Separation and Detection of Peroxynitrite Using Microchip Electrophoresis with Amperometric Detection

Peroxynitrite (ONOO-) is a highly reactive species implicated in the pathology of several cardiovascular and neurodegenerative diseases. It is generated in vivo by the diffusion-limited reaction of nitric oxide (NO•) and superoxide anion (•O2-) and is known to be produced during periods of inflammation. Detection of ONOO- is made difficult by its short half-life under physiological conditions (∼1 s). Here we report a method for the separation and detection of ONOOfrom other electroactive species utilizing a microchip electrophoresis device incorporating an amperometric detection scheme. Microchip electrophoresis permits shorter separation times (∼25 s for ONOO-) and higher temporal resolution than conventional capillary electrophoresis (several minutes). This faster analysis allows ONOO- to be detected before substantial degradation occurs, and the increased temporal resolution permits more accurate tracking of dynamic changes in chemical systems

Salisapiliaceae – a new family of oomycetes from marsh grass litter of southeastern North America

Several filamentous oomycete species of the genus Halophytophthora have recently been described from marine environments, mostly from subtropical and tropical ecosystems. During a survey of oomycetes from leaf litter of Spartina alterniflora in salt marshes of southeastern Georgia, isolates of four taxa were recovered that bore similarity to some members of Halophytophthora but were highly divergent from isolates of Halophytophthora s.str. based on a combined sequence analysis of two nuclear loci. In phylogenetic analyses, these isolates were placed basal to a monophyletic group comprised of Pythium of the Pythiaceae and the Peronosporaceae. Sequence and morphology of these taxa diverged from the type species Halophytophthora vesicula, which was placed within the Peronosporaceae with maximum support. As a consequence a new family, the Salisapiliaceae, and a new genus, Salisapilia, are described to accommodate the newly discovered species, along with one species previously classified within Halophytophthora. Morphological features that separate these taxa from Halophytophthora are a smaller hyphal diameter, oospore production, lack of vesicle formation during sporulation, and a plug of hyaline material at the sporangial apex that is displaced during zoospore release. Our findings offer a first glance at the presumably much higher diversity of oomycetes in estuarine environments, of which ecological significance requires further exploration

Microchip Electrophoresis with Amperometric Detection Method for Profiling Cellular Nitrosative Stress Markers

The overproduction of nitric oxide (NO) in cells results in nitrosative stress due to the generation of highly reactive species such as peroxynitrite and N2O3. These species disrupt the cellular redox processes through the oxidation, nitration, and nitrosylation of important biomolecules. Microchip electrophoresis (ME) is a fast separation method that can be used to profile cellular nitrosative stress through the separation of NO and nitrite from other redox-active intracellular components such as cellular antioxidants. This paper describes a ME method with electrochemical detection (ME-EC) for the separation of intracellular nitrosative stress markers in macrophage cells. The separation of nitrite, azide (interference), iodide (internal standard), tyrosine, glutathione, and hydrogen peroxide (neutral marker) was achieved in under 40 s using a run buffer consisting of 7.5 to 10 mM NaCl, 10 mM boric acid, and 2 mM TTAC at pH 10.3 to 10.7. Initially, NO production was monitored by the detection of nitrite (NO2−) in cell lysates. There was a 2.5- to 4-fold increase in NO2− production in lipopolysaccharide (LPS)-stimulated cells. The concentration of NO2− inside a single unstimulated macrophage cell was estimatedto be 1.41 mM using the method of standard additions. ME-EC was then used for the direct detection of NO and glutathione in stimulated and native macrophage cell lysates. NO was identified in these studies based on its migration time and rapid degradation kinetics. The intracellular levels of glutathione in native and stimulated macrophages were also compared, and no significant difference was observed between the two conditions

Microchip electrophoresis with amperometric detection for the study of the generation of nitric oxide by NONOate salts

Microchip electrophoresis (ME) with electrochemical detection was used to monitor nitric oxide (NO) production from diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA/NO) and 1-(hydroxyl-NNO-azoxy)-L-proline disodium salt (PROLI/NO). NO was generated through acid hydrolysis of these NONOate salts. The products of acid hydrolysis were introduced into a 5-cm separation channel using gated injection. The separation was accomplished using reverse polarity and a background electrolyte consisting of 10 mM boric acid and 2 mM tetradecyltrimethylammonium bromide, pH 11. Electrochemical detection was performed using an isolated potentiostat in an in-channel configuration. Potentials applied to the working electrode, typically higher than +1.0 V vs. Ag/AgCl, allowed the direct detection of nitrite, NO, DEA/NO, and PROLI/NO. Baseline resolution was achieved for the separation of PROLI/NO and NO while resolution between DEA/NO and NO was poor (1.0±0.2). Nitrite was present in all samples tested

Performance of van der Waals Corrected Functionals for Guest Adsorption in the M-2(dobdc) Metal-Organic Frameworks

Small-molecule binding in metal–organic frameworks (MOFs) can be accurately studied both experimentally and computationally, provided the proper tools are employed. Herein, we compare and contrast properties associated with guest binding by means of density functional theory (DFT) calculations using nine different functionals for the M2(dobdc) (dobdc4– = 2,5-dioxido,1,4-benzenedicarboxylate) series, where M = Mg, Mn, Fe, Co, Ni, Cu, and Zn. Additionally, we perform Quantum Monte Carlo (QMC) calculations for one system to determine if this method can be used to assess the performance of DFT. We also make comparisons with previously published experimental results for carbon dioxide and water and present new methane neutron powder diffraction (NPD) data for further comparison. All of the functionals are able to predict the experimental variation in the binding energy from one metal to the next; however, the interpretation of the performance of the functionals depends on which value is taken as the reference. On the one hand, if we compare against experimental values, we would conclude that the optB86b-vdW and optB88-vdW functionals systematically overestimate the binding strength, while the second generation of van der Waals (vdW) nonlocal functionals (vdw-DF2 and rev-vdW-DF2) correct for this providing a good description of binding energies. On the other hand, if the QMC calculation is taken as the reference then all of the nonlocal functionals yield results that fall just outside the error of the higher-level calculation. The empirically corrected vdW functionals are in reasonable agreement with experimental heat of adsorptions but under bind when compared with QMC, while Perdew–Burke–Ernzerhof fails by more than 20 kJ/mol regardless of which reference is employed. All of the functionals, with the exception of vdW-DF2, predict reasonable framework and guest binding geometries when compared with NPD measurements. The newest of the functionals considered, rev-vdW-DF2, should be used in place of vdW-DF2, as it yields improved bond distances with similar quality binding energies

An Integrated Microfluidic Device for Monitoring Changes in Nitric Oxide Production in Single T-Lymphocyte (Jurkat) Cells

A considerable amount of attention has been focused on the analysis of single cells in an effort to better understand cell heterogeneity in cancer and neurodegenerative diseases. Although microfluidic devices have several advantages for single cell analysis, few papers have actually demonstrated the ability of these devices to monitor chemical changes in perturbed biological systems. In this paper, a new microfluidic channel manifold is described that integrates cell transport, lysis, injection, electrophoretic separation, and fluorescence detection into a single device, making it possible to analyze individual cells at a rate of 10 cells/min in an automated fashion. The system was employed to measure nitric oxide (NO) production in single T-lymphocytes (Jurkat cells) using a fluorescent marker, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA). The cells were also labeled with 6-carboxyfluorescein diacetate (6-CFDA) as an internal standard. The NO production by control cells was compared to that of cells stimulated using lipopolysaccharide (LPS), which is known to cause the expression of inducible nitric oxide synthase (iNOS) in immune-type cells. Statistical analysis of the resulting electropherograms from a population of cells indicated a twofold increase in NO production in the induced cells. These results compare nicely to a recently published bulk cell analysis of NO

Contributions of a global network of tree diversity experiments to sustainable forest plantations

status: publishe

A million and more trees for science

TreeDivNet is the largest network of biodiversity experiments worldwide, but needs to expand. We encourage colleagues to establish new experiments on the relation between tree species diversity and forest ecosystem functioning, and to make use of the platform for collaborative research