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1. Dual cationic–anionic profiling of metabolites in a single identified cell in a live Xenopus laevis embryo by microprobe CE-ESI-MS

   https://doi.org/10.1039/C8AN01999A  

   Portero, Erika P. ; Nemes, Peter ( January 2019 , The Analyst)

   In situ capillary microsampling with capillary electrophoresis (CE) electrospray ionization (ESI) mass spectrometry (MS) enabled the characterization of cationic metabolites in single cells in complex tissues and organisms. For deeper coverage of the metabolome and metabolic networks, analytical approaches are needed that provide complementary detection for anionic metabolites, ideally using the same instrumentation. Described here is one such approach that enables sequential cationic and anionic (dual) analysis of metabolites in the same identified cell in a live vertebrate embryo. A calibrated volume was microaspirated from the animal-ventral cell in a live 8-cell embryo of Xenopus laevis , and cationic and anionic metabolites were one-pot microextracted from the aspirate, followed by CE-ESI-MS analysis of the same extract. A laboratory-built CE-ESI interface was reconfigured to enable dual cationic–anionic analysis with ∼5–10 nM (50–100 amol) lower limit of detection and a capability for quantification. To provide robust separation and efficient ion generation, the CE-ESI interface was enclosed in a nitrogen gas filled chamber, and the operational parameters were optimized for the cone-jet spraying regime in both the positive and negative ion mode. A total of ∼250 cationic and ∼200 anionic molecular features were detected from the cell between m / z 50–550, including 60 and 24 identified metabolites, respectively. With only 11 metabolites identified mutually, the duplexed approach yielded complementary information on metabolites produced in the cell, which in turn deepened network coverage for several metabolic pathways. With scalability to smaller cells and adaptability to other types of tissues and organisms, dual cationic–anionic detection with in situ microprobe CE-ESI-MS opens a door to better understand cell metabolism. 

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2. Florida mangrove saltmarsh reference surface soils

   https://doi.org/10.6073/pasta/0e08cbe07c84488cb7b9dd16669946d0  

   Lewis, David Bruce ( January 2021 , Environmental Data Initiative)

   Site description. This data package consists of data obtained from sampling surface soil (the 0-7.6 cm depth profile) in black mangrove (Avicennia germinans) dominated forest and black needlerush (Juncus roemerianus) saltmarsh along the Gulf of Mexico coastline in peninsular west-central Florida, USA. This location has a subtropical climate with mean daily temperatures ranging from 15.4 °C in January to 27.8 °C in August, and annual precipitation of 1336 mm. Precipitation falls as rain primarily between June and September. Tides are semi-diurnal, with 0.57 m median amplitudes during the year preceding sampling (U.S. NOAA National Ocean Service, Clearwater Beach, Florida, station 8726724). Sea-level rise is 4.0 ± 0.6 mm per year (1973-2020 trend, mean ± 95 % confidence interval, NOAA NOS Clearwater Beach station). The A. germinans mangrove zone is either adjacent to water or fringed on the seaward side by a narrow band of red mangrove (Rhizophora mangle). A near-monoculture of J. roemerianus is often adjacent to and immediately landward of the A. germinans zone. The transition from the mangrove to the J. roemerianus zone is variable in our study area. An abrupt edge between closed-canopy mangrove and J. roemerianus monoculture may extend for up to several hundred meters in some locations, while other stretches of ecotone present a gradual transition where smaller, widely spaced trees are interspersed into the herbaceous marsh. Juncus roemerianus then extends landward to a high marsh patchwork of succulent halophytes (including Salicornia bigellovi, Sesuvium sp., and Batis maritima), scattered dwarf mangrove, and salt pans, followed in turn by upland vegetation that includes Pinus sp. and Serenoa repens. Field design and sample collection. We established three study sites spaced at approximately 5 km intervals along the western coastline of the central Florida peninsula. The sites consisted of the Salt Springs (28.3298°, -82.7274°), Energy Marine Center (28.2903°, -82.7278°), and Green Key (28.2530°, -82.7496°) sites on the Gulf of Mexico coastline in Pasco County, Florida, USA. At each site, we established three plot pairs, each consisting of one saltmarsh plot and one mangrove plot. Plots were 50 m^2 in size. Plots pairs within a site were separated by 230-1070 m, and the mangrove and saltmarsh plots composing a pair were 70-170 m apart. All plot pairs consisted of directly adjacent patches of mangrove forest and J. roemerianus saltmarsh, with the mangrove forests exhibiting a closed canopy and a tree architecture (height 4-6 m, crown width 1.5-3 m). Mangrove plots were located at approximately the midpoint between the seaward edge (water-mangrove interface) and landward edge (mangrove-marsh interface) of the mangrove zone. Saltmarsh plots were located 20-25 m away from any mangrove trees and into the J. roemerianus zone (i.e., landward from the mangrove-marsh interface). Plot pairs were coarsely similar in geomorphic setting, as all were located on the Gulf of Mexico coastline, rather than within major sheltering formations like Tampa Bay, and all plot pairs fit the tide-dominated domain of the Woodroffe classification (Woodroffe, 2002, "Coasts: Form, Process and Evolution", Cambridge University Press), given their conspicuous semi-diurnal tides. There was nevertheless some geomorphic variation, as some plot pairs were directly open to the Gulf of Mexico while others sat behind keys and spits or along small tidal creeks. Our use of a plot-pair approach is intended to control for this geomorphic variation. Plot center elevations (cm above mean sea level, NAVD 88) were estimated by overlaying the plot locations determined with a global positioning system (Garmin GPS 60, Olathe, KS, USA) on a LiDAR-derived bare-earth digital elevation model (Dewberry, Inc., 2019). The digital elevation model had a vertical accuracy of ± 10 cm (95 % CI) and a horizontal accuracy of ± 116 cm (95 % CI). Soil samples were collected via coring at low tide in June 2011. From each plot, we collected a composite soil sample consisting of three discrete 5.1 cm diameter soil cores taken at equidistant points to 7.6 cm depth. Cores were taken by tapping a sleeve into the soil until its top was flush with the soil surface, sliding a hand under the core, and lifting it up. Cores were then capped and transferred on ice to our laboratory at the University of South Florida (Tampa, Florida, USA), where they were combined in plastic zipper bags, and homogenized by hand into plot-level composite samples on the day they were collected. A damp soil subsample was immediately taken from each composite sample to initiate 1 y incubations for determination of active C and N (see below). The remainder of each composite sample was then placed in a drying oven (60 °C) for 1 week with frequent mixing of the soil to prevent aggregation and liberate water. Organic wetland soils are sometimes dried at 70 °C, however high drying temperatures can volatilize non-water liquids and oxidize and decompose organic matter, so 50 °C is also a common drying temperature for organic soils (Gardner 1986, "Methods of Soil Analysis: Part 1", Soil Science Society of America); we accordingly chose 60 °C as a compromise between sufficient water removal and avoidance of non-water mass loss. Bulk density was determined as soil dry mass per core volume (adding back the dry mass equivalent of the damp subsample removed prior to drying). Dried subsamples were obtained for determination of soil organic matter (SOM), mineral texture composition, and extractable and total carbon (C) and nitrogen (N) within the following week. Sample analyses. A dried subsample was apportioned from each composite sample to determine SOM as mass loss on ignition at 550 °C for 4 h. After organic matter was removed from soil via ignition, mineral particle size composition was determined using a combination of wet sieving and density separation in 49 mM (3 %) sodium hexametaphosphate ((NaPO_3)_6) following procedures in Kettler et al. (2001, Soil Science Society of America Journal 65, 849-852). The percentage of dry soil mass composed of silt and clay particles (hereafter, fines) was calculated as the mass lost from dispersed mineral soil after sieving (0.053 mm mesh sieve). Fines could have been slightly underestimated if any clay particles were burned off during the preceding ignition of soil. An additional subsample was taken from each composite sample to determine extractable N and organic C concentrations via 0.5 M potassium sulfate (K_2SO_4) extractions. We combined soil and extractant (ratio of 1 g dry soil:5 mL extractant) in plastic bottles, reciprocally shook the slurry for 1 h at 120 rpm, and then gravity filtered it through Fisher G6 (1.6 μm pore size) glass fiber filters, followed by colorimetric detection of nitrite (NO_2^-) + nitrate (NO_3^-) and ammonium (NH_4^+) in the filtrate (Hood Nowotny et al., 2010,Soil Science Society of America Journal 74, 1018-1027) using a microplate spectrophotometer (Biotek Epoch, Winooski, VT, USA). Filtrate was also analyzed for dissolved organic C (referred to hereafter as extractable organic C) and total dissolved N via combustion and oxidation followed by detection of the evolved CO_2 and N oxide gases on a Formacs HT TOC/TN analyzer (Skalar, Breda, The Netherlands). Extractable organic N was then computed as total dissolved N in filtrate minus extractable mineral N (itself the sum of extractable NH_4-N and NO_2-N + NO_3-N). We determined soil total C and N from dried, milled subsamples subjected to elemental analysis (ECS 4010, Costech, Inc., Valencia, CA, USA) at the University of South Florida Stable Isotope Laboratory. Median concentration of inorganic C in unvegetated surface soil at our sites is 0.5 % of soil mass (Anderson, 2019, Univ. of South Florida M.S. thesis via methods in Wang et al., 2011, Environmental Monitoring and Assessment 174, 241-257). Inorganic C concentrations are likely even lower in our samples from under vegetation, where organic matter would dilute the contribution of inorganic C to soil mass. Nevertheless, the presence of a small inorganic C pool in our soils may be counted in the total C values we report. Extractable organic C is necessarily of organic C origin given the method (sparging with HCl) used in detection. Active C and N represent the fractions of organic C and N that are mineralizable by soil microorganisms under aerobic conditions in long-term soil incubations. To quantify active C and N, 60 g of field-moist soil were apportioned from each composite sample, placed in a filtration apparatus, and incubated in the dark at 25 °C and field capacity moisture for 365 d (as in Lewis et al., 2014, Ecosphere 5, art59). Moisture levels were maintained by frequently weighing incubated soil and wetting them up to target mass. Daily CO_2 flux was quantified on 29 occasions at 0.5-3 week intervals during the incubation period (with shorter intervals earlier in the incubation), and these per day flux rates were integrated over the 365 d period to compute an estimate of active C. Observations of per day flux were made by sealing samples overnight in airtight chambers fitted with septa and quantifying headspace CO_2 accumulation by injecting headspace samples (obtained through the septa via needle and syringe) into an infrared gas analyzer (PP Systems EGM 4, Amesbury, MA, USA). To estimate active N, each incubated sample was leached with a C and N free, 35 psu solution containing micronutrients (Nadelhoffer, 1990, Soil Science Society of America Journal 54, 411-415) on 19 occasions at increasing 1-6 week intervals during the 365 d incubation, and then extracted in 0.5 M K_2SO_4 at the end of the incubation in order to remove any residual mineral N. Active N was then quantified as the total mass of mineral N leached and extracted. Mineral N in leached and extracted solutions was detected as NH_4-N and NO_2-N + NO_3-N via colorimetry as above. This incubation technique precludes new C and N inputs and persistently leaches mineral N, forcing microorganisms to meet demand by mineralizing existing pools, and thereby directly assays the potential activity of soil organic C and N pools present at the time of soil sampling. Because this analysis commences with disrupting soil physical structure, it is biased toward higher estimates of active fractions. Calculations. Non-mobile C and N fractions were computed as total C and N concentrations minus the extractable and active fractions of each element. This data package reports surface-soil constituents (moisture, fines, SOM, and C and N pools and fractions) in both gravimetric units (mass constituent / mass soil) and areal units (mass constituent / soil surface area integrated through 7.6 cm soil depth, the depth of sampling). Areal concentrations were computed as X × D × 7.6, where X is the gravimetric concentration of a soil constituent, D is soil bulk density (g dry soil / cm^3), and 7.6 is the sampling depth in cm. 

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3. Improved identification and quantitation of mature endogenous peptides in the rodent hypothalamus using a rapid conductive sample heating system

   https://doi.org/10.1039/C7AN01358B  

   Yang, Ning ; Anapindi, Krishna D. ; Romanova, Elena V. ; Rubakhin, Stanislav S. ; Sweedler, Jonathan V. ( January 2017 , The Analyst)

   Measurement, identification, and quantitation of endogenous peptides in tissue samples by mass spectrometry (MS) contribute to our understanding of the complex molecular mechanisms of numerous biological phenomena. For accurate results, it is essential to arrest the postmortem degradation of ubiquitous proteins in samples prior to performing peptidomic measurements. Doing so ensures that the detection of endogenous peptides, typically present at relatively low levels of abundance, is not overwhelmed by protein degradation products. Heat stabilization has been shown to inactivate the enzymes in tissue samples and minimize the presence of protein degradation products in the subsequent peptide extracts. However, the efficacy of different heat treatments to preserve the integrity of full-length endogenous peptides has not been well documented; prior peptidomic studies of heat stabilization methods have not distinguished between the full-length (mature) and numerous truncated (possible artifacts of sampling) forms of endogenous peptides. We show that thermal sample treatment via rapid conductive heat transfer is effective for detection of mature endogenous peptides in fresh and frozen rodent brain tissues. Freshly isolated tissue processing with the commercial Stabilizor T1 heat stabilization system resulted in the confident identification of 65% more full-length mature neuropeptides compared to widely used sample treatment in a hot water bath. This finding was validated by a follow-up quantitative multiple reaction monitoring MS analysis of select neuropeptides. The rapid conductive heating in partial vacuum provided by the Stabilizor T1 effectively reduces protein degradation and decreases the chemical complexity of the sample, as assessed by determining total protein content. This system enabled the detection, identification, and quantitation of neuropeptides related to 22 prohormones expressed in individual rat hypothalami and suprachiasmatic nuclei. 

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4. Expedition 357 Preliminary Report: Atlantis Massif Serpentinization and Life

   https://doi.org/10.14379/iodp.pr.357.2016  

   Früh-Green, G.L. ; Orcutt, B.N. ; Green, S. ; Cotterill, C. ( May 2016 , Preliminary report)

   null (Ed.)

   International Ocean Discovery Program (IODP) Expedition 357 successfully cored an east–west transect across the southern wall of Atlantis Massif on the western flank of the Mid-Atlantic Ridge to study the links between serpentinization processes and microbial activity in the shallow subsurface of highly altered ultramafic and mafic sequences that have been uplifted to the seafloor along a major detachment fault zone. The primary goals of this expedition were to (1) examine the role of serpentinization in driving hydrothermal systems, sustaining microbial communities, and sequestering carbon; (2) characterize the tectonomagmatic processes that lead to lithospheric heterogeneities and detachment faulting; and (3) assess how abiotic and biotic processes change with variations in rock type and progressive exposure on the seafloor. To accomplish these objectives, we developed a coring and sampling strategy based around the use of seabed rock drills—the first time that such systems have been used in the scientific ocean drilling programs. This technology was chosen in hopes of achieving high recovery of the carbonate cap sequences and intact contact and deformation relationships. The expedition plans also included several engineering developments to assess geochemical parameters during drilling; sample bottom water before and after drilling; supply synthetic tracers during drilling for contamination assessment; gather downhole electrical resistivity and magnetic susceptibility logs for assessing fractures, fluid flow, and extent of serpentinization; and seal boreholes to provide opportunities for future experiments. Seventeen holes were drilled at nine sites across Atlantis Massif, with two sites on the eastern end of the southern wall (Sites M0068 and M0075), three sites in the central section of the southern wall north of the Lost City hydrothermal field (Sites M0069, M0072, and M0076), two sites on the western end (Sites M0071 and M0073), and two sites north of the southern wall in the direction of the central dome of the massif and Integrated Ocean Drilling Program Site U1309 (Sites M0070 and M0074). Use of seabed rock drills enabled collection of more than 57 m of core, with borehole penetration ranging from 1.3 to 16.44 meters below seafloor and core recoveries as high as 75% of total penetration. This high level of recovery of shallow mantle sequences is unprecedented in the history of ocean drilling. The cores recovered along the southern wall of Atlantis Massif have highly heterogeneous lithologies, types of alteration, and degrees of deformation. The ultramafic rocks are dominated by harzburgites with intervals of dunite and minor pyroxenite veins, as well as gabbroic rocks occurring as melt impregnations and veins, all of which provide information about early magmatic processes and the magmatic evolution in the southernmost portion of Atlantis Massif. Dolerite dikes and basaltic rocks represent the latest stage of magmatic activity. Overall, the ultramafic rocks recovered during Expedition 357 revealed a high degree of serpentinization, as well as metasomatic talc-amphibole-chlorite overprinting and local rodingitization. Metasomatism postdates an early phase of serpentinization but predates late-stage intrusion and alteration of dolerite dikes and the extrusion of basalt. The intensity of alteration is generally lower in the gabbroic and doleritic rocks. Chilled margins in dolerite intruded into talc-amphibole-chlorite schists are observed at the most eastern Site M0075. Deformation in Expedition 357 cores is variable and dominated by brecciation and formation of localized shear zones; the degree of carbonate veining was lower than anticipated. All types of variably altered and deformed ultramafic and mafic rocks occur as components in sedimentary breccias and as fault scarp rubble. The sedimentary cap rocks include basaltic breccias with a carbonate sand matrix and/or fossiliferous carbonate. Fresh glass on basaltic components was observed in some of the breccias. The expedition also successfully applied new technologies, namely (1) extensively using an in situ sensor package and water sampling system on the seabed drills for evaluating real-time dissolved oxygen and methane, pH, oxidation-reduction potential, temperature, and conductivity during drilling; (2) deploying a borehole plug system for sealing seabed drill boreholes at four sites to allow access for future sampling; and (3) proving that tracers can be delivered into drilling fluids when using seabed drills. The rock drill sensor packages and water sampling enabled detection of elevated dissolved methane and hydrogen concentrations during and/or after drilling, with “hot spots” of hydrogen observed over Sites M0068–M0072 and methane over Sites M0070–M0072. Shipboard determination of contamination tracer delivery confirmed appropriate sample handling procedures for microbiological and geochemical analyses, which will aid all subsequent microbiological investigations that are part of the science party sampling plans, as well as verify this new tracer delivery technology for seabed drill rigs. Shipboard investigation of biomass density in select samples revealed relatively low and variable cell densities, and enrichment experiments set up shipboard reveal growth. Thus, we anticipate achieving many of the deep biosphere–related objectives of the expedition through continued scientific investigation in the coming years. Finally, although not an objective of the expedition, we were serendipitously able to generate a high-resolution (20 m per pixel) multibeam bathymetry map across the entire Atlantis Massif and the nearby fracture zone, Mid-Atlantic Ridge, and eastern conjugate, taking advantage of weather and operational downtime. This will assist science party members in evaluating and interpreting tectonic and mass-wasting processes at Atlantis Massif. 

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5. North Temperate Lakes LTER: Phytoplankton - Madison Lakes Area 1995 - current

   https://doi.org/10.6073/pasta/bd03330e83fc52a5442bb39e131c95e9  

   Magnuson, John J. ; Carpenter, Stephen R. ; Stanley, Emily H. ( January 2022 , Environmental Data Initiative)

   Phytoplankton samples for the 4 southern Wisconsin LTER lakes (Mendota, Monona, Wingra, Fish) have been collected for analysis by LTER since 1995 (1996 Wingra, Fish) when the southern Wisconsin lakes were added to the North Temperate Lakes LTER project. Samples are collected as a composite whole-water sample and are preserved in gluteraldehyde. Composite sample depths are 0-8 meters for Lake Mendota (to conform to samples collected and analyzed since 1990 for a UW/DNR food web research study), and 0-2 meters for the other three lakes. A tube sampler is used for the 0-8 m Lake Mendota samples; samples for the other lakes are obtained by collecting water at 1-meter intervals using a Kemmerer water sampler and compositing the samples in a bucket. Samples are taken in the deep hole region of each lake at the same time and location as other limnological sampling. Phytoplankton samples are analyzed by PhycoTech, Inc., a private lab specializing in phytoplankton analyses (see data protocol for procedures). Samples for Wingra and Fish lakes are archived but not routinely counted. Permanent slide mounts (3 per sample) are prepared for all analyzed Mendota and Monona samples as well as 6 samples per year for Wingra and Fish; the slide mounts are archived at the University of Wisconsin - Madison Zoology Museum. Phytoplankton are identified to species using an inverted microscope (Utermohl technique) and are reported as natural unit (i.e., colonies, filaments, or single cells) densities per mL, cell densities per mL, and algal biovolume densities per mL. Multiple entries for the same species on the same date may be due to different variants or vegetative states - (e.g., colonial or attached vs. free cell.) Biovolumes for individual cells of each species are determined during the counting procedure by obtaining cell measurements needed to calculate volumes for geometric solids (e.g., cylinders, spheres, truncated cones) corresponding to actual cell shapes. Biovolume concentrations are then computed by mulitplying the average cell biovolume by the cell densities in the water sample. Note that one million cubicMicrometers of biovolume PerMilliliter of water are equal to a biovolume concentration of one cubicMillimeterPerMilliliter. Assuming a cell density equal to water, a cubicMillimeterPerMilliliter of biovolume converts to a biomass concentration of one milligramPerLiter. Sampling Frequency: bi-weekly during ice-free season from late March or early April through early September, then every 4 weeks through late November; sampling is conducted usually once during the winter (depending on ice conditions). Number of sites: 4 Several taxonomic updates have been made to this dataset February 2013, see methods for details. 

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