CHIP-mediated hyperubiquitylation of tau promotes its self-assembly into the insoluble tau filaments

The tau protein is a highly soluble and natively unfolded protein. Under pathological conditions, tau undergoes multiple post-translational modifications (PTMs) and conformational changes to form insoluble filaments, which are the proteinaceous signatures of tauopathies. To dissect the crosstalk among tau PTMs during the aggregation process, we phosphorylated and ubiquitylated recombinant tau in vitro using GSK3β and CHIP, respectively. The resulting phospho–ub-tau contained conventional polyubiquitin chains with lysine 48 linkages, sufficient for proteasomal degradation, whereas unphosphorylated ub-tau species retained only one–three ubiquitin moieties. Mass-spectrometric analysis of in vitro reconstituted phospho–ub-tau revealed seven additional ubiquitylation sites, some of which are known to stabilize tau protofilament stacking in the human brain with tauopathy. When the ubiquitylation reaction was prolonged, phospho–ub-tau transformed into insoluble hyperubiquitylated tau species featuring fibrillar morphology and in vitro seeding activity. We developed a small-molecule inhibitor of CHIP through biophysical screening; this effectively suppressed tau ubiquitylation in vitro and delayed its aggregation in cultured cells including primary cultured neurons. Our biochemical findings point to a “multiple-hit model,” where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process, thus indicating that targeting tau ubiquitylation may be an effective strategy to alleviate the course of tauopathies.

Multiple-hit model for tau aggregation, where sequential events of tau phosphorylation and hyperubiquitylation function as a key driver of the fibrillization process.     

A supramolecular microfluidic optical chemosensor

A supramolecular microfluidic optical chemosensor (muFOC) has been fabricated. A serpentine channel has been patterned with a sol-gel film that incorporates a cyclodextrin supramolecule modified with a Tb(3+) macrocycle. Bright emission from the Tb(3+) ion is observed upon exposure of the (mu)FOC to biphenyl in aqueous solution. The signal transduction mechanism was elucidated by undertaking steady-state and time-resolved spectroscopic measurements directly on the optical chemosensor patterned within the microfluidic network. The presence of biphenyl in the cyclodextrin receptor site triggers Tb(3+) emission by an absorption-energy transfer-emission process. These results demonstrate that the intricate signal transduction mechanisms of supramolecular optical chemosensors are successfully preserved in microfluidic environments.     

Dielectrophoretic force on a sphere near a planar boundary

The small gap distance separating a spherical colloidal particle in electrophoretic motion from a planar nonconducting surface is a required parameter for calculating its electrophoretic mobility. In the presence of an externally applied electric field, this gap distance is determined by balancing the van der Waals, electrical double layer interaction, and gravitational forces with a dielectrophoretic (DEP) force. Here, the DEP force was determined analytically by integration of the Maxwell stress over the surface of the particle. The account of this force showed that its previous omission from the analysis always resulted in underpredicted gap distances. Furthermore, the DEP force dominated under conditions of low particle density or high electric field strength and led to much higher gap distances on the order of a few microns. In one particular case, a combination of low particle density and small particle size produced two possible equilibrium gap distances for the particle. However, the particle was unstable in the second equilibrium position when subjected to small perturbations. In general, larger particles had smaller gap sizes. The effects of four other parameters on gap distance were studied, and gap distances were found to increase with lower particle density, higher electric field strength, higher particle and wall zeta potentials, and lower Hamaker constants. Retardation effects on van der Waals attraction were considered.     

Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry

The structural determination of sn-1 and sn-2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB-MS/MS). The collision-induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn-1 and sn-2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H](+) ions was shown to be greatly different. Moreover, the CID-MS/MS spectra of sodium-adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na - 103](+), [M + Na - 85](+), and [M + Na - 59](+), by which their regio-specificity can be differentiated.     

Microwave synthesis,characterization and catalytic properties of titanium-incorporated ZSM-5 zeolite

Titanium-incorporated ZSM-5 zeolites (Si/Al = 50–200 and Si/Ti = 70) were successfully synthesized in a one-step sol-gel process under microwave irradiation. The characteristics of Ti-ZSM-5 zeolites were investigated using X-ray power diffraction, UV/Vis-DRS, FT-IR spectroscopy and solid-state ²⁷Al-NMR to monitor the physico-chemical properties. Simultaneously, the acidic properties were characterized by the NH₃-TPD profile. The characterization results revealed that the Ti⁴⁺ and Al³⁺ ions were well incorporated into the framework of Ti-ZSM-5 zeolite. The prepared zeolite was moderately active but selective in the dehydration of methanol to dimethyl ether.     

Thermally produced biodegradable scaffolds for cartilage tissue engineering

A novel process was developed to fabricate biodegradable polymer scaffolds for tissue engineering applications, without using organic solvents. Solvent residues in scaffolds fabricated by processes involving organic solvents may damage cells transplanted onto the scaffolds or tissue near the transplantation site. Poly(L-lactic acid) (PLLA) powder and NaCl particles in a mold were compressed and subsequently heated at 180 degrees C (near the PLLA melting temperature) for 3 min. The heat treatment caused the polymer particles to fuse and form a continuous matrix containing entrapped NaCl particles. After dissolving the NaCl salts, which served as a porogen, porous biodegradable PLLA scaffolds were formed. The scaffold porosity and pore size were controlled by adjusting the NaCl/PLLA weight ratio and the NaCl particle size. The characteristics of the scaffolds were compared to those of scaffolds fabricated using a conventional solvent casting/particulate leaching (SC/PL) process, in terms of pore structure, pore-size distribution, and mechanical properties. A scanning electron microscopic examination showed highly interconnected and open pore structures in the scaffolds fabricated using the thermal process, whereas the SC/PL process yielded scaffolds with less interconnected and closed pore structures. Mercury intrusion porosimetry revealed that the thermally produced scaffolds had a much more uniform distribution of pore sizes than the SC/PL process. The utility of the thermally produced scaffolds was demonstrated by engineering cartilaginous tissues in vivo. In summary, the thermal process developed in this study yields tissue-engineering scaffolds with more favorable characteristics, with respect to, freedom from organic solvents, pore structure, and size distribution than the SC/PL process. Moreover, the thermal process could also be used to fabricate scaffolds from polymers that are insoluble in organic solvents, such as poly(glycolic acid). Cartilage tissue regenerated from thermally produced PLLA scaffold.     

Fabrication of hollow palladium spheres and their successful application to the recyclable heterogeneous catalyst for suzuki coupling reactions

Novel palladium hollow spheres were synthesized using silica spheres as a template, and they were successfully applied as recyclable heterogeneous catalysts for Suzuki cross coupling reactions.     

The potential of serially coupled alkyl-bonded silica monolithic columns for high resolution separations of pharmaceutical compounds in biological fluids

Summary In this paper we investigate the potential of alkyl-bonded silica monolithic columns for the isolation and identification of drug-related components in biological fluids. Up to 6 columns have been connected in series to produce a chromatographic system with up to 40,000 plates. This high-resolution chromatography system has been coupled to both MS and NMR to enable efficient detection and characterisation of drug-related components in biological fluids. The use of six coupled columns has been shown to give enhanced resolution over a high quality silica particulate column packed with 3 μm material which exhibits the same back pressure. The effect of volume and mass load on the performance of monolithic columns for semi-preparative chromatography of biological fluids has also been investigated. In these studies it was possible to inject up to 100 mL of neat urine with no loss of chromatographic performance. Furthermore, upon re-testing, the columns showed similar chromatographic performance. Again several columns were serially connected, producing enhanced resolution in the semi-preparative mode.     

Highly sensitive and simple fluorescence staining of proteins in sodium dodecyl sulfate-polyacrylamide-based gels by using hydrophobic tail-mediated enhancement of fluorescein luminescence

Fluorescein has an extremely low luminescence intensity in acidic aqueous media. However, when it was bound to proteins, subsequent increase of luminescence intensity took place. Furthermore, when a hydrophobic tail, such as aliphatic hydrocarbons, was introduced to fluorescein, more dramatic increase of luminescence intensity was observed upon binding to proteins. In the present study, by utilizing this luminescence enhancement, three hydrophobic fluorescein dyes (5-dodecanoyl amino fluorescein, 5-hexadecanoyl amino fluorescein, and 5-octadecanoyl amino fluorescein) were examined as noncovalent fluorescent stains of protein bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Effective incorporation of the dyes to proteins in gels was accomplished either simply by adding dyes at the protein fixation step, or by treating gels with a staining solution after the fixation. The sensitivity of this staining method using the fluorescein derivatives was approximately 1 ng/band for most proteins. For some cases, protein bands containing as low as 0.1 ng were successfully visualized. In addition, the detection sensitivity showed much less protein-to-protein variation than silver staining. This new staining method was also successfully applied to two-dimensional electrophoresis of rat brain proteins. Its overall sensitivity was comparable to that of silver staining.     

Catalytic carbonylation for the synthesis of chemical intermediates

Chemistry related to three catalytic carbonylation reactions is discussed. Synthesis of diphenylurea from nitrobenzene, aniline, and CO gives isolated yields above 98% at 100–120 °C and 15–60 bar of CO in the presence of a palladium (II) complex, PPh₃ and NEt₄Cl. Experimental evidence was provided to prove a new reaction stoichiometry and involvement of a carbamoyl intermediate. In carbonylation of HCHO over ion exchange resin catalysts, reaction temperature, time, pressure, and solvent were important variables to obtain high yields of methyl glycolate. Carbonylation of isobutylphenylethanol at 120°C and 40 bar of CO in the presence of PdCl₂−PPh₃−HCl gives 98% yield of α-(4-isobutylphenyl) propionic acid (ibuprofen). Each catalyst component had a definite role that is indispensable for an efficient overall reaction.