Preparative isolation of (+)-beta-eudesmol fromAmyris balsamifera

Summary Optically pure (+)-beta-eudesmol is a possible starting material for the synthesis of several termite defense compounds. A two step procedure for the isolation of gram quantities of (+)-beta-eudesmol from commercially availableAmyris balsamifera oil (syn. West Indian sandalwood oil), containing 8% beta-eudesmol, was developed. Step one consisted of an efficient vacuum distillation of the total oil. Step two was a medium pressure LC separation with an AgNO₃ impregnated silica gel stationary phase. Several other separation procedures failed due to the presence of many closely related sesquiterpene alcohols (75% of the oil).     

Sequence-specific single-molecule analysis of 8-oxo-7,8-dihydroguanine lesions in DNA based on unzipping kinetics of complementary probes in ion channel recordings

Translocation measurements of intact DNA strands with the ion channel α-hemolysin (α-HL) are limited to single-stranded DNA (ssDNA) experiments as the dimensions of the channel prevent double-stranded DNA (dsDNA) translocation; however, if a short oligodeoxynucleotide is used to interrogate a longer ssDNA strand, it is possible to unzip the duplex region when it is captured in the α-HL vestibule, allowing the longer strand to translocate through the α-HL channel. This unzipping process has a characteristic duration based on the stability of the duplex. Here, ion channel recordings are used to detect the presence and relative location of the oxidized damage site 8-oxo-7,8-dihydroguanine (OG) in a sequence-specific manner. OG engages in base pairing to C or A with unique stabilities relative to native base Watson-Crick pairings, and this phenomenon is used here to engineer probe sequences (10-15mers) that, when base-paired with a 65mer sequence of interest, containing either G or OG at a single site, produce characteristic unzipping times that correspond well with the duplex melting temperature (T(m)). Unzipping times also depend on the direction from which the duplex enters the vestibule if the stabilities of leading base pairs at the ends of the duplex are significantly different. It is shown here that the presence of a single DNA lesion can be distinguished from an undamaged sequence and that the relative location of the damage site can be determined based on the duration of duplex unzipping.     

Nanopore detection of 8-oxo-7,8-dihydro-2'-deoxyguanosine in immobilized single-stranded DNA via adduct formation to the DNA damage site

The ability to detect DNA damage within the context of the surrounding sequence is an important goal in medical diagnosis and therapies, but there are no satisfactory methods available to detect a damaged base while providing sequence information. One of the most common base lesions is 8-oxo-7,8-dihydroguanine, which occurs during oxidation of guanine. In the work presented here, we demonstrate the detection of a single oxidative damage site using ion channel nanopore methods employing α-hemolysin. Hydantoin lesions produced from further oxidation of 8-oxo-7,8-dihydroguanine, as well as spirocyclic adducts produced from covalently attaching a primary amine to the spiroiminodihydantoin lesion, were detected by tethering the damaged DNA to streptavidin via a biotin linkage and capturing the DNA inside an α-hemolysin ion channel. Spirocyclic adducts, in both homo- and heteropolymer background single-stranded DNA sequences, produced current blockage levels differing by almost 10% from those of native base current blockage levels. These preliminary studies show the applicability of ion channel recordings not only for DNA sequencing, which has recently received much attention, but also for detecting DNA damage, which will be an important component to any sequencing efforts.     

Investigations into beam monitors at the AEg ̄ IS experiment

Detailed diagnostic of antiproton beams at low energies is required for essentially all experiments at the Antiproton Decelerator (AD), but will be particularly important for the future Extra Low ENergy Antiproton ring (ELENA) and its keV beam lines to the different experiments. Many monitors have been successfully developed and operated at the AD, but in particular beam profile monitoring remains a challenge. A dedicated beam instrumentation and detector test stand has recently been setup at the AE \(\bar {g}\) IS experiment (Antimatter Experiment: Gravity, Interferometry, Spectroscopy). Located behind the actual experiment, it allows for parasitic use of the antiproton beam at different energies for testing and calibration. With the aim to explore and validate different candidate technologies for future low energy beam lines, as well as the downstream antihydrogen detector in AE \(\bar {g}\) IS, measurements have been carried out using Silicon strip and pixel detectors, a purpose-built secondary emission monitor and emulsions. Here, results from measurements and characterization of the different detector types with regard to their future use at the AD complex are presented.     

Fluorescence microscopy of the pressure-dependent structure of lipid bilayers suspended across conical nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.