Increased levels of choline metabolites are an early marker of docetaxel treatment response in BRCA1-mutated mouse mammary tumors: an assessment by ex vivo proton magnetic resonance spectroscopy.

BACKGROUND Docetaxel is one of the most frequently used drugs to treat breast cancer. However, resistance or incomplete response to docetaxel is a major challenge. The aim of this study was to utilize MR metabolomics to identify potential biomarkers of docetaxel resistance in a mouse model for BRCA1-mutated breast cancer. METHODOLOGY High resolution magic angle spinning (HRMAS) (1)H MR spectroscopy was performed on tissue samples obtained from docetaxel-sensitive or -resistant BRCA1-mutated mammary tumors in mice. Measurements were performed on samples obtained before treatment and at 1-2, 3-5 and 6-7 days after a 25 mg/kg dose of docetaxel. The MR spectra were analyzed by multivariate analysis, followed by analysis of the signals of individual compounds by peak fitting and integration with normalization to the integral of the creatine signal and of all signals between 2.9 and 3.6 ppm. RESULTS The HRMAS spectra revealed significant metabolic differences between sensitive and resistant tissue samples. In particular choline metabolites were higher in resistant tumors by more than 50% with respect to creatine and by more than 30% with respect to all signals between 2.9 and 3.6 ppm. Shortly after treatment (1-2 days) the normalized choline metabolite levels were significantly increased by more than 30% in the sensitive group coinciding with the time of highest apoptotic activity induced by docetaxel. Thereafter, choline metabolites in these tumors returned towards pre-treatment levels. No change in choline compounds was observed in the resistant tumors over the whole time of investigation. CONCLUSIONS Relative tissue concentrations of choline compounds are higher in docetaxel resistant than in sensitive BRCA1-mutated mouse mammary tumors, but in the first days after docetaxel treatment only in the sensitive tumors an increase of these compounds is observed. Thus both pre- and post-treatment tissue levels of choline compounds have potential to predict response to docetaxel treatment.

A dual resonance, image -guided, volume localization technique for magnetic resonance spectroscopy

An interleaved, dual resonance, volume localization technique for $\sp1$H/$\sp{31}$P magnetic resonance spectroscopy has been designed, implemented on a 2 T imager/spectrometer, and verified with phantom studies.^ Localization techniques, including several single voxel techniques and spectroscopic imaging, were implemented, and studies were performed to compare the efficiency of each sequence of $\sp1$H/$\sp{31}$P spectral acquisitions. The sequence chosen was a hybrid of the stimulated echo single voxel technique and the spectroscopic imaging technique.^ Water suppression during the $\sp1$H spectral acquisitions was accomplished by the use of three narrow bandwidth RF saturation pulses in combination with three spoiler gradients. The spoiler gradient amplitudes were selected on the basis of a numerical solution of the Bloch equations. A post-acquisition water suppression algorithm was used to minimize any residual water signal.^ For interleaved $\sp1$H/$\sp{31}$P acquisitions, a dual resonance RF coil was constructed and interfaced to the existing RF detection system via a custom-designed dual resonance transcoupler and switching system. Programmable attenuators were incorporated to allow for changes in receiver and transmitter attenuation "on the fly".^ To provide the rapidly switched gradient fields required for the $\sp1$H/$\sp{31}$P acquisitions, an actively screened gradient coil system was designed and implemented. With this system, gradient field rise times on the order of 100 $\mu$s were obtained. These rapid switching times were necessary for minimizing intrasequence delays and for improving localization quality and water suppression efficiency.^ The interleaved $\sp1$H/$\sp{31}$P volume localization technique was tested using a two-compartment phantom. Analysis of the data showed that the spectral contamination was less than three percent. One-to-one spatial correspondence of the $\sp1$H and $\sp{31}$P spectra was verified and allowed for direct correlation of the spectral data with a standard magnetic resonance image. ^

TROSY in triple-resonance experiments: New perspectives for sequential NMR assignment of large proteins

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below ∼25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.

Object-related activity revealed by functional magnetic resonance imaging in human occipital cortex.

The stages of integration leading from local feature analysis to object recognition were explored in human visual cortex by using the technique of functional magnetic resonance imaging. Here we report evidence for object-related activation. Such activation was located at the lateral-posterior aspect of the occipital lobe, just abutting the posterior aspect of the motion-sensitive area MT/V5, in a region termed the lateral occipital complex (LO). LO showed preferential activation to images of objects, compared to a wide range of texture patterns. This activation was not caused by a global difference in the Fourier spatial frequency content of objects versus texture images, since object images produced enhanced LO activation compared to textures matched in power spectra but randomized in phase. The preferential activation to objects also could not be explained by different patterns of eye movements: similar levels of activation were observed when subjects fixated on the objects and when they scanned the objects with their eyes. Additional manipulations such as spatial frequency filtering and a 4-fold change in visual size did not affect LO activation. These results suggest that the enhanced responses to objects were not a manifestation of low-level visual processing. A striking demonstration that activity in LO is uniquely correlated to object detectability was produced by the "Lincoln" illusion, in which blurring of objects digitized into large blocks paradoxically increases their recognizability. Such blurring led to significant enhancement of LO activation. Despite the preferential activation to objects, LO did not seem to be involved in the final, "semantic," stages of the recognition process. Thus, objects varying widely in their recognizability (e.g., famous faces, common objects, and unfamiliar three-dimensional abstract sculptures) activated it to a similar degree. These results are thus evidence for an intermediate link in the chain of processing stages leading to object recognition in human visual cortex.

Rapid Scan Electron Paramagnetic Resonance (EPR) and Digital EPR Development

Rapid scan electron paramagnetic resonance (EPR) was developed in the Eaton laboratory at the University of Denver. Applications of rapid scan to wider spectra, such as for immobilized nitroxides, spin-labeled proteins, irradiated tooth and fingernail samples were demonstrated in this dissertation. The scan width has been increased from 55 G to 160 G. The signal to noise (S/N) improvement for slowly tumbling spin-labeled protein samples that is provided by rapid scan EPR will be highly advantageous for biophysical studies. With substantial improvement in S/N by rapid scan, the dose estimation for irradiated tooth enamels became more reliable than the traditional continuous wave (CW) EPR. An alternate approach of rapid scan, called field-stepped direct detection EPR, was developed to reconstruct wider EPR signals. A Mn2+ containing crystal was measured by field-stepped direct detection EPR, which had a spectrum more than 6000 G wide. Since the field-stepped direct detection extends the advantages of rapid scan to much wider scan ranges, this methodology has a great potential to replace the traditional CW EPR. With recent advances in digital electronics, a digital rapid scan spectrometer was built based on an arbitrary waveform generator (AWG), which can excite spins and detect EPR signals with a fully digital system. A near-baseband detection method was used to acquire the in-phase and quadrature signals in one physical channel. The signal was analyzed digitally to generate ideally orthogonal quadrature signals. A multiharmonic algorithm was developed that employed harmonics of the modulation frequencies acquired in the spectrometer transient mode. It was applied for signals with complicated lineshapes, and can simplify the selection of modulation amplitude. A digital saturation recovery system based on an AWG was built at X-band (9.6 GHz). To demonstrate performance of the system, the spin-lattice relaxation time of a fused quartz rod was measured at room temperature with fully digital excitation and detection.

Iminopropadienones R-N=C=C=C=O and carbon suboxide, C3O2. Theoretical and experimental 13C NMR spectra

The C-13 NMR data of five iminopropadienones R-N=C=C=C=O as well as carbon suboxide, C3O2, have been examined theoretically and experimentally. The best theoretical results were obtained using the GIAO/B3LYP/6-31 +G**//MP2/6-31G* level of theory, which reproduces the chemical shifts of the iminopropadienone substituents extremely well while underestimating those of the cumulenic carbons by 5-10 ppm. The computationally faster GIAO/HF/6-31 + G**//B3LYP/6-31 G* level is also adequate. (C) 2004 Elsevier B.V. All rights reserved.

The use of simultaneous H-1 & P-31 magic angle spinning nuclear magnetic resonance measurements to characterize energy metabolism during the conversion of muscle to meat

In order to investigate the potential of magic angle spinning nuclear magnetic resonance (MAS NMR) in the elucidation of post-mortem metabolism in muscle biopsies, simultaneous H-1 and (31)p MAS NMR measurements were made continuously on postmortem (20 min to 24 h) muscle longissimus samples from rabbits. The animals had either been or not been given adrenaline (0.5 mg kg(-1) 4 h pre-slaughter) to deplete stores of muscle glycogen. The intracellular pH was calculated from H-1 spectra, and the post-mortem rate of formation of lactate was followed and quantified. Comparison of measurements made on muscle samples from rabbits treated with adrenaline with measurements made on muscle samples from untreated' rabbits revealed significant effects of adrenaline treatment on both pH (pH24 h = 6.42 vs. pH24 It = 5.60) and formation of lactate (16 mmol g(-1) vs. 65 mmol g(-1)). The P-31 NMR spectra were used to follow the rate of degradation of ATP and phosphocreatine. The present study clearly shows that MAS NMR has potential for the study of post-mortem energy metabolism.

Matrix-assisted diffusion-ordered spectroscopy:application of surfactant solutions to the resolution of isomer spectra

The component spectra of a mixture of isomers with nearly identical diffusion coefficients cannot normally be distinguished in a standard diffusion-ordered spectroscopy (DOSY) experiment but can often be easily resolved using matrix-assisted DOSY, in which diffusion behaviour is manipulated by the addition of a co-solute such as a surfactant. Relatively little is currently known about the conditions required for such a separation, for example, how the choice between normal and reverse micelles affects separation or how the isomer structures themselves affect the resolution. The aim of this study was to explore the application of sodium dodecyl sulfate (SDS) normal micelles in aqueous solution and sodium 1,4-bis(2-ethylhexyl)sulfosuccinate (AOT) aggregates in chloroform, at a range of concentrations, to the diffusion resolution of some simple model sets of isomers such as monomethoxyphenols and short chain alcohols. It is shown that SDS micelles offer better resolution where these isomers differ in the position of a hydroxyl group, whereas AOT aggregates are more effective for isomers differing in the position of a methyl group. For both the normal SDS micelles and the less well-defined AOT aggregates, differences in the resolution of the isomers can in part be rationalised in terms of differing degrees of hydrophobicity, amphiphilicity and steric effects. Copyright © 2012 John Wiley & Sons, Ltd.

Investigation of the chemical origin and evidential value of differences in the SERS spectra of blue gel inks

Highly swellable polymer films doped with Ag nanoparticle aggregates (poly-SERS films) have been used to record very high signal:noise ratio, reproducible surface-enhanced (resonance) Raman (SER(R)S) spectra of in situ dried ink lines and their constituent dyes using both 633 and 785 nm excitation. These allowed the chemical origins of differences in the SERRS spectra of different inks to be determined. Initial investigation of pure samples of the 10 most common blue dyes showed that the dyes which had very similar chemical structures such as Patent Blue V and Patent Blue VF (which differ only by a single OH group) gave SERRS spectra in which the only indications that the dye structure had been changed were small differences in peak positions or relative intensities of the bands. SERRS studies of 13 gel pen inks were consistent with this observation. In some cases inks from different types of pens could be distinguished even though they were dominated by a single dye such as Victoria Blue B (Zebra Surari) or Victoria Blue BO (Pilot Acroball) because their predominant dye did not appear in other inks. Conversely, identical spectra were also recorded from different types of pens (Pilot G7, Zebra Z-grip) because they all had the same dominant Brilliant Blue G dye. Finally, some of the inks contained mixtures of dyes which could be separated by TLC and removed from the plate before being analysed with the same poly-SERS films. For example, the Pentel EnerGel ink pen was found to give TLC spots corresponding to Erioglaucine and Brilliant Blue G. Overall, this study has shown that the spectral differences between different inks which are based on chemically similar, but nonetheless distinct dyes, are extremely small, so very close matches between SERRS spectra are required for confident identification. Poly-SERS substrates can routinely provide the very stringent reproducibility and sensitivity levels required. This, coupled with the awareness of the reasons underlying the observed differences between similarly coloured inks allows a more confident assessment of the evidential value of inks SERS and should underpin adoption of this approach as a routine method for the forensic examination of inks.

Analysis and control of transient spectra using time-dependent density functional theory.

215 p.

Electronic and vibrational spectra of gaspeite

Visible, near-infrared, IR and Raman spectra of magnesian gaspeite are presented. Nickel ion is the main source of the electronic bands as it is the principal component in the mineral where as the bands in IR and Raman spectra are due to the vibrational processes in the carbonate ion as an entity. The combination of electronic absorption and vibrational spectra (including near-infrared, FTIR and Raman) of magnesian gaspeite are explained in terms of the cation co-ordination and the behaviour of CO32– anion in the Ni–Mg carbonate. The electronic absorption spectrum consists of three broad and intense bands at 8130, 13160 and 22730 cm–1 due to spin-allowed transitions and two weak bands at 20410 and 30300 cm–1 are assigned to spin-forbidden transitions of Ni2+ in an octahedral symmetry. The crystal field parameters evaluated from the observed bands are Dq = 810; B = 800 and C = 3200 cm–1. The two bands in the near-infrared spectrum at 4330 and 5130 cm–1 are overtone and combination of CO32– vibrational modes. For the carbonate group, infrared bands are observed at 1020 cm–1(1 ), 870 cm–1 (2), 1418 cm–1 (3) and 750 cm–1 (4), of which3, the asymmetric stretching mode is most intense. Three well resolved Raman bands at 1571, 1088 and 331 cm–1 are assigned to 3, 1 and MO stretching vibrations.