The Benthic Exchange of O2, N2 and Dissolved Nutrients Using Small Core Incubations

The measurement of sediment-water exchange of gases and solutes in aquatic sediments provides data valuable for understanding the role of sediments in nutrient and gas cycles. After cores with intact sediment-water interfaces are collected, they are submerged in incubation tanks and kept under aerobic conditions at in situ temperatures. To initiate a time course of overlying water chemistry, cores are sealed without bubbles using a top cap with a suspended stirrer. Time courses of 4-7 sample points are used to determine the rate of sediment water exchange. Artificial illumination simulates day-time conditions for shallow photosynthetic sediments, and in conjunction with dark incubations can provide net exchanges on a daily basis. The net measurement of N₂ is made possible by sampling a time course of dissolved gas concentrations, with high precision mass spectrometric analysis of N₂:Ar ratios providing a means to measure N₂ concentrations. We have successfully applied this approach to lakes, reservoirs, estuaries, wetlands and storm water ponds, and with care, this approach provides valuable information on biogeochemical balances in aquatic ecosystems.     

Assessment of Isolated Wetland Condition in Florida Using Epiphytic Diatoms at Genus, Species, and Subspecies Taxonomic Resolution

Diatoms are useful indicators of aquatic conditions, and metrics based on published autecological indicator values have been developed utilizing their sensitivities to various ambient physical and chemical conditions. The autecological values often differ within genera, and indeed within species taxonomic levels, requiring identification to subspecies taxonomic level for accurate application. This study was conducted to determine if autecological metrics, and ultimately indices of biotic integrity, could be developed using mean autecological values at the genus, species, and subspecies taxonomic levels, and to investigate the potential benefits of increased taxonomic resolution. Sixty-nine isolated herbaceous wetlands in various land use modalities in peninsular Florida were sampled a single time for epiphytic diatoms, and soil/water physicochemical parameters. Thirty genera, 148 species, and 26 subspecies were identified. The proportional matrices at each taxonomic level were highly similar (Mantel’s r > 0.75, P < 0.0001). Autecological metrics and two sensitive or tolerant measures were developed at each taxonomic level. Wetland condition, as determined by summed metric values, was strongly correlated across taxonomic level (r ² > 0.83, P < 0.0001), and no significant difference was found when sites were placed into bins of excellent, good, fair, or poor, based on quartile scoring, for each taxonomic level. Specific conductance, soil pH, soil and water total phosphorous, and water color were significantly related to site non-metric multidimensional scaling (NMDS) ordination scores at each taxonomic level. This study concludes that indices of biotic integrity, when developed using autecological indices, provide similar qualitative conditional information across taxonomic levels for isolated herbaceous wetlands. Electronic supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.     

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.