Optimization of unnatural base pair packing for polymerase recognition

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.     

Spectroscopic characterization of copper(II) binding to the immunosuppressive drug mycophenolic acid

Mycophenolic acid (MPA) is a drug that has found widespread use as an immunosuppressive agent which limits rejection of transplanted organs. Optimal use of this drug is hampered by gastrointestinal side effects which can range in severity. One mechanism by which MPA causes gastropathy may involve a direct interaction between the drug and gastric phospholipids. To combat this interaction we have investigated the potential of MPA to coordinate Cu(II), a metal which has been used to inhibit gastropathy associated with use of the NSAID indomethacin. Using a range of spectroscopic techniques we show that Cu(II) is coordinated to two MPA molecules via carboxylates and, at low pH, water ligands. The copper complex formed is stable in solution as assessed by mass spectrometry and 1H NMR diffusion experiments. Competition studies with glycine and albumin indicate that the copper-MPA complex will release Cu(II) to amino acids and proteins thereby allowing free MPA to be transported to its site of action. Transfer to serum albumin proceeds via a Cu(MPA)(albumin) ternary complex. These results raise the possibility that copper complexes of MPA may be useful in a therapeutic situation.     

Optimisation and characterisation of biosensors based on polyaniline

With lower limits of detection and increased stability constantly being demanded of biosensor devices, characterisation of the constituent layers that make up the sensor has become unavoidable, since this is inextricably linked with its performance. This work describe the optimisation and characterisation of two aspects of sensor performance: a conductive polymer layer (polyaniline) and the immobilised protein layer. The influence of the thickness of polyaniline films deposited electrochemically onto screen-printed electrode surfaces is described in this work in terms of its influence on a variety of amperometric sensor performance characteristics: time to reach steady state, charging current, catalytic current, background current and signal/background ratios. The influence of polymer film thickness on the conductivity and morphology of finished films is also presented.

An electrostatic method of protein immobilisation is used in this work and scanning electron microscopy in conjunction with gold-labelled antibodies and back-scattered electron detection has enabled the direct visualisation of individual groups of proteins on the sensor surface. Such information can provide an insight into the performance of sensors under influence of increasing protein concentrations.     

Reactive quenching of OH(A 2Sigma+) by D2 studied using crossed molecular beams

Reactive quenching of OH(A 2Sigma+,v=0) by D2 forming HOD+D was studied in crossed molecular beams. The D atom products are primarily forward scattered relative to the incident D2. The dominant mechanism involves a direct reaction from relatively large impact parameters with approximately 88% of the available energy appearing in HOD internal excitation.     

Weak lensing of the CMB

The cosmic microwave background (CMB) represents a unique source for the study of gravitational lensing. It is extended across the entire sky, partially polarized, located at the extreme distance of z = 1,100, and is thought to have the simple, underlying statistics of a Gaussian random field. Here we review the weak lensing of the CMB, highlighting the aspects which differentiate it from the weak lensing of other sources, such as galaxies. We discuss the statistics of the lensing deflection field which remaps the CMB, and the corresponding effect on the power spectra. We then focus on methods for reconstructing the lensing deflections, describing efficient quadratic maximum-likelihood estimators and delensing. We end by reviewing recent detections and observational prospects.     

Second-generation aerosol shock tube: an improved design

An improved, second-generation aerosol shock tube (AST II) has been developed for the study of the chemical kinetics of low-vapor-pressure fuels. These improvements enable a wider range of fuel concentrations and enhanced spatial uniformity relative to our initial aerosol shock tube (AST I). In addition, the design of AST II limits the aerosol loading zone in the shock tube to a fixed region (1.2 m in length adjacent to the shock tube endwall). AST II achieves these improvements using a separate holding tank to prepare the aerosol mixture and a slightly under-pressure dump tank to carefully pull the aerosol mixture into the tube in a plug-flow. This filling method is capable of producing room temperature test gas mixtures of n-dodecane with equivalence ratios of up to 3.0 in 21 % O₂, three times the loading achievable in the earlier AST I that used a flow-through strategy. Improvements in aerosol uniformity were quantified by measuring the liquid volume concentration at multiple locations in the shock tube. The measurements made over a length of 1.1 m of shock tube indicate that the AST II method of filling produces non-uniformities in liquid volume concentration of less than 2 %, whereas in the AST I method of filling the non-uniformities reached 16 %. The improved uniformity can also be seen in measurement of gas-phase fuel concentration behind the incident shock wave after the liquid droplets have evaporated. Significant reduction in the scatter of ignition delay times measured using AST II have also been achieved, confirming the importance of uniform loading of the aerosol in making high-quality combustion measurements.     

Use of a kinetic energy orifice as a probe of metastable dissociation in Fourier transform ion cyclotron resonance mass spectrometry

Although Fourier transform ion cyclotron resonance mass spectrometry is a powerful tool in the qualitative observation of gas phase reactions, ion detection is on the millisecond time scale, orders of magnitude longer than typically found when using a sector instrument. Observations of short-lived species such as chemically activated adduct ions can be accomplished using selective ion excitation as a probe of intermediate lifetime. Whereas ion elimination has been shown to be effective in monitoring ion lifetimes on the microsecond time scale, problems associated with detecting ions produced with high kinetic energies limits the technique. Use of a kinetic energy orifice as an ion skimmer effectively eliminates ions near the center of the ion cell at relatively low kinetic energies. By modifying a single section cell to include a kinetic energy orifice, the lifetimes of chemically activated adduct ions have been investigated.     

Dynamic fragmentation of powders in spherical geometry

Experimental evidence from a wide range of sources shows that the expanding cloud of explosively disseminated material comprises of “particles” or fragments which have different dimensions from those associated with the original material. Photographic evidence shows jets or fingers behind these expanding fragments. Powders and liquids have often been used to surround explosives to act as blast mitigants; this is the main driver for our research. Other examples of areas where these features are observed include fuel air explosives and enhanced blast explosives as well as quasi-static pressure mitigation systems. In this paper, we consider the processes occurring when an explosive interacts with a surrounding layer of powder in spherical geometry. Results from explosive experiments designed to investigate the effects of powder grain size and powder fill-to-burster charge mass ratio ( \(F\) / \(B\) ) are presented and compared with results from numerical modelling to explore what determines the primary fragment size distribution resulting from explosive dissemination of a layer of material and when this process begins. The evidence clearly shows that the process starts during the first wave transit period of the powder material and, despite the surrounding material initially being a loose powder, shows the characteristics of a brittle fracture mechanism. Later time video evidence shows the same number of jets or fingers as are identified by X-rays of the early, primary fragmentation process. The number of fragments is only a very weak function of the initial grain size of the powder.     

CC or co bond cleavage in a phenolic lignin model compound: selectivity depends on vanadium catalyst

The aerobic oxidation of a phenolic lignin model compound with a vanadium catalyst results in the oxidative cleavage of the C?C bond between the aryl ring and the adjacent hydroxy-substituted carbon atom. Labeling experiments indicate key mechanistic differences to a previously reported related C?O bond cleavage reaction. The selectivity in C?C versus C?O bond cleavage depends on the choice of the vanadium catalyst.     

Wavelength-modulation-spectroscopy for real-time, in situ NO detection in combustion gases with a 5.2 μm quantum-cascade laser

A mid-infrared absorption strategy with calibration-free wavelength-modulation-spectroscopy (WMS) has been developed and demonstrated for real-time, in situ detection of nitric oxide in particulate-laden combustion-exhaust gases up to temperatures of 700 K. An external-cavity quantum-cascade laser (ECQCL) near 5.2 μm accessed the fundamental absorption band of NO, and a wavelength-scanned, 1f-normalized WMS with second-harmonic detection (WMS-2f/1f) strategy was developed. Due to the external-cavity laser architecture, large nonlinear intensity modulation (IM) was observed when the wavelength was modulated by injection-current modulation, and the IM indices were also found to be strongly wavelength-dependent as the center wavelength was scanned with piezoelectric tuning of the cavity. A quantitative model of the 1f-normalized WMS-2f signal was developed and validated under laboratory conditions. A sensor was subsequently designed, built and demonstrated for real-time, in situ measurements of NO across a 3 m path in the particulate-laden exhaust of a pulverized-coal-fired power plant boiler. The 1f-normalized WMS-2f method proved to have better noise immunity for non-absorption transmission, than wavelength-scanned direct absorption. A 0.3 ppm-m detection limit was estimated using the R15.5 transition near 1927 cm⁻¹ with 1 s averaging. Mid-infrared QCL-based NO absorption with 1f-normalized WMS-2f detection shows excellent promise for practical sensing in the combustion exhaust.