Retroviral display and high throughput screening

Retroviruses distinguish themselves from all other mammalian viruses by their abilities to infect and propagate in mammalian cells without causing a cytopathic effect and to stably integrate their genetic information into the genome of the host cell. These unique properties make them an ideal platform for the display and directed evolution of proteins in a mammalian cell environment. This review will describe the essentials about retrovirus biology and then discuss in detail display and screening strategies that have been developed during the past 15 years of retroviral display technology.     

Translational diffusion of macromolecules and nanoparticles modeled as non-overlapping bead arrays in an effective medium

There are three objectives to the present work. First, starting from a boundary element (BE) formulation of low Reynolds number hydrodynamics, model the translational diffusion of macromolecules modeled as an array of non-overlapping beads, and show how this approach is equivalent to previous formulations of "bead hydrodynamics" and under what conditions. Second, show how this approach can be improved upon by accounting for the variation in forces over the surfaces of individual beads and also extending the approach to a gel modeled as an effective medium, EM. Third, develop a "combined obstruction and hydrodynamic effect" model of the translational diffusion of irregularly shaped macromolecules in a gel. In one of the cases studied, the BE approach is shown to be equivalent to previous "bead model" formulations in which intersubunit hydrodynamic interaction is modeled using the Rotne-Prager tensor. A bead model that accounts for the variation in hydrodynamic stress forces over the individual bead surfaces is shown to be in best agreement with exact results for simple bead arrays made up of 2-4 subunits. The translational diffusion of rods, modeled as strings of from 2 to 100 touching beads in dilute gels is examined. Interpolation formulas valid over a range of gel concentrations and rod lengths are derived for the parallel and perpendicular components of the diffusion tensor as well as the orientationally averaged diffusion tensor. The EM model accounts for the long-range hydrodynamic interaction exerted by the gel support matrix on the diffusing particle of interest but does not account for the reduction in diffusion caused by the direct obstruction of the gel, or steric effect. Both effects are accounted for by writing the translational diffusion in a gel as the product of two terms representing long-range hydrodynamic interaction and steric effects. Finally, the diffusion of a 564 base pair DNA in a 2% agarose gel is examined and model results are compared to experiment (Pluen, A.; Netti, P. A.; Jain, R. K.; Berk, D. A. Biophys. J. 1999, 77, 542-552). For reasonable choices of model parameters, fair agreement between theory and experiment is achieved.     

Unfolding ESIPT in Bis-2,5-(2-benzoxazolyl) Hydroquinone and 2,5-Bis(benzo[d]oxazol-2-yl)-4-methoxyphenol: a Comprehensive Computational Approach

The photo-physical behaviour of bis-2,5-(2-benzoxazolyl) hydroquinone and 2,5-bis (benzo[d]oxazol-2-yl)-4-methoxyphenol was studied using the Density Functional Theory (DFT) and Time-Dependent Density Functional Theory (TD-DFT). All the possible rotamers were optimized to obtain global minimum optimized structure. The theoretical absorption and emission values of rotamers estimated by using TD-DFT [TD-B3LYP/6–31G(d)] are in good agreement with experimental absorption and emission wavelengths. Based on the absorption values, the contribution of respective rotamer is determined theoretically.     

Dioxidomolybdenum(VI) complexes of allyl N'-2-hydroxy-3-methoxybenzylidenecarbamohydrazonothioate: synthesis,spectral, and theoretical investigations

The new dibasic NNO ligand H₂L (H₂L = allyl N′-2-hydroxy-3-methoxybenzylidenecarbamohydrazonothioate) was synthesized by condensation of 2-hydroxy-3-methoxybenzaldehyde with the product resulting from the reaction of thiosemicarbazide with allyl bromide. Four dioxidomolybdenum(VI) complexes with the general formula [MoO₂L(S)] (S=MeOH, EtOH, DMSO, and 1-methylimidazole) were synthesized and characterized by elemental analysis, FT-IR, EI-MS and UV-Vis spectroscopy, and by X-ray crystallography. Spectroscopic evidence indicates that the cis-MoO₂ chelates have octahedral geometry in which H₂L coordinates via the phenolate oxygen, azomethine nitrogen and deprotonated thioamide nitrogen. The other sites are occupied by two oxido and an additional ligand (S). Density functional theory calculations of spectral parameters were also carried out for these systems.     

Parents’ Attitudes Toward Mathematics and the Influence on Their Students’ Attitudes toward Mathematics: A Quantitative Study

The purpose of this study was to investigate parents’ attitudes toward mathematics, their students' attitude toward mathematics, and the influence of the parents’ attitude on the students' attitude toward mathematics. Data analyses revealed statistically significant positive correlations between parents’ and students’ attitudes toward mathematics. Additionally, parents’ mathematics attitude significantly predicted students’ attitudes toward mathematics (n=146). By understanding the influence of parents’ attitudes on students’ attitudes toward mathematics, school efforts can be geared toward fostering favorable attitudes toward mathematics among parents.     

Normal‐to‐Abnormal NHC Rearrangement of AlIII,GaIII, and InIII Trialkyl Complexes: Scope,Mechanism, Reactivity Studies,and H2 Activation

The present contribution reports experimental and theoretical mechanistic investigations on a normal‐to‐abnormal (C2‐to‐C4‐bonded) NHC rearrangement processes occurring with bulky group 13 metal NHC adducts, including the scope of such a reactivity for Al compounds. The sterically congested adducts (nItBu)MMe₃ (nItBu=1,3‐di‐tert‐butylimidazol‐2‐ylidene; M=Al, Ga, In; 1 a – c ) readily rearrange to quantitatively afford the corresponding C4‐bonded complexes (aItBu)MMe₃ ( 4 a – c ), a reaction that may be promoted by THF. Thorough experimental data and DFT calculations were performed on the nNHC‐to‐aNHC process converting the Al‐nNHC ( 1 a ) to its aNHC analogue 4 a . A nItBu/aItBu isomerization is proposed to account for the formation of the thermodynamic product 4 a through reaction of transient aItBu with THF–AlMe₃. The reaction of benzophenone with (nItBu)AlMe₃ afforded the zwitterionic species (aItBu)(CPh₂‐O‐AlMe₃) ( 6 ), reflecting the unusual reactivity that such bulky adducts may display. Interestingly, the nItBu/Al(iBu)₃ Lewis pair behaves like a frustrated Lewis pair (FLP) since it readily reacts with H₂ under mild conditions. This may open the way to future reactivity developments involving commonly used trialkylaluminum precursors.     

A time-resolved luminescent competitive assay to detect L-selectin using aptamers as recognition elements

L-selectin is a protein with potential importance for numerous diseases and clinical disorders. In this paper, we present a new aptamer-based luminescent assay developed to detect L-selectin. The sensing system working principle is based on Förster Resonance Energy Transfer (FRET) from a donor terbium complex (TbC) to an acceptor cyanine dye (Cy5). In the present approach, the biotinylated aptamer is combined with Cy5-labelled streptavidin (Cy5-Strep) to yield an aptamer-based acceptor construct (Apta-Cy5-Strep), while L-selectin is conjugated using luminescent TbC. Upon aptamer binding to the TbC-labelled L-selectin (L-selectin-TbC), permanent donor-acceptor proximity is established which allows for radiationless energy transfer to occur. However, when unlabelled L-selectin is added, it competes with the L-selectin-TbC and the FRET signal decreases as the L-selectin concentration increases. FRET from the TbC to Cy5 was observed with time-gated time-resolved luminescence spectroscopy. A significant change in the corrected luminescence signal was observed in the dynamic range of 10–500 ng/mL L-selectin, the concentration range relevant for accelerated cognitive decline of Alzheimer's disease, with a limit of detection (LOD) equal to 10 ng/mL. The aptasensor-based assay is homogeneous and can be realized within one hour. Therefore, this method has the potential to become an alternative to tedious heterogeneous analytical methods, e.g. based on enzyme-linked immunosorbent assay (ELISA).     

Towards Structural‐Functional Mimics of Acetylene Hydratase: Reversible Activation of Acetylene using a Biomimetic Tungsten Complex

The synthesis and characterization of a biomimetic system that can reversibly bind acetylene (ethyne) is reported. The system has been designed to mimic catalytic intermediates of the tungstoenzyme acetylene hydratase. The thiophenyloxazoline ligand S‐Phoz (2‐(4′,4′‐dimethyloxazolin‐2′‐yl)thiophenolate) is used to generate a bioinspired donor environment around the W center, facilitating the stabilization of W–acetylene adducts. The featured complexes [W(C₂H₂)(CO)(S‐Phoz)₂] ( 2 ) and [WO(C₂H₂)(S‐Phoz)₂] ( 3 ) are extremely rare from a synthetic and structural point of view as very little is known about W–C₂H₂ adducts. Upon exposure to visible light, 3 can release C₂H₂ from its coordination sphere to yield the 14‐electron species [WO(S‐Phoz)₂] ( 4 ). Under light‐exclusion 4 re‐activates C₂H₂ making this the first fully characterized system for the reversible activation of acetylene.     

Photochemical Strain-Release-Driven Cyclobutylation of C(sp3)-Centered Radicals

A new photoredox-catalyzed decarboxylative radical addition approach to functionalized cyclobutanes is described. The reaction involves an unprecedented formal Giese-type addition of C(sp³)-centered radicals to highly strained bicyclo[1.1.0]butanes. The mild photoredox conditions, which make use of a readily available and bench stable phenyl sulfonyl bicyclo[1.1.0]butane, proved to be amenable to a diverse range of α-amino and α-oxy carboxylic acids, providing a concise route to 1,3-disubstituted cyclobutanes. Furthermore, kinetic studies and DFT calculations unveiled mechanistic details on bicyclo[1.1.0]butane reactivity relative to the corresponding olefin system.     

Structure, electronic properties and catalytic behaviour of an activity-enhancing CYP102A1 (P450(BM3)) variant

The substrate-free crystal structure of a five-mutation directed evolution variant of CYP102A1 (P450(BM3)) with generic activity-enhancing properties ("KT2") has been determined to 1.9-? resolution. There is a close resemblance to substrate-bound structures of the wild-type enzyme (WT). The disruption of two salt bridges that link the G- and I-helices in WT causes conformational changes that break several hydrogen bonds and reduce the angle of the kink in the I-helix where dioxygen activation is thought to take place. The side-chain of a key active site residue, Phe87, is rotated in one molecule of the asymmetric unit, and the side-chains of Phe158 and Phe261 cascade into the orientations found in fatty-acid-bound forms of the enzyme. The iron is out of the porphyrin plane, towards the proximal cysteine. Unusually, the axial water ligand to the haem iron is not hydrogen-bonded to Ala264. The first electron transfer from the reductase domain to the haem domain of substrate-free KT2 is almost as fast as in palmitate-bound WT even though the reduction potential of the haem domain is only slightly more oxidising than that of substrate-free WT. However, NADPH is turned over slowly in the absence of substrate, so the catalytic cycle is gated by a step subsequent to the first electron transfer-a contrast to WT. Propylbenzene binding slightly raises the first electron transfer rate in WT but not in KT2. It is proposed that the generic rate accelerating properties of KT2 arise from the substrate-free form being in a catalytically ready conformation, such that substrate-induced changes to the structure play a less significant role in promoting the first electron transfer than in WT.