Development of recombinant protein-based influenza vaccine. Expression and affinity purification of H1N1 influenza virus neuraminidase

The influenza virus surface glycoprotein antigen neuraminidase (NA) is a crucial viral enzyme with many potential medical applications; therefore, the development of efficient upstream and downstream processing strategy for the expression and purification of NA is of high importance. In the present work the NA gene from the H1N1 influenza virus strain A/Beijing/262/95 was cloned from viral RNA and expressed in expresSF+ insect cells using the baculovirus expression vector system (BVES). A limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify the recombinant H1N1 neuraminidase. Affinity-ligand design was based on mimicking the interactions of the lock-and-key (LAK) motif (Phe-Gly-Gln), a common structural moiety found in the subunit interface of glutathione S-transferase I (GST I), and plays an important structural role in subunit-subunit recognition. Solid-phase combinatorial chemistry was used to synthesize 13 variants of the lock-and-key lead ligand (Phe-Trz-X, where X was selected alpha-amino acid) using the 1,3,5-triazine moiety (Trz) as the scaffold for assembly. One immobilized ligand, bearing phenylalanine and isoleucine linked on the chlorotriazine ring (Phe-Trz-Ile), displayed high affinity for NA. Absorption equilibrium and molecular modeling studies were carried out to provide a detailed picture of Phe-Trz-Ile interaction with NA. This LAK-mimetic affinity adsorbent was exploited in the development of a facile purification protocol for NA, which led to 335-fold purification in a single-step. The present purification procedure is the most efficient reported so far for recombinant NA.     

Diffusion damping during adiabatic z-rotation pulses for NMR spectroscopy in inhomogeneous magnetic fields

High-resolution nuclear magnetic resonance spectra from samples located in inhomogeneous static and radio frequency magnetic fields can be obtained by applying a train of z-rotation radio frequency pulses to repeatedly refocus the inhomogeneous broadening during signal detection. z-rotation pulses based on an adiabatic double passage are effective over wide bandwidths using a limited amount of radio frequency power at the expense of being time consuming and, consequently, sensitive to motion of the spin bearing molecules. The signal damping resulting from molecular self-diffusion during the pulse was studied experimentally and using Brownian dynamics simulations. The results show that the analytical expression for diffusion damping during a double spin echo is a reasonable approximation for the signal decay during an adiabatic z-rotation pulse. Methods to alleviate the effects of diffusion are discussed.     

Enhancing the acquisition efficiency of fast magnetic resonance imaging via broadband encoding of signal content

Current efficient magnetic resonance imaging (MRI) methods such as parallel-imaging and k-t methods encode MR signals using a set of effective encoding functions other than the Fourier basis. This work revisits the proposition of directly manipulating the set of effective encoding functions at the radiofrequency excitation step in order to increase MRI efficiency. This approach, often termed "broadband encoding," enables the application of algebraic matrix factorization technologies to extract efficiency by representing and encoding MR signal content in a compacted form. Broadband imaging equivalents of fast multiecho, parallel and k-t MRI are developed and analyzed. The potential of these techniques to increase the time efficiency of data acquisition is experimentally verified on a commercial MRI scanner using simple spin-echo imaging. A three-dimensional gradient-echo dynamic imaging application that demonstrates the potential benefits of this approach compared to the present state of the art for certain applications is also presented.     

Enzymatic reductions for the regio- and stereoselective synthesis of hydroxy-keto esters and dihydroxy esters

Ketoreductases were utilized for the stereoselective synthesis of δ-hydroxy-β-keto esters, β-hydroxy-δ-keto esters, and β,δ-dihydroxy esters. Seven out of eight possible stereoisomers were obtained from the enzymatic reduction of the corresponding β,δ-diketo ester in high enantio- and diastereomeric excess.     

Instabilities in quantum-dot spin-VCSELs

A modified spin-flip model is used to investigate the stability of optically-pumped quantum-dot spin-vertical cavity surface-emitting lasers. in terms of pump intensity and polarization. The stability maps exhibit pronounced polarization switching in the stable regions. Periodic oscillations in unstable regions are attributed to competition between spin relaxation and birefringence.     

One‐Pot Synthesis of O‐Allylhydroxylamines through the Organocatalytic Oxidation of Tertiary Allylic Amines Followed by a [2,3]‐Meisenheimer Rearrangement

A cheap, green, and highly efficient one‐pot method for the synthesis of O‐protected allylic alcohols is described. By utilizing 2,2,2‐trifluoroacetophenone as the organocatalyst and H₂O₂ as the oxidant, a variety of allylic amine N‐oxides were synthesized, which upon heating are converted to the final products through a [2,3]‐Meisenheimer rearrangement.     

New mesomorphic organocyclosiloxanes II. Thermal behaviour and mesophase structure of organocyclohexasiloxanes

Investigations of thermotropic phase transitions performed on organocyclosiloxanes [PhSi(O)OSiR]₆, where R is Me₃, Me₂(CH₂Cl) or Me₂(CH≃CH₂), have revealed that all these hexamers are mesomorphic compounds. The hexamers exhibit uncommon polymesomorphic behaviour forming two quite different mesomorphic structures. The molecular arrangement in the low temperature (LT) modification is characterized by two-dimensional (2D) long-range order with hexagonal packing. The X-ray diffraction pattern and peculiarities of molecular packing in the crystal lead us to suggest that the LT-mesophase is columnar, presumably of the Col_hd type. The LT-mesophase is formed by dimeric moieties, which associate with each other in column-like substructures, the ring planes not orthogonal to the stack axis. The high temperature (HT) mesophase is a plastic crystal (3D-order), where molecules take up positions in a face-centred cubic lattice. This is a very uncommon example of thermal behaviour for plastic crystals that provides a unique opportunity to bridge the gap between plastic crystalline and liquid crystalline mesomorphic behaviour. The thermal and structural properties of the mesophases depend upon the type of side groups of the hexamers. The size of the ring also affects the phase behaviour and the mesomorphic structure. This conclusion is consistent with data obtained by us earlier for cyclotetrasiloxanes.     

Controlled modulation of mesoporosities in metal (M = Al, Fe) phosphate solids

Mesoporous solids which possess average pore diameters between 7 and 20 nm, depending on the composition, have been prepared. The solids have the general formula Al₁₀₀P_χM₂₀ where M = Al or Fe, and χ = 0, 4.5, 9, 18, 36, 72 or 144. The initial addition of phosphorus as phosphate transforms the originally crystalline oxide/oxides into amorphous solids. These amorphous materials possess a narrow pore size distribution: 80–90% of the pores lie within 1–2 nm of the average pore diameter. Subsequent incremental amounts of phosphorus transform the material into a crystalline solid whilst the pore size distribution becomes much wider and the maximum moves towards larger pore diameters. Substitution of 20% of the aluminium by iron results, at a low phosphorus content, in pores with smaller pore volumes and smaller surface areas.

The data in the dV_p/dD_p = > D_p) graphs, where V_p is the incremental pore volume and D_p is the average pore diameter, can be approximated using an admixture of Gaussian and Lorentzian curves. For low phosphorus contents the dV_p/dD_p = (D_p) curves have a mainly Gaussian profile but the gradual addition of phosphorus transforms them to Lorentzian-type curves. An attempt to approximate the histograms dV_p =(D_p) with the minimum number of distribution curves made up of the corresponding Gaussian and Lorentzian components indicates that each successive addition of phosphorus creates a dominant new pore component at a larger pore diameter. At the same time, the components at smaller pore diameters are diminished and eventually disappear as more phosphorus is added.