Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging

Metals have a number of important roles within the brain. We used laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to map the three-dimensional concentrations and distributions of transition metals, in particular iron (Fe), copper (Cu) and zinc (Zn) within the murine brain. LA-ICP-MS is one of the leading analytical tools for measuring metals in tissue samples. Here, we present a complete data reduction protocol for measuring metals in biological samples, including the application of a pyramidal voxel registration technique to reproducibly align tissue sections. We used gold (Au) nanoparticle and ytterbium (Yb)-tagged tyrosine hydroxylase antibodies to assess the co-localisation of Fe and dopamine throughout the entire mouse brain. We also examined the natural clustering of metal concentrations within the murine brain to elucidate areas of similar composition. This clustering technique uses a mathematical approach to identify multiple ‘elemental clusters’, avoiding user bias and showing that metal composition follows a hierarchical organisation of neuroanatomical structures. This work provides new insight into the distinct compartmentalisation of metals in the brain, and presents new avenues of exploration with regard to region-specific, metal-associated neurodegeneration observed in several chronic neurodegenerative diseases.     

Synthesis and preliminary cytotoxicity studies of achiral pyrrolyl-substituted titanocenes

From the reaction of various 6-pyrrolylfulvenes (3a–3d) with Super Hydride (LiBEt₃H), lithiated cyclopentadienide intermediates (4a–4d) were synthesised. These intermediates were then transmetallated with titanium tetrachloride TiCl₄ to yield the pyrrolyl-substituted titanocenes bis-[((1-(4-methoxybenzyl)-pyrrole)2-)cyclopentadienyl]titanium(IV) dichloride (5a), bis-[((1-(4-methoxyphenyl)-pyrrole)2-)cyclopentadienyl]titanium(IV) dichloride (5b), bis-[((2,4-bis(4-methoxyphenyl)-1-methyl-pyrrole)2-)cyclopentadienyl]titanium(IV) dichloride (5c), bis-[((2-(4-methoxyphenyl)-1-methyl-pyrrole)2-)cyclopentadienyl]titanium(IV) dichloride (5d). Titanocene 5b crystallised and was characterised by X-ray crystallography. The four titanocenes 5a–5d were tested for their cytotoxicity through MTT-based in vitro tests on CAKI-1 cell lines in order to determine their IC₅₀ values. Titanocenes 5a–5d were found to have IC₅₀ values of 440 (±35), 68 (±14), 105 (±30), and 36 (±7) μM.     

Optimization of two methods for the analysis of hydrogen peroxide: high performance liquid chromatography with fluorescence detection and high performance liquid chromatography with electrochemical detection in direct current mode

Two complementary methods were optimized for the separation and detection of trace levels of hydrogen peroxide. The first method utilized reversed-phase high-performance liquid chromatography with fluorescence detection (HPLC-FD). With this approach, hydrogen peroxide was detected based upon its participation in the hemin-catalyzed oxidation of p-hydroxyphenylacetic acid to yield the fluorescent dimer. The second method utilized high performance liquid chromatography with electrochemical detection (HPLC-ED). With this approach, hydrogen peroxide was detected based upon its oxidation at a gold working electrode at an applied potential of 400 mV vs. hydrogen reference electrode (Pd/H(2)). Both methods were linear across the range of 15-300 μM, and the electrochemical method was linear across a wider range of 7.4-15,000 μM. The limit of detection for hydrogen peroxide was 6 μM by HPLC/FD, and 0.6 μM by HPLC/ED. A series of organic peroxides and inorganic ions were evaluated for their potential to interfere with the detection of hydrogen peroxide. Studies investigating the recovery of hydrogen peroxide with three different extraction protocols were also performed. Post-blast debris from the detonation of a mixture of concentrated hydrogen peroxide with nitromethane was analyzed on both systems. Hydrogen peroxide residues were successfully detected on this post-blast debris.     

Diblock copolymer surfactant transport across the interface between two homopolymers

Dynamics of adsorption and desorption of a diblock copolymer to an interface between two homopolymers was measured using dynamic secondary-ion mass spectrometry (SIMS). Thin films were constructed consisting of a layer of saturated polybutadiene with 90% 1,2-addition (sPB90), followed by a layer of saturated polybutadiene with 63% 1,2-addition (sPB63), and finally by another layer of the sPB90 homopolymer. A sPB90-sPB63 diblock copolymer was initially included only in the top sPB90 layer of the film at a volume fraction of 0.05. The thin films were annealed at ambient temperature for times ranging between 0.2 and 108 h, and the concentration profiles of the diblock copolymer through the films were measured using SIMS. The dynamics of adsorption and desorption of the diblock copolymer at the two sPB90-sPB63 interfaces was gauged by comparing the different transient concentration profiles. The sorption process was modeled as diffusion in an external field, generated from self-consistent field theory (SCFT). All parameters for the model were determined independently. Although the model neglects the dynamics of conformational change, experimental results matched theory very well.     

Exploring the interaction of ruthenium(II) polypyridyl complexes with DNA using single-molecule techniques

Here we explore DNA binding by a family of ruthenium(II) polypyridyl complexes using an atomic force microscope (AFM) and optical tweezers. We demonstrate using AFM that Ru(bpy)2dppz2+ intercalates into DNA (K(b) = 1.5 x 10(5) M(-1)), as does its close relative Ru(bpy)2dppx2+ (K(b) = 1.5 x 10(5) M(-1)). However, intercalation by Ru(phen)3(2+) and other Ru(II) complexes with K(b) values lower than that of Ru(bpy)2dppz2+ is difficult to determine using AFM because of competing aggregation and surface-binding phenomena. At the high Ru(II) concentrations required to evaluate intercalation, most of the DNA strands acquire a twisted, curled conformation that is impossible to measure accurately. The condensation of DNA on mica in the presence of polycations is well known, but it clearly precludes the accurate assessment by AFM of DNA intercalation by most Ru(II) complexes, though not by ethidium bromide and other monovalent intercalators. When stretching individual DNA molecules using optical tweezers, the same limitation on high metal concentration does not exist. Using optical tweezers, we show that Ru(phen)2dppz2+ intercalates avidly (K(b) = 3.2 x 10(6) M(-1)) whereas Ru(bpy)3(2+) does not intercalate, even at micromolar ruthenium concentrations. Ru(phen)3(2+) is shown to intercalate weakly (i.e., at micromolar concentrations (K(b) = 8.8 x 10(3) M(-1))). The distinct differences in DNA stretching behavior between Ru(phen)3(2+) and Ru(bpy)3(2+) clearly illustrate that intercalation can be distinguished from groove binding by pulling the DNA with optical tweezers. Our results demonstrate both the benefits and challenges of two single-molecule methods of exploring DNA binding and help to elucidate the mode of binding of Ru(phen)3(2+).     

Synthesis of structurally diverse bis-peptide oligomers

We have developed second-generation monomers 1 and 2 and improved conditions for rapidly and simultaneously closing multiple diketopiperazines on solid support. These new conditions involve either the microwave heating of a suspension of solid-supported amino-tetrafluoropropyl esters in acetic acid/triethylamine catalyst solution or continuous flow of catalyst solution through the resin, heated in a flow cell apparatus. We demonstrate that the new monomers 1 and 2 can be combined with the new conditions easily to synthesize previously inaccessible hetero and homo spiro ladder oligomers 3 and 4 and others.     

Colchicine glycorandomization influences cytotoxicity and mechanism of action

The reaction of 70 unprotected, diversely functionalized free reducing sugars with methoxyamine-appended colchicine led to the production of a 58-member glycorandomized library. High-throughput cytotoxicity assays revealed glycosylation to modulate specificity and potency. Library members were also identified which, unlike the parent natural product (a destabilizer), stabilized in vitro tubulin polymerization in a manner similar to taxol. This study highlights a simple extension of neoglycorandomization toward amine-bearing scaffolds and the potential benefit of glycosylating nonglycosylated natural products.     

Discovery of fluorescent cyanopyridine and deazalumazine dyes using small molecule macroarrays

[structure: see text] Small molecule macroarrays of cyanopyridines and deazalumazines were generated in high purities via spatially addressed synthesis on planar cellulose supports. Examination of the spectral properties of the heterocycles both on and off of the planar support revealed a set of promising new fluorescent dyes that exhibit high quantum yields, low pH dependence, and high sensitivity to solvent polarity.     

A multichannel gel electrophoresis and continuous fraction collection apparatus for high‐throughput protein separation and characterization

To facilitate a direct interface between protein separation by PAGE and protein identification by mass spectrometry, we developed a multichannel system that continuously collects fractions as protein bands migrate off the bottom of gel electrophoresis columns. The device was constructed using several short linear gel columns, each of a different percent acrylamide, to achieve a separation power similar to that of a long gradient gel. A “Counter Free‐Flow” elution technique then allows continuous and simultaneous fraction collection from multiple channels at low cost. We demonstrate that rapid, high‐resolution separation of a complex protein mixture can be achieved on this system using SDS‐PAGE. In a 2.5 h electrophoresis run, for example, each sample was separated and eluted into 48–96 fractions over a mass range of ～10–150 kDa; sample recovery rates were 50% or higher; each channel was loaded with up to 0.3 mg of protein in 0.4 mL; and a purified band was eluted in two to three fractions (200 μL/fraction). Similar results were obtained when running native gel electrophoresis, but protein aggregation limited the loading capacity to about 50 μg per channel and reduced resolution.