A Simple, Rapid Method for Extracting Large Plasmid DNA from Bacteria

We are studying the lateral transfer of transmissible antibiotic resistance plasmids among stream bacteria impacted by fecal runoff from poultry and cattle. Such plasmids are typically large (ca. 40 – 100 kb) and occur in low copy numbers in the cell and have therefore typically been difficult to isolate and therefore to study. Traditional protocols, based upon variations of the standard alkaline-lysis method, are long (ca. 1 1/2 to 2 days) and difficult. Commercial kits designed for the isolation of Baterial Artificial Chromosomes (BACs) can be used and are an improvement; however, these are expensive and still require hours of sustained effort. We have adapted a method published by Rondon et al. (1999), originally designed for the isolation of BAC DNA, for the rapid isolation of large plasmid DNA. In this method, lysis and alkaline denaturation steps are combined, incubation steps are vastly reduced, proteins are removed via a simple ammonium acetate/chloroform step, and the DNA precipitated using a plyethylene glycol/NaCl step. No ethanol precipitation is required. If additional purification is required, extracted DNA can be further processed through a Qiagen Plasmid Mini or Midi column (Qiagen Inc., Valencia CA). The method is rapid (under 1 hour), easy, very inexpensive and has been reliably used by undergraduate students to isolate large (up to 200 kb) native plasmids from a variety of both Gram-positive and Gram-negative genera including _Shigella_, _Klebsiella_, _E. coli_, _Pseudomonas_, _Bacills_, _Streptococcus_, _Staphylococcus_, and _Enterococcus_, as well as BACs from _E. coli_. The protocol is simple and reliable enough to be used for the rapid large-scale visualization of native plasmids and we have used it to visualize and isolate DNA from hundreds of multidrug resistance plasmids exogenously captured from stream sediments, soils, and beach sands.&#xa

The MethaneSAT Mission

The MethaneSAT mission is expected to launch in Q4 of 2022 with a primary goal of providing systematic monitoring of methane emissions from regions accounting for more than 80% of global oil and gas production. High precision measurements from the sensor will allow quantification and mapping of essentially all methane emissions from these regions and identify the location of major point sources and quantify those methane emissions. MethaneSAT has a wide observing swath (~200km), high spatial resolution (~100m x400m), low detection threshold (~2pbb @ 1.5 km2), and targeting capability up to 40° off nadir, enabling it to fill a critical data and observing gap in obtaining quantitative measurements of methane emissions that can be detected by current and planned satellites that either focus on point sources or map the globe. MethaneSAT data will be publicly available and will provide companies, countries and the civil society at-large, the capability to quantify total methane emissions over time and map where they occur, resulting in improved capacity to manage, and reduce those emissions. The science and policy objectives were used to derive the mission architecture that consists of a single space craft in a sun-synchronous low-Earth orbit with agility to meet the frequent site revisit requirements through off-nadir pointing. MethaneSAT consists of two spectrometers, one covering 1249 – 1305nm wavelengths for Oxygen detection and one covering 1605 –1683 nm wavelengths for Methane and Carbon Dioxide retrievals, with 0.1 nm spectral sampling and 0.3 nm spectral resolution. MethaneSAT strongly leverages Ball Aerospace’s heritage designs and spectrometer technologies developed for Landsat, Ozone Mapping & Profiler Suite (OMPS), and the Tropospheric Emissions: Monitoring Pollution (TEMPO)/Geostationary Environmental Monitoring Spectrometer (GEMS) instruments, with new designs developed a s necessary to meet mission needs. MethaneSAT is packaged into a SmallSat and will launch as a secondary payload on a Falcon 9 rocket

Biosynthetic Gene Cluster of the Glycopeptide Antibiotic Teicoplanin Characterization of Two Glycosyltransferases and the Key Acyltransferase

AbstractThe gene cluster encoding biosynthesis of the clinically important glycopeptide antibiotic teicoplanin has been cloned from Actinoplanes teichomyceticus. Forty-nine putative open reading frames (ORFs) were identified within an 89 kbp genetic locus and assigned roles in teicoplanin biosynthesis, export, resistance, and regulation. Two ORFs, designated orfs 1 and 10*, showed significant homology to known glycosyltransferases. When heterologously expressed in Escherichia coli, these glycosyltransferases were shown to catalyze the transfer of UDP-(N-acetyl)-glucosamine onto, respectively, 3-chloro-β-hydroxytyrosine-6 (3-Cl6βHty) and 4-hydroxyphenylglycine-4 (4Hpg) of the teicoplanin heptapeptide aglycone. The product of another ORF, orf11*, was demonstrated in vitro to transfer n-acetyl-, n-butyryl-, and n-octanoyl-groups from acyl-CoA donors either to a free UDP-aminosugar or to an aminosugar moiety in the teicoplanin pseudoaglycone, thus identifying Orf11* as the key acyltransferase in teicoplanin maturation. These findings should accelerate the combinatorial engineering of new and improved glycopeptide drugs

In situ measurements of tropospheric volcanic plumes in Ecuador and Colombia during TC

A NASA DC‐8 research aircraft penetrated tropospheric gas and aerosol plumes sourced from active volcanoes in Ecuador and Colombia during the Tropical Composition, Cloud and Climate Coupling (TC4 ) mission in July–August 2007. The likely source volcanoes were Tungurahua (Ecuador) and Nevado del Huila (Colombia). The TC4 data provide rare insight into the chemistry of volcanic plumes in the tropical troposphere and permit a comparison of SO2 column amounts measured by the Ozone Monitoring Instrument (OMI) on the Aura satellite with in situ SO2 measurements. Elevated concentrations of SO2, sulfate aerosol, and particles were measured by DC‐8 instrumentation in volcanic outflow at altitudes of 3–6 km. Estimated plume ages range from ∼2 h at Huila to ∼22–48 h downwind of Ecuador. The plumes contained sulfate‐rich accumulation mode particles that were variably neutralized and often highly acidic. A significant fraction of supermicron volcanic ash was evident in one plume. In‐plume O3 concentrations were ∼70%–80% of ambient levels downwind of Ecuador, but data are insufficient to ascribe this to O3 depletion via reactive halogen chemistry. The TC4 data record rapid cloud processing of the Huila volcanic plume involving aqueous‐phase oxidation of SO2 by H2O2, but overall the data suggest average in‐plume SO2 to sulfate conversion rates of ∼1%–2% h−1 . SO2 column amounts measured in the Tungurahua plume (∼0.1–0.2 Dobson units) are commensurate with average SO2 columns retrieved from OMI measurements in the volcanic outflow region in July 2007. The TC4 data set provides further evidence of the impact of volcanic emissions on tropospheric acidity and oxidizing capacit

Climate Change and Biosphere Response: Unlocking the Collections Vault

Natural history collections (NHCs) are an important source of the long-term data needed to understand how biota respond to ongoing anthropogenic climate change. These include taxon occurrence data for ecological modeling, as well as information that can be used to reconstruct mechanisms through which biota respond to changing climates. The full potential of NHCs for climate change research cannot be fully realized until high-quality data sets are conveniently accessible for research, but this requires that higher priority be placed on digitizing the holdings most useful for climate change research (e.g., whole-biota studies, time series, records of intensively sampled common taxa). Natural history collections must not neglect the proliferation of new information from efforts to understand how present-day ecosystems are responding to environmental change. These new directions require a strategic realignment for many NHC holders to complement their existing focus on taxonomy and systematics. To set these new priorities, we need strong partnerships between NHC holders and global change biologists

Cytokine Response to Traditional and Cluster Sets in Resistance-trained Women

Resistance exercise that incorporates intra-set rest between repetition blocks (i.e., cluster sets [CS]) can produce a smaller metabolic stress and endocrine response than traditional sets (TS). PURPOSE: To examine the effect of CS on the acute cytokine response in resistance trained women. METHODS: 12 resistance-trained women (mean ± SE; 23.7 ± 1.1 years; 160.1 ± 1.5 cm; 62.5 ± 1.7 kg; 5 ± 1 years training) completed 3 sessions in the follicular phase. One-repetition maximum (1RM) back squat (BS) (98.7 ± 4.1 kg), and BS:body mass (1.6 ± 0.1) were determined in Session 1. For Session 2 (3 days post Session 1) and Session 3 (7 days post Session 2), subjects were randomly assigned to either 4 sets of 10 reps with 120 seconds (s) inter-set rest (TS) or 4 x (2 x 5 reps) with 30s intra-set rest and 90s inter-set rest (CS). All performed both protocols at 70% 1RM BS. Instructions were to perform every rep “as explosively as possible”. Blood was collected pre-exercise (PRE), immediately after sets 1, 2, 3, 4 (IP), and at 5 (+5), 15 (+15), 30 (+30), and 60 (+60) min post-exercise and analyzed for interleukin (IL)-1β, IL-2, IL-6, IL-8, IL 10, and IL-15. Data were analyzed using repeated measures ANOVAs (2 × 9). RESULTS: A significant main effect of time (p\u3c0.05) was found for IL-1β, IL-2, IL-8, IL-10, and IL-15. Concentration of IL-1β was smaller at +5 (3.9 ± 0.4 ng/mL), +15 (3.6 ± 0.4) +30 (3.5 ± 0.3), and +60 (3.7 ± 0.4) compared to IP (4.1 ± 0.4). IL-2 was greater after set 1 (10.8 ± 1.0 ng/mL), and set 2 (11.0 ± 1.2) compared to PRE (10.2 ± 1.0), and smaller at +30 (9.9 ± 1.0) compared to IP (11.0 ± 1.0). IL-8 was greater after set 1 (8.4 ± 0.6 ng/mL), set 2 (8.6 ± 0.7), and set 3 (8.5 ± 0.7) compared to PRE (8.0 ± 0.6). IL-10 was smaller at +30 (31.3 ± 7.4 ng/mL) compared to PRE (34.0 ± 7.4), and also smaller at +15 (32.6 ± 7.9) +30 (31.3 ± 7.4), and +60 (33.4 ± 8.6) compared to IP (38.0 ± 8.6). IL-15 was greater at IP (15.5 ± 4.0 ng/mL) compared to PRE (13.4 ± 3.5), and smaller at PRE (13.4 ± 3.5), +30 (11.9 ± 3.3), and +60 (11.6 ± 3.2) compared to IP (15.5 ± 4.0). No condition × time point effects were observed. CONCLUSION: Both TS and CS induced an acute cytokine response in resistance-trained women; incorporating intra-set rest (CS) did not appear to affect this cytokine response

The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A

SummaryA genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5′-fluoro-5′-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5′-fluoro-5′-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate

Sediment structure and physicochemical changes following tidal inundation at a large open coast managed realignment site

Managed realignment (MR) schemes are being implemented to compensate for the loss of intertidal saltmarsh habitats by breaching flood defences and inundating the formerly defended coastal hinterland. However, studies have shown that MR sites have lower biodiversity than anticipated, which has been linked with anoxia and poor drainage resulting from compaction and the collapse of sediment pore space caused by the site's former terrestrial land use. Despite this proposed link between biodiversity and soil structure, the evolution of the sediment sub-surface following site inundation has rarely been examined, particularly over the early stages of the terrestrial to marine or estuarine transition. This paper presents a novel combination of broad- and intensive-scale analysis of the sub-surface evolution of the Medmerry Managed Realignment Site (West Sussex, UK) in the three years following site inundation. Repeated broad-scale sediment physiochemical datasets are analysed to assess the early changes in the sediment subsurface and the preservation of the former terrestrial surface, comparing four locations of different former land uses. Additionally, for two of these locations, high-intensity 3D-computed X-ray microtomography and Itrax micro-X-ray fluorescence spectrometry analyses are presented. Results provide new data on differences in sediment properties and structure related to the former land use, indicating that increased agricultural activity leads to increased compaction and reduced porosity. The presence of anoxic conditions, indicative of poor hydrological connectivity between the terrestrial and post-inundation intertidal sediment facies, was only detected at one site. This site has experienced the highest rate of accretion over the terrestrial surface (ca. 7 cm over 36 months), suggesting that poor drainage is caused by the interaction (or lack of) between sediment facies rather than the former land use. This has significant implications for the design of future MR sites in terms of preparing sites, their anticipated evolution, and the delivery of ecosystem services

Activation of intestinal spinal afferent endings by changes in intra‐mesenteric arterial pressure

Author manuscript made available following 12 month embargo from date of publication (25 June 2015) in accordance with publisher copyright policy.KEY POINTS: A major class of mechano-nociceptors to the intestine have mechanotransduction sites on extramural and intramural arteries and arterioles ('vascular afferents'). These sensory neurons can be activated by compression or axial stretch of vessels. Using isolated preparations we showed that increasing intra-arterial pressure, within the physiological range, activated mechano-nociceptors on vessels in intact mesenteric arcades, but not in isolated arteries. This suggests that distortion of the branching vascular tree is the mechanical adequate stimulus for these sensory neurons, rather than simple distension. The same rises in pressure also activated intestinal peristalsis in a partially capsaicin-sensitive manner indicating that pressure-sensitive vascular afferents influence enteric circuits. The results identify the mechanical adequate stimulus for a major class of mechano-nociceptors with endings on blood vessels supplying the gut wall; these afferents have similar endings to ones supplying other viscera, striated muscle and dural vessels. ABSTRACT: Spinal sensory neurons innervate many large blood vessels throughout the body. Their activation causes the hallmarks of neurogenic inflammation: vasodilatation through the release of the neuropeptide calcitonin gene-related peptide and plasma extravasation via tachykinins. The same vasodilator afferent neurons show mechanical sensitivity, responding to crushing, compression or axial stretch of blood vessels - responses which activate pain pathways and which can be modified by cell damage and inflammation. In the present study, we tested whether spinal afferent axons ending on branching mesenteric arteries ('vascular afferents') are sensitive to increased intravascular pressure. From a holding pressure of 5 mmHg, distension to 20, 40, 60 or 80 mmHg caused graded, slowly adapting increases in firing of vascular afferents. Many of the same afferent units showed responses to axial stretch, which summed with responses evoked by raised pressure. Many vascular afferents were also sensitive to raised temperature, capsaicin and/or local compression with von Frey hairs. However, responses to raised pressure in single, isolated vessels were negligible, suggesting that the adequate stimulus is distortion of the arterial arcade rather than distension per se. Increasing arterial pressure often triggered peristaltic contractions in the neighbouring segment of intestine, an effect that was mimicked by acute exposure to capsaicin (1 μm) and which was reduced after desensitisation to capsaicin. These results indicate that sensory fibres with perivascular endings are sensitive to pressure-induced distortion of branched arteries, in addition to compression and axial stretch, and that they contribute functional inputs to enteric motor circuits

Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity