A methodology for radiolabeling of the endocannabinoid 2-arachidonoylglycerol (2-AG)

The metabolic intermediate and endocannabinoid signaling lipid 2-arachidonoylglycerol (2-AG) has not been readily labeled, primarily because of its instability toward rearrangement. We now detail a synthetic method that easily gives tritiated 2-AG from [5,6,8,9,11,12,14,15-(3)H(N)]arachidonic acid in two steps. We utilized a short chain 1,3-diacylglycerol and proceeded through the "structured lipid" [5',6',8',9',11',12',14',15'-(3)H(N)]2-arachidonoyl-1,3-dibutyrylglycerol, a triacylglycerol that was conveniently deprotected in ethanol with acrylic beads containing Candida antarctica lipase B to give [5',6',8',9',11',12',14',15'-(3)H(N)]2-arachidonoylglycerol ([(3)H]2-AG). The flash chromatographic separation necessary to isolate the labeled 2-acylglycerol [(3)H]2-AG resulted in only 4% of the rearrangement byproducts that have been a particular problem with previous methodologies. This reliable "kit" method to prepare the radiolabeled endocannabinoid as needed gave tritiated 2-arachidonoylglycerol [(3)H]2-AG with a specific activity of 200 Ci/mmol for enzyme assays, metabolic studies, and tissue imaging. It has been run on unlabeled materials on over 10 mg scales and should be generally applicable to other 2-acylglycerols.     

Real-time TEM imaging of the formation of crystalline nanoscale gaps

We present real-time transmission electron microscopy of nanogap formation by feedback controlled electromigration that reveals a remarkable degree of crystalline order. Crystal facets appear during feedback controlled electromigration indicating a layer-by-layer, highly reproducible electromigration process avoiding thermal runaway and melting. These measurements provide insight into the electromigration induced failure mechanism in sub-20 nm size interconnects, indicating that the current density at failure increases as the width decreases to approximately 1 nm.     

Observation of spin-lattice relaxations of dilute Fe3+ in MgO by Mössbauer spectroscopy

We present a method to describe the temperature dependence of emission Mössbauer spectra showing slow spin-lattice relaxations of Fe^3?+? in MgO single crystals, obtained after implantation of ⁵⁷Mn at ISOLDE/CERN. The analysis is based on the Blume-Tjon model for the line-shape of relaxing paramagnetic sextets with the spin relaxation rate, τ ^???1 as a parameter. The temperature dependent spin relaxation rate of Fe^3?+? in MgO is found to increase to ~10⁸ s^???1 at 647 K by assuming a relaxation rate of τ ^???1?< 10⁶ s^???1 at 77 K. The results are in accordance with those obtained by electron paramagnetic resonance spectroscopy demonstrating the possibility of retrieving spin-lattice relaxation rates of dilute Fe^3?+? from emission Mössbauer spectroscopy of Mn/Fe-implanted oxides.     

Theoretical investigation of the structures of unsupported 38-atom CuPt clusters

A genetic algorithm has been used to perform a global sampling of the potential energy surface in the search for the lowest-energy structures of unsupported 38-atom Cu–Pt clusters. Structural details of bimetallic Cu–Pt nanoparticles are analyzed as a function of their chemical composition and the parameters of the Gupta potential, which is used to mimic the interatomic interactions. The symmetrical weighting of all parameters used in this work strongly influences the chemical ordering patterns and, consequently, cluster morphologies. The most stable structures are those corresponding to potentials weighted toward Pt characteristics, leading to Cu–Pt mixing for a weighting factor of 0.7. This reproduces density functional theory (DFT) results for Cu–Pt clusters of this size. For several weighting factor values, the Cu₃₀Pt₈ cluster exhibits slightly higher relative stability. The copper-rich Cu₃₂Pt₆ cluster was reoptimized at the DFT level to validate the reliability of the empirical approach, which predicts a Pt@Cu core-shell segregated cluster. A general increase of interatomic distances is observed in the DFT calculations, which is greater in the Pt core. After cluster relaxation, structural changes are identified through the pair distribution function. For the majority of weighting factors and compositions, the truncated octahedron geometry is energetically preferred at the Gupta potential level of theory.     

Anionic cobalt-platinum-ethynyl (CoPt–C<Subscript>2</Subscript>H) metal-organic subnanoparticles: a DFT modeling study

Anionic CoPt-ethynyl metal-organic clusters have been investigated comprehensively. The lowest energetic of anionic Co _n Pt _m (ethynyl) clusters have been generally found as 3D structure but with low symmetrical point groups. Our results indicate that the most preferred dissociation channel of the studied clusters is Co atom ejection and the favorable dissociation channel is independent of cluster size. The anionic Pt₅C₂H cluster shows the highest chemical stability according to the HOMO-LUMO Gap analysis. The C₂H generally prefers to bind on a bridge site with a few exceptions. The Co_4?5 nanoparticles have a lengthening effect on the C≡C bond of the ethynyl molecule, which may be valuable for C≡C bond activation. In addition, the lowest and the highest vibrational frequencies are reported to guide further experimental studies.     

Fingerprinting proteins coupled with polymers by mass spectrometry: Investigation of polyethylene glycol-conjugated superoxide dismutase

Matrix-assisted laser desorption-ionization (MALDI) mass spectrometry was investigated as a method for the rapid determination of the extent of polymer coupling in polyethylene glycol- (PEG) conjugated superoxide dismutase (SOD). PEG-conjugated SOD, an antioxidant with an extended in vivo circulation lifetime compared to that of superoxide dismutase, is being evaluated as an effective therapeutic agent for the treatment of injuries and arthritis. The mass spectra of a standard batch of PEG-conjugated bovine SOD showed the presence of identifiable and well resolved peaks that correspond to 0–7 PEG molecules attached to bovine SOD. The area of each of the peaks provides a determination of the amount of PEG-conjugated SOD with a given number of bound PEG groups. SOD is a noncovalent dimer of two identical subunits that dissociates in MALDI. The information obtained in the mass spectra thus corresponds to a monomer of SOD. Each SOD monomer contains 10 lysines, which are the sites of PEG-conjugation. Multiple MALDI determinations of two batches of samples indicated good reproducibility for routine determination of the extent of polymer content. The amount of PEG-conjugated SOD that contained a given number of PEG molecules, determined by MALDI, was compared with the value deduced from the amount of PEG-conjugation at each attachment site measured by a peptide mapping method. Agreement between the data obtained in the two techniques (MALDI and peptide mapping) indicates that MALDI may be used to obtain quantitative information on PEG-conjugated SOD to determine the amounts of PEG-conjugated protein each with a different number of PEG groups attached. Measurement of several batches of samples stored at a higher temperature showed a lower extent of PEG-conjugation in PEG-conjugated SOD. This reduction in the PEG content resulted from the PEG-deconjugation of PEG-conjugated SOD at a higher temperature. Thus, MALDI can be used to examine the stability of PEG-conjugated SOD. The high sensitivity, relatively straightforward data interpretation, speed of analyses, and good reproducibility in measurements make this technique a useful analytical tool for fingerprinting PEG-conjugated SOD as well as potentially other polymer-conjugated proteins.