Generalized phase diversity for wave-front sensing

Phase diversity is a phase-retrieval algorithm that uses a pair of intensity images taken symmetrically about the wave front to be determined. If these images are taken about the system input pupil this is equivalent to a curvature-sensing algorithm. Traditionally a defocus aberration kernel is used to produce the phase-diverse data. We present a generalization of this method to allow the use of other functions as the diversity kernel. We discuss the necessary and sufficient conditions that such a function must satisfy for use in a null wave-front sensor. Computer simulations were used to validate these results.     

Vowel enhancement effects in cochlear-implant users

Auditory enhancement of certain frequencies can occur through prior stimulation of surrounding frequency regions. The underlying neural mechanisms are unknown, but may involve stimulus-driven changes in cochlear gain via the medial olivocochlear complex (MOC) efferents. Cochlear implants (CIs) bypass the cochlea and stimulate the auditory nerve directly. If the MOC plays a critical role in enhancement then CI users should not exhibit this effect. Results using vowel stimuli, with and without preceding sounds designed to enhance formants, provided evidence of auditory enhancement in both normal-hearing listeners and CI users, suggesting that vowel enhancement is not mediated solely by cochlear effects.     

Metal-salen-base-pair complexes inside DNA: complexation overrides sequence information

Two isomeric salicylic aldehyde nucleobases have been prepared and incorporated into various DNA duplexes. Reaction with ethylenediamine leads to formation of the well-known salen ligand inside the DNA double helix. Addition of transition-metal ions such as Cu(2+), Mn(2+), Ni(2+), Fe(2+), or VO(2+) results in the formation of metal-salen-base-pair complexes, which were studied by using UV and circular dichroism (CD) spectroscopy. HPLC and ESI mass spectrometric measurements reveal an unusually high stability of the DNA-metal system. These metal-salen complexes act as interstrand cross-links and thereby lead to a strong stabilization of the DNA duplexes, as studied by thermal de- and renaturing experiments. Complex formation is strong enough to override sequence information even when the preorganization of the ligand precursors is unfavorable and the DNA duplex is distorted by the metal complexation. Furthermore, melting-point studies show that the salen complex derived from ligand 2 fits better into the DNA duplex, in accordance with results obtained from the crystal structure of the corresponding copper-salen complex 8.     

Submicron streptavidin patterns for protein assembly

Micron and submicron-scale features of aldehyde functionality were fabricated in polymer films by photolithography to develop a platform for protein immobilization and assembly at a biologically relevant scale. Films containing the pH-reactive polymer poly(3,3'-diethoxypropyl methacrylate) and a photoacid generator (PAG) were patterned from 500 nm to 40 mum by exposure to 365 nm (i-line) light. Upon PAG activation and hydrolysis of acetals, aldehyde groups formed. After the films were incubated with a biotinylated aldehyde reactive probe, the X-ray photoelectron spectroscopy results were consistent with biotin being attached to the surface. The background was subsequently passivated by flood exposure and incubation with an aminooxy-terminated poly(ethylene glycol), resulting in a 98% reduction in nonspecific protein adsorption. Protein patterning and assembly was demonstrated using streptavidin, biotinylated anthrax toxin receptor-1, and the protective antigen moiety of anthrax toxin and confirmed by fluorescence microscopy and atomic force microscopy (AFM). AFM demonstrated that 500 nm protein features were achieved. Because of the abundance of biotinylated proteins, this methodology provides a platform for protein immobilization and assembly for various applications in biotechnology.     

Sample complexity reduction for two-dimensional electrophoresis using solution isoelectric focusing prefractionation

Despite its excellent resolving power, 2-DE is of limited use when analyzing cellular proteomes, especially in differential expression studies. Frequently, fewer than 2000 protein spots are detected on a single 2-D gel (a fraction of the total proteome) regardless of the gel platform, sample, or detection method used. This is due to the vast number of proteins expressed and their equally vast dynamic range. To exploit 2-DE unique ability as both an analytical and a preparative tool, the significant sample prefractionation is necessary. We have used solution isoelectric focusing (sIEF) via the ZOOM IEF Fractionator (Invitrogen) to generate sample fractions from complex bacterial lysates, followed by parallel 2-DE, using narrow-range IPG strips that bracket the sIEF fractions. The net result of this process is a significant enrichment of the bacterial proteome resolved on multiple 2-D gels. After prefractionation, we detected 5525 spots, an approximate 3.5-fold increase over the 1577 spots detected in an unfractionated gel. We concluded that sIEF is an effective means of prefractionation to increase depth of field and improve the analysis of low-abundance proteins.     

Real-time investigation of lung surfactant respreading with surface vibrational spectroscopy

The respreading of a lung surfactant monolayer at the air-water interface is investigated with broad bandwidth sum frequency generation (BBSFG) spectroscopy. The lung surfactant mixture contains chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoylphosphatidylglycerol (POPG), palmitic acid (PA), and KL4 (a 21-residue polypeptide analogue to the surfactant protein SP-B). DPPC-d62 serves as a probe molecule for the spectroscopic investigation. The BBSFG spectra of DPPC-d62 in the lung surfactant mixture are obtained in the C-D stretching region in real-time during film compression and expansion in a Langmuir trough. The BBSFG intensity of the CD3 stretch peak from DPPC-d62 terminal methyl groups is used as a measure of the interfacial density of DPPC-d62 after careful consideration of orientation effects. For the first time, the interfacial loss of DPPC in a complex lung surfactant mixture is quantified. Spectroscopic results reveal that there is an 18% DPPC-d62 interfacial loss during film respreading. However, the surface pressure-area isotherm measurements demonstrate that there is a rather large trough area reduction (37%) during film expansion. The relatively small interfacial loss of DPPC-d62 and the rather large trough area reduction indicate that the respreading of DPPC and non-DPPC components in the lung surfactant is not uniform and a surface refinement process exists during film compression and expansion. This refinement process results in a DPPC-enriched monolayer with a significant depletion of non-DPPC components after film respreading. Implication for replacement surfactant design from this work is discussed.     

Lithol Red: a systematic structural study on salts of a sulfonated azo pigment

The first systematic series of single-crystal diffraction structures of azo lake pigments is presented (Lithol Red with cations=Mg(II), Ca(II), Sr(II), Ba(II), Na(I) and Cd(II)) and includes the only known structures of non-Ca examples of these pigments. It is shown that these commercially and culturally important species show structural behaviour that can be predicted from a database of structures of related sulfonated azo dyes, a database that was specifically constructed for this purpose. Examples of the successful structural predictions from the prior understanding of the model compounds are that 1) the Mg salt is a solvent-separated ion pair, whereas the heavier alkaline-earth elements Ca, Sr and Ba form contact ion pairs, namely, low-dimensional coordination complexes; 2) all of the Lithol Red anions exist as the hydrazone tautomer and have planar geometries; and 3) the commonly observed packing mode of alternating inorganic layers and organic bilayers is as expected for an ortho-sulfonated azo species with a planar anion geometry. However, the literature database of dye structures has no predictive use for organic solvate structures, such as that of the observed Na Lithol Red DMF solvate. Interestingly, the Cd salt is isostructural with the Mg salt and not with the Ca salt. It is also observed that linked eight-membered [MOSO](2) rings are the basic coordination motif for all of the known structures of Ca, Sr and Ba salts of sulfonated azo pigments in which competing carboxylate groups are absent.     

Quantifying Electronic Effects in QM and QM/MM Biomolecular Modeling with the Fukui Function

Multi-scale quantum-mechanical/molecular-mechanical(QM/MM) and large-scale QM simulation provide valuable insight into enzyme mechanism and structure-property relationships. Analysis of the electron density afforded through these methods can enhance our understanding of how the enzyme environment modulates reactivity at the enzyme active site. From this perspective, tools from conceptual density functional theory to interrogate electron densities can provide added insight into enzyme function. We recently introduced the highly parallelizable Fukui shift analysis(FSA) method, which identifies how frontier states of an active site are altered by the presence of an additional QM residue to identify when QM treatment of a residue is essential as a result of quantum-mechanically affecting the behavior of the active site. We now demonstrate and analyze distance and residue dependence of Fukui function shifts in pairs of residues representing different non-covalent interactions. We also show how the interpretation of the Fukui function as a measure of relative nucleophilicity provides insight into enzymes that carry out S_N2 methyl transfer. The FSA method represents a promising approach for the systematic, unbiased determination of quantum mechanical effects in enzymes and for other complex systems that necessitate multi-scale modeling.     

Heterotelechelic Polymers for Capture and Release of Protein-Polymer Conjugates

A heterotelechelic biotin-maleimide polymer containing a cleavable disulfide bond was synthesized by RAFT polymerization and used to reversibly modify surfaces with proteins.