in vivo and test changes in neuronal excitability and metabolism when delivered to the OB. Using patch-clamp electrophysiology to measure Kv1.3 properties in heterologously expressed cells and native mitral cells in OB slices, we found that QDMgTx had a fast rate of inhibition, but with a reduced IC50, and increased action potential firing frequency. QDMgTx was capable of labeling cloned Kv1.3 channels but was not visible when delivered to native Kv1.3 in the OB. Diet-induced obese mice were observed to reduce body weight and clear glucose more quickly following osmotic mini-pump delivery of QDMgTx/MgTx to the OB, and following MgTx delivery, they increased the use of fats as fuels (reduced respiratory exchange ratio). These results suggest that enhanced excitability of bulbar output neurons can drive metabolic responses.

Molecular Evidence for an Active Microbial Methane Cycle in Subsurface Serpentinite-Hosted Groundwaters in the Samail Ophiolite, Oman.

Serpentinization can generate highly reduced fluids replete with hydrogen (H2) and methane (CH4), potent reductants capable of driving microbial methanogenesis and methanotrophy, respectively. However, CH4 in serpentinized waters is thought to be primarily abiogenic, raising key questions about the relative importance of methanogens and methanotrophs in the production and consumption of CH4 in these systems. Herein, we apply molecular approaches to examine the functional capability and activity of microbial CH4 cycling in serpentinization-impacted subsurface waters intersecting multiple rock and water types within the Samail Ophiolite of Oman. Abundant 16S rRNA genes and transcripts affiliated with the methanogenic genus Methanobacterium were recovered from the most alkaline (pH, >10), H2- and CH4-rich subsurface waters. Additionally, 16S rRNA genes and transcripts associated with the aerobic methanotrophic genus Methylococcus were detected in wells that spanned varied fluid geochemistry. Metagenomic sequencing yielded genes encoding homologs of proteins involved in the hydrogenotrophic pathway of microbial CH4 production and in microbial CH4 oxidation. Transcripts of several key genes encoding methanogenesis/methanotrophy enzymes were identified, predominantly in communities from the most hyperalkaline waters. These results indicate active methanogenic and methanotrophic populations in waters with hyperalkaline pH in the Samail Ophiolite, thereby supporting a role for biological CH4 cycling in aquifers that undergo low-temperature serpentinization.IMPORTANCE Serpentinization of ultramafic rock can generate conditions favorable for microbial methane (CH4) cycling, including the abiotic production of hydrogen (H2) and possibly CH4 Systems of low-temperature serpentinization are geobiological targets due to their potential to harbor microbial life and ubiquity throughout Earth's history. Biomass in fracture waters collected from the Samail Ophiolite of Oman, a system undergoing modern serpentinization, yielded DNA and RNA signatures indicative of active microbial methanogenesis and methanotrophy. Intriguingly, transcripts for proteins involved in methanogenesis were most abundant in the most highly reacted waters that have hyperalkaline pH and elevated concentrations of H2 and CH4 These findings suggest active biological methane cycling in serpentinite-hosted aquifers, even under extreme conditions of high pH and carbon limitation. These observations underscore the potential for microbial activity to influence the isotopic composition of CH4 in these systems, which is information that could help in identifying biosignatures of microbial activity on other planets.

Structure comparison of the chimeric AAV2.7m8 vector with parental AAV2.

The AAV2.7m8 vector is an engineered capsid with a 10-amino acid insertion in adeno-associated virus (AAV) surface variable region VIII (VR-VIII) resulting in the alteration of an antigenic region of AAV2 and the ability to efficiently transduce retina cells following intravitreal administration. Directed evolution and in vivo screening in the mouse retina isolated this vector. In the present study, we sought to identify the structural differences between a recombinant AAV2.7m8 (rAAV2.7m8) vector packaging a GFP genome and its parental serotype, AAV2, by cryo-electron microscopy (cryo-EM) and image reconstruction. The structures of rAAV2.7m8 and AAV2 were determined to 2.91 and 3.02 Å resolution, respectively. The rAAV2.7m8 amino acid side-chains for residues 219-745 (the last C-terminal residue) were interpretable in the density map with the exception of the 10 inserted amino acids. While observable in a low sigma threshold density, side-chains were only resolved at the base of the insertion, likely due to flexibility at the top of the loop. A comparison to parental AAV2 (ordered from residues 217-735) showed the structures to be similar, except at some side-chains that had different orientations and, in VR-VIII containing the 10 amino acid insertion. VR-VIII is part of an AAV2 antigenic epitope, and the difference is consistent with rAAV2.7m8's escape from a known AAV2 monoclonal antibody, C37-B. The observations provide valuable insight into the configuration of inserted surface peptides on the AAV capsid and structural differences to be leveraged for future AAV vector rational design, especially for retargeted tropism and antibody escape.

The deep, hot biosphere: Twenty-five years of retrospection.

Twenty-five years ago this month, Thomas Gold published a seminal manuscript suggesting the presence of a "deep, hot biosphere" in the Earth's crust. Since this publication, a considerable amount of attention has been given to the study of deep biospheres, their role in geochemical cycles, and their potential to inform on the origin of life and its potential outside of Earth. Overwhelming evidence now supports the presence of a deep biosphere ubiquitously distributed on Earth in both terrestrial and marine settings. Furthermore, it has become apparent that much of this life is dependent on lithogenically sourced high-energy compounds to sustain productivity. A vast diversity of uncultivated microorganisms has been detected in subsurface environments, and we show that H2, CH4, and CO feature prominently in many of their predicted metabolisms. Despite 25 years of intense study, key questions remain on life in the deep subsurface, including whether it is endemic and the extent of its involvement in the anaerobic formation and degradation of hydrocarbons. Emergent data from cultivation and next-generation sequencing approaches continue to provide promising new hints to answer these questions. As Gold suggested, and as has become increasingly evident, to better understand the subsurface is critical to further understanding the Earth, life, the evolution of life, and the potential for life elsewhere. To this end, we suggest the need to develop a robust network of interdisciplinary scientists and accessible field sites for long-term monitoring of the Earth's subsurface in the form of a deep subsurface microbiome initiative.

Probing the geological source and biological fate of hydrogen in Yellowstone hot springs.

Hydrogen (H2 ) is enriched in hot springs and can support microbial primary production. Using a series of geochemical proxies, a model to describe variable H2 concentrations in Yellowstone National Park (YNP) hot springs is presented. Interaction between water and crustal iron minerals yields H2 that partition into the vapour phase during decompressional boiling of ascending hydrothermal fluids. Variable vapour input leads to differences in H2 concentration among springs. Analysis of 50 metagenomes from a variety of YNP springs reveals that genes encoding oxidative hydrogenases are enriched in communities inhabiting springs sourced with vapour-phase gas. Three springs in the Smokejumper (SJ) area of YNP that are sourced with vapour-phase gas and with the most H2 in YNP were examined to determine the fate of H2 . SJ3 had the most H2 , the most 16S rRNA gene templates and the greatest abundance of culturable hydrogenotrophic and autotrophic cells of the three springs. Metagenomics and transcriptomics of SJ3 reveal a diverse community comprised of abundant populations expressing genes involved in H2 oxidation and carbon dioxide fixation. These observations suggest a link between geologic processes that generate and source H2 to hot springs and the distribution of organisms that use H2 to generate energy.

Structural Insights into Human Bocaparvoviruses.

Bocaparvoviruses are emerging pathogens of the Parvoviridae family. Human bocavirus 1 (HBoV1) causes severe respiratory infections and HBoV2 to HBoV4 cause gastrointestinal infections in young children. Recent reports of life-threatening cases, lack of direct treatment or vaccination, and a limited understanding of their disease mechanisms highlight the need to study these pathogens on a molecular and structural level for the development of therapeutics. Toward this end, the capsid structures of HBoV1, HBoV3, and HBoV4 were determined to a resolution of 2.8 to 3.0 Å by cryo-electron microscopy and three-dimensional image reconstruction. The bocaparvovirus capsids, which display different tissue tropisms, have features in common with other parvoviruses, such as depressions at the icosahedral 2-fold symmetry axis and surrounding the 5-fold symmetry axis, protrusions surrounding the 3-fold symmetry axis, and a channel at the 5-fold symmetry axis. However, unlike other parvoviruses, densities extending the 5-fold channel into the capsid interior are conserved among the bocaparvoviruses and are suggestive of a genus-specific function. Additionally, their major viral protein 3 contains loops with variable regions at their apexes conferring capsid surface topologies different from those of other parvoviruses. Structural comparisons at the strain (HBoV) and genus (bovine parvovirus and HBoV) levels identified differences in surface loops that are functionally important in host/tissue tropism, pathogenicity, and antigenicity in other parvoviruses and likely play similar roles in these viruses. This study thus provides a structural framework to characterize determinants of host/tissue tropism, pathogenicity, and antigenicity for the development of antiviral strategies to control human bocavirus infections.IMPORTANCE Human bocaviruses are one of only a few members of the Parvoviridae family pathogenic to humans, especially young children and immunocompromised adults. There are currently no treatments or vaccines for these viruses or the related enteric bocaviruses. This study obtained the first high-resolution structures of three human bocaparvoviruses determined by cryo-reconstruction. HBoV1 infects the respiratory tract, and HBoV3 and HBoV4 infect the gastrointestinal tract, tissues that are likely targeted by the capsid. Comparison of these viruses provides information on conserved bocaparvovirus-specific features and variable regions resulting in unique surface topologies that can serve as guides to characterize HBoV determinants of tissue tropism and antigenicity in future experiments. Based on the comparison to other existing parvovirus capsid structures, this study suggests capsid regions that likely control successful infection, including determinants of receptor attachment, host cell trafficking, and antigenic reactivity. Overall, these observations could impact efforts to design antiviral strategies and vaccines for HBoVs.

Metabolic Capability and Phylogenetic Diversity of Mono Lake during a Bloom of the Eukaryotic Phototroph Picocystis sp. Strain ML.

Algal blooms in lakes are often associated with anthropogenic eutrophication; however, they can occur without the human introduction of nutrients to a lake. A rare bloom of the alga Picocystis sp. strain ML occurred in the spring of 2016 at Mono Lake, a hyperalkaline lake in California, which was also at the apex of a multiyear-long drought. These conditions presented a unique sampling opportunity to investigate microbiological dynamics and potential metabolic function during an intense natural algal bloom. We conducted a comprehensive molecular analysis along a depth transect near the center of the lake from the surface to a depth of 25 m in June 2016. Across sampled depths, rRNA gene sequencing revealed that Picocystis-associated chloroplasts were found at 40 to 50% relative abundance, greater than values recorded previously. Despite high relative abundances of the photosynthetic oxygenic algal genus Picocystis, oxygen declined below detectable limits below a depth of 15 m, corresponding with an increase in microorganisms known to be anaerobic. In contrast to previously sampled years, both metagenomic and metatranscriptomic data suggested a depletion of anaerobic sulfate-reducing microorganisms throughout the lake's water column. Transcripts associated with photosystem I and II were expressed at both 2 m and 25 m, suggesting that limited oxygen production could occur at extremely low light levels at depth within the lake. Blooms of Picocystis appear to correspond with a loss of microbial activity such as sulfate reduction within Mono Lake, yet microorganisms may survive within the sediment to repopulate the lake water column as the bloom subsides.IMPORTANCE Mono Lake, California, provides a habitat to a unique ecological community that is heavily stressed due to recent human water diversions and a period of extended drought. To date, no baseline information exists from Mono Lake to understand how the microbial community responds to human-influenced drought or algal bloom or what metabolisms are lost in the water column as a consequence of such environmental pressures. While previously identified anaerobic members of the microbial community disappear from the water column during drought and bloom, sediment samples suggest that these microorganisms survive at the lake bottom or in the subsurface. Thus, the sediments may represent a type of seed bank that could restore the microbial community as a bloom subsides. Our work sheds light on the potential photosynthetic activity of the halotolerant alga Picocystis sp. strain ML and how the function and activity of the remainder of the microbial community responds during a bloom at Mono Lake.

Disturbance opens recruitment sites for bacterial colonization in activated sludge.

Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems.

Interfacing Microfluidics with Negative Stain Transmission Electron Microscopy.

A microfluidic platform is presented for preparing negatively stained grids for use in transmission electron microscopy (EM). The microfluidic device is composed of glass etched with readily fabricated features that facilitate the extraction of the grid poststaining and maintains the integrity of the sample. Utilization of this device simultaneously reduced environmental contamination on the grids and improved the homogeneity of the heavy metal stain needed to enhance visualization of biological specimens as compared to conventionally prepared EM grids. This easy-to-use EM grid preparation device provides the basis for future developments of systems with more integrated features, which will allow for high-throughput and dynamic structural biology studies.

New insights into rotating frame relaxation at high field.

Measurements of spin-lock relaxation rates in the rotating frame (R1ρ ) at high magnetic fields afford the ability to probe not only relatively slow molecular motions, but also other dynamic processes, such as chemical exchange and diffusion. In particular, measurements of the variation (or dispersion) of R1ρ with locking field allow the derivation of quantitative parameters that describe these processes. Measurements in deuterated solutions demonstrate the manner and degree to which exchange dominates relaxation at high fields (4.7 T, 7 T) in simple solutions, whereas temperature and pH are shown to be very influential factors affecting the rates of proton exchange. Simulations and experiments show that multiple exchanging pools of protons in realistic tissues can be assumed to behave independently of each other. R1ρ measurements can be combined to derive an exchange rate contrast (ERC) that produces images whose intensities emphasize protons with specific exchange rates rather than chemical shifts. In addition, water diffusion in the presence of intrinsic susceptibility gradients may produce significant effects on R1ρ dispersions at high fields. The exchange and diffusion effects act independently of each other, as confirmed by simulation and experimentally in studies of red blood cells at different levels of oxygenation. Collectively, R1ρ measurements provide an ability to quantify exchange processes, to provide images that depict protons with specific exchange rates and to describe the microstructure of tissues containing magnetic inhomogeneities. As such, they complement traditional T1 or T2 measurements and provide additional insights from measurements of R1ρ at a single locking field. Copyright © 2016 John Wiley & Sons, Ltd.

The influence of frame alignment with dose compensation on the quality of single particle reconstructions.

As direct electron detection devices in cryo-electron microscopy become ubiquitous, the field is now ripe for new developments in image analysis techniques that take advantage of their increased SNR coupled with their high-throughput frame collection abilities. In approaching atomic resolution of native-like biomolecules, the accurate extraction of structural locations and orientations of side-chains from frames depends not only on the electron dose that a sample receives but also on the ability to accurately estimate the CTF. Here we use a new 2.8Å resolution structure of a recombinant gene therapy virus, AAV-DJ with Arixtra, imaged on an FEI Titan Krios with a DE-20 direct electron detector to probe new metrics including relative side-chain density and ResLog analysis for optimizing the compensation of electron beam damage and to characterize the factors that are limiting the resolution of the reconstruction. The influence of dose compensation on the accuracy of CTF estimation and particle classifiability are also presented. We show that rigorous dose compensation allows for better particle classifiability and greater recovery of structural information from negatively charged, electron-sensitive side-chains, resulting in a more accurate macromolecular model.

Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a.

BACKGROUND: Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3. RESULTS: We have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins. CONCLUSION: These results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a.

Microbially influenced corrosion communities associated with fuel-grade ethanol environments.

Microbially influenced corrosion (MIC) is a costly problem that impacts hydrocarbon production and processing equipment, water distribution systems, ships, railcars, and other types of metallic infrastructure. In particular, MIC is known to cause considerable damage to hydrocarbon fuel infrastructure including production, transportation, and storage systems, often times with catastrophic environmental contamination results. As the production and use of alternative fuels such as fuel-grade ethanol (FGE) increase, it is important to consider MIC of engineered materials exposed to these "newer fuels" as they enter existing infrastructure. Reports of suspected MIC in systems handling FGE and water prompted an investigation of the microbial diversity associated with these environments. Small subunit ribosomal RNA gene pyrosequencing surveys indicate that acetic-acid-producing bacteria (Acetobacter spp. and Gluconacetobacter spp.) are prevalent in environments exposed to FGE and water. Other microbes previously implicated in corrosion, such as sulfate-reducing bacteria and methanogens, were also identified. In addition, acetic-acid-producing microbes and sulfate-reducing microbes were cultivated from sampled environments containing FGE and water. Results indicate that complex microbial communities form in these FGE environments and could cause significant MIC-related damage that may be difficult to control. How to better manage these microbial communities will be a defining aspect of improving mitigation of global infrastructure corrosion.

Dispersion of relaxation rates in the rotating frame under the action of spin-locking pulses and diffusion in inhomogeneous magnetic fields.

PURPOSE: A method is described for characterizing magnetically inhomogeneous media and the spatial scales of intrinsic susceptibility variations within samples. The rate of spin-lattice relaxation in the rotating frame, R1ρ , is affected by diffusion effects to a degree that depends on the magnitude of an applied spin-locking field. Appropriate analysis of the dispersion of R1ρ with locking field may be used to characterize susceptibility variations in inhomogeneous tissues. THEORY AND METHODS: The contribution of diffusion to R1ρ is quantified by an analytic expression derived by analyzing of the effects of diffusion through periodic variations of magnetic susceptibility and is used to predict the effects of inhomogeneities in simple phantoms. The theory is further applied to imaging to derive parametric images that portray the dimensions of susceptibility inhomogeneities independent of their magnitude. RESULTS: Significant dispersion of R1ρ with locking field was predicted and measured experimentally for suspensions of microspheres ranging from 1 to 90 µm in diameter. For scales of practical interest, these dispersion effects occur at much lower locking fields than the range in which chemical exchange effects cause similar dispersion. CONCLUSION: There is good agreement between theory and experiment, and the method has potential for quantitative tissue characterization and functional imaging.