The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath) Is a Novel Copper-containing Three-subunit Enzyme: isolation and charactization

The particulate methane monooxygenase (pMMO) is known to be very difficult to study mainly due to its unusual activity instability in vitro. By cultivating Methylococcus capsulatus (Bath) under methane stress conditions and high copper levels in the growth medium, membranes highly enriched in the pMMO with exceptionally stable activity can be isolated from these cells. Purified and active pMMO can be subsequently obtained from these membrane preparations using protocols in which an excess of reductants and anaerobic conditions were maintained during membrane solubilization by dodecyl beta-D-maltoside and purification by chromatography. The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins. The dominant species of the pMMO was found to consist of three subunits, alpha, beta, and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, respectively. A second species of the pMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, alpha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively. The alpha and alpha' subunits from these two forms of the pMMO contain identical N-terminal sequences. The gamma subunit, however, exhibits variation in its N-terminal sequence. The pMMO is a copper-containing protein only and shows a requirement for Cu(I) ions. Approximately 12-15 Cu ions per 94-kDa monomeric unit were observed. The pMMO is sensitive to dioxygen tension. On the basis of dioxygen sensitivity, three kinetically distinct forms of the enzyme can be distinguished. A slow but air-stable form, which is converted into a "pulsed" state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO. This form was the subject of our pMMO isolation effort. Other forms (types II and III) are deactivated to various extents upon exposure to atmospheric dioxygen pressure. Under inactivating conditions, these unstable forms release protons to the buffer (~10 H+/94-kDa monomeric unit) and eventually become completely inactive

A Case-Study Application of the Experimental Watershed Study Design to Advance Adaptive Management of Contemporary Watersheds

settings Open AccessFeature PaperArticle A Case-Study Application of the Experimental Watershed Study Design to Advance Adaptive Management of Contemporary Watersheds by Jason A. Hubbart 1,*,Elliott Kellner 2 andSean J. Zeiger 3 1 West Virginia University, Institute of Water Security and Science, Davis College of Agriculture, Natural Resources and Design, Schools of Agriculture and Food, and Natural Resources, 3109 Agricultural Sciences Building, Morgantown, WV 26506, USA 2 West Virginia University, Institute of Water Security and Science, Davis College of Agriculture Natural Resources and Design, Division of Plant and Soil Sciences, 3011 Agricultural Sciences Building, Morgantown, WV 26506, USA 3 School of Natural Resources, University of Missouri, 203-T ABNR Building, Columbia, MO 65211, USA * Author to whom correspondence should be addressed. Water 2019, 11(11), 2355; https://doi.org/10.3390/w11112355 Received: 14 September 2019 / Revised: 30 October 2019 / Accepted: 6 November 2019 / Published: 9 November 2019 (This article belongs to the Special Issue Integrated Water Resources Research: Advancements in Understanding to Improve Future Sustainability) Download PDF Browse Figure Review Reports Cite This Paper Abstract Land managers are often inadequately informed to make management decisions in contemporary watersheds, in which sources of impairment are simultaneously shifting due to the combined influences of land use change, rapid ongoing human population growth, and changing environmental conditions. There is, thus, a great need for effective collaborative adaptive management (CAM; or derivatives) efforts utilizing an accepted methodological approach that provides data needed to properly identify and address past, present, and future sources of impairment. The experimental watershed study design holds great promise for meeting such needs and facilitating an effective collaborative and adaptive management process. To advance understanding of natural and anthropogenic influences on sources of impairment, and to demonstrate the approach in a contemporary watershed, a nested-scale experimental watershed study design was implemented in a representative, contemporary, mixed-use watershed located in Midwestern USA. Results identify challenges associated with CAM, and how the experimental watershed approach can help to objectively elucidate causal factors, target critical source areas, and provide the science-based information needed to make informed management decisions. Results show urban/suburban development and agriculture are primary drivers of alterations to watershed hydrology, streamflow regimes, transport of multiple water quality constituents, and stream physical habitat. However, several natural processes and watershed characteristics, such as surficial geology and stream system evolution, are likely compounding observed water quality impairment and aquatic habitat degradation. Given the varied and complicated set of factors contributing to such issues in the study watershed and other contemporary watersheds, watershed restoration is likely subject to physical limitations and should be conceptualized in the context of achievable goals/objectives. Overall, results demonstrate the immense, globally transferrable value of the experimental watershed approach and coupled CAM process to address contemporary water resource management challenges

A Ferredoxin Disulfide Reductase Delivers Electrons to the to the Methanosarcina barkeri Class III Ribonucleotide Reductase

Two subtypes of class III anaerobic ribonucleotide reductases (RNRs) studied so far couple the reduction of ribonucleotides to the oxidation of formate, or the oxidation of NADPH via thioredoxin and thioredoxin reductase. Certain methanogenic archaea contain a phylogenetically distinct third subtype of class III RNR, with distinct active-site residues. Here we report the cloning and recombinant expression of the Methanosarcina barkeri class III RNR and show that the electrons required for ribonucleotide reduction can be delivered by a [4Fe-4S] protein ferredoxin disulfide reductase, and a conserved thioredoxin-like protein NrdH present in the RNR operon. The diversity of class III RNRs reflects the diversity of electron carriers used in anaerobic metabolism.Singapore. Agency for Science, Technology and ResearchNational Institutes of Health (U.S.) (Grant GM081393

Renewing the Exploration Approach for Mid-Enthalpy Systems: Examples from Northern England and Scotland

After a promising start in the 1970s and 80s, the UK rather fell behind other countries in the search for viable mid-enthalpy geothermal resources. This situation began to turn around in 2004, when the first of three deep geothermal exploration boreholes were drilled in northern England. What distinguished these from earlier drilling in Cornwall was the deliberate search for naturallyhigh permeability associated with major faults, especially those that have undergone strike-slip reactivation during the Cenozoic. Boreholes at Eastgate in the North Pennines targeted buried radiothermal granite, whereas the 1,821m-deep Science Central Borehole in Newcastle upon Tyne targeted a postulated deep sedimentary aquifer (the Fell Sandstones), which were inferred to be connected laterally to the granitic heat source by a major fault (the reactivation of the Iapetus geo-suture). The drilling was in both cases rewarded with impressive heat flows, and in the case of Eastgate with what is believed to be the highest permeability yet found in a deep granite batholith anywhere in the world. In parallel with these developments, a re-assessment was made of the preexisting geothermal heat flow database for the UK, applying newly-standardised correction protocols for palaeoclimatic and topographic distortions, which were found to be particularly marked in Scotland (where only shallow boreholes had been used to establish geothermal gradients in the original 1980s analysis), Similar prospects in northern England (similar to that drilled at Science Central) are now the focus of commercial exploration efforts. Appraisal of fault dispositions relative to the present-day maximum compressive stress azimuth are being used to identify the most promising areas for intersecting fault-related permeability at depth. New geophysical tools – most notably atomic dielectric resonance scanning – are also being appraised for their ability to directly detect features (such as hot brines) which are indicative of localised convection in target fault zones and aquifers

Molecular basis of cobalamin-dependent RNA modification

Queuosine (Q) was discovered in the wobble position of a transfer RNA (tRNA) 47 years ago, yet the final biosynthetic enzyme responsible for Q-maturation, epoxyqueuosine (oQ) reductase (QueG), was only recently identified. QueG is a cobalamin (Cbl)-dependent, [4Fe-4S] cluster-containing protein that produces the hypermodified nucleoside Q in situ on four tRNAs. To understand how QueG is able to perform epoxide reduction, an unprecedented reaction for a Cbl-dependent enzyme, we have determined a series of high resolution structures of QueG from Bacillus subtilis. Our structure of QueG bound to a tRNA[superscript Tyr] anticodon stem loop shows how this enzyme uses a HEAT-like domain to recognize the appropriate anticodons and position the hypermodified nucleoside into the enzyme active site. We find Q bound directly above the Cbl, consistent with a reaction mechanism that involves the formation of a covalent Cbl-tRNA intermediate. Using protein film electrochemistry, we show that two [4Fe-4S] clusters adjacent to the Cbl have redox potentials in the range expected for Cbl reduction, suggesting how Cbl can be activated for nucleophilic attack on oQ. Together, these structural and electrochemical data inform our understanding of Cbl dependent nucleic acid modification.National Science Foundation (U.S.) (MCB 1122977)National Institutes of Health (U.S.) (GM72623 S01, GM120283, and GM17151

The streptococcal collagen-like protein-1 (Scl1) is a significant determinant for biofilm formation by group a Streptococcus

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an <it>in vitro </it>equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1) plays an important role in GAS biofilm formation.</p> <p>Results</p> <p>Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm <it>in vitro</it>. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, <it>Lactococcus lactis</it>, was sufficient to induce biofilm formation by this organism.</p> <p>Conclusions</p> <p>Overall, this work (i) identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii) identifies GAS surface properties that may aid in biofilm stability and, (iii) establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host <it>Lactococcus</it>. In summary, the GAS surface adhesin Scl1 may have an important role in biofilm-associated pathogenicity.</p

Contemporary multicenter outcomes of continent cutaneous ileocecocystoplasty in the adult population over a 10-year period: A Neurogenic Bladder Research Group study

AIMS: Evidence is sparse on the long-term outcomes of continent cutaneous ileocecocystoplasty (CCIC). We hypothesized that obesity, laparoscopic/robotic approach, and concomitant surgeries would affect morbidity after CCIC and aimed to evaluate the outcomes of CCIC in adults in a multicenter contemporary study. METHODS: We retrospectively reviewed the charts of adult patients from sites in the Neurogenic Bladder Research Group undergoing CCIC (2007-2017) who had at least 6 months of follow-up. We evaluated patient demographics, surgical details, 90-day complications, and follow-up surgeries. the Mann-Whitney U test was used to compare continuous variables and χ² and Fisher\u27s Exact tests were used to compare categorical variables. RESULTS: We included 114 patients with a median age of 41 years. The median postoperative length of stay was 8 days. At 3 months postoperatively, major complications occurred in 18 (15.8%), and 24 patients (21.1%) were readmitted. During a median follow-up of 40 months, 48 patients (42.1%) underwent 80 additional related surgeries. Twenty-three patients (20.2%) underwent at least one channel revision, most often due to obstruction (15, 13.2%) or incontinence (4, 3.5%). Of the channel revisions, 10 (8.8%) were major and 14 (12.3%) were minor. Eleven patients (9.6%) abandoned the catheterizable channel during the follow-up period. Obesity and laparoscopic/robotic surgical approach did not affect outcomes, though concomitant surgery was associated with a higher rate of follow-up surgeries. CONCLUSIONS: In this contemporary multicenter series evaluating CCIC, we found that the short-term major complication rate was low, but many patients require follow-up surgeries, mostly related to the catheterizable channel

Ultrasound Mediated Gemcitabine Delivery Reduces the Normal-Tissue Toxicity of Chemoradiation Therapy in a Muscle-Invasive Bladder Cancer Model

Purpose: Chemoradiation is the standard of care in muscle-invasive bladder. While agents such as gemcitabine can enhance tumour radiosensitisation, their side effects can limit patient eligibility and treatment efficacy. Here we investigate ultrasound and microbubbles for targeting gemcitabine delivery to reduce normal tissue toxicity in a murine orthotopic MIBC model. Materials and Methods: CD1-nude mice were injected orthotopically with RT112 bladder tumour cells. Conventional chemoradiation involved injecting gemcitabine (10 mg/kg) before 6 Gy targeted irradiation of the bladder area using a Small Animal Radiation Research Platform (SARRP). Ultrasound-mediated gemcitabine delivery (10 mg/kg gemcitabine) involved either co-administration of microbubbles with gemcitabine or conjugating gemcitabine onto microbubbles followed by exposure to ultrasound (1.1 MHz centre frequency, 1 MPa peak negative pressure, 1% duty cycle and 0.5 Hz pulse repetition frequency), prior to SARRP irradiation. The effect of ultrasound and microbubbles alone was also tested. Tumour volumes were measured by 3D ultrasound imaging. Acute normal tissue toxicity from 12 Gy to the lower bowel area was assessed using an intestinal crypt assay in mice culled. 3.75 days post-treatment. Results: Significant tumour growth delay was observed with conventional chemoradiation and both microbubble groups (p Conclusions: Ultrasound and microbubbles offer a promising new approach for improving chemoradiation therapy in muscle-invasive bladder cancer, maintaining tumour growth delay but with reduced acute intestinal toxicity compared to conventional chemoradiation therapy.</p

Validation of Twelve Small Kepler Transiting Planets in the Habitable Zone

We present an investigation of twelve candidate transiting planets from Kepler with orbital periods ranging from 34 to 207 days, selected from initial indications that they are small and potentially in the habitable zone (HZ) of their parent stars. Few of these objects are known. The expected Doppler signals are too small to confirm them by demonstrating that their masses are in the planetary regime. Here we verify their planetary nature by validating them statistically using the BLENDER technique, which simulates large numbers of false positives and compares the resulting light curves with the Kepler photometry. This analysis was supplemented with new follow-up observations (high-resolution optical and near-infrared spectroscopy, adaptive optics imaging, and speckle interferometry), as well as an analysis of the flux centroids. For eleven of them (KOI-0571.05, 1422.04, 1422.05, 2529.02, 3255.01, 3284.01, 4005.01, 4087.01, 4622.01, 4742.01, and 4745.01) we show that the likelihood they are true planets is far greater than that of a false positive, to a confidence level of 99.73% (3 sigma) or higher. For KOI-4427.01 the confidence level is about 99.2% (2.6 sigma). With our accurate characterization of the GKM host stars, the derived planetary radii range from 1.1 to 2.7 R_Earth. All twelve objects are confirmed to be in the HZ, and nine are small enough to be rocky. Excluding three of them that have been previously validated by others, our study doubles the number of known rocky planets in the HZ. KOI-3284.01 (Kepler-438b) and KOI-4742.01 (Kepler-442b) are the planets most similar to the Earth discovered to date when considering their size and incident flux jointly.Comment: 27 pages in emulateapj format, including tables and figures. To appear in The Astrophysical Journa