Dissociating EEG sources linked to stimulus and response evaluation in numerical Stroop task using Independent Component Analysis.

OBJECTIVES: Independent Component Analysis (ICA) is a powerful data-driven technique, which separates EEG signals into functionally and physiologically distinct source activities. The aim of this study was to identify the neural sources, which contribute to scalp ERPs including N450. METHODS: Dense-array EEG data were obtained from 20 participants performing numerical Stroop task. By applying ICA, artifacts were identified and removed. The remaining neural sources underwent clustering and further clusters' ERP analysis. RESULTS: While the traditional channels' ERP analysis confirmed the occurrence of conflict-related N450 potential, the ICA results revealed two sources contributing to its variance: the mid-parietal cluster with source estimated in posterior cingulate cortex (PCC) and fronto-central cluster with source in anterior cingulate cortex (ACC). The former showed increased (prolonged) activity before the response for cognitively demanding trials, whereas the latter showed negative deflection after the response. PCC activity was decreased (shortened) before erroneous responses, while ACC showed strong error-related negativity. CONCLUSIONS: PCC is responsible for stimulus evaluation, while ACC is responsible for evaluating the action-outcome. Moreover, errors are committed due to insufficient stimuli processing within PCC. SIGNIFICANCE: ICA proved to be reliable and effective method for ERP analysis, which shed new light into the brain potentials evoked by the numerical Stroop task.

Contributive sources analysis: a measure of neural networks' contribution to brain activations.

General linear model (GLM) is a standard and widely used fMRI analysis tool. It enables the detection of hypothesis-driven brain activations. In contrast, Independent Component Analysis (ICA) is a powerful technique, which enables the detection of data-driven spatially independent networks. Hybrid approaches that combine and take advantage of GLM and ICA have been proposed. Yet the choice of the best method is still a challenge, considering that the techniques may yield slightly different results regarding the number of brain regions involved in a task. A poor statistical power or the deviance from the predicted hemodynamic response functions is possible cause for GLM failures in extracting some activations picked by ICA. However, there might be another explanation for different results obtained with GLM and ICA approaches, such as networks cancelation. In this paper, we propose a new supplementary method that can give more insight into the functional data as well as help to clarify inconsistencies between the results of studies using GLM and ICA. We introduce a contributive sources analysis (CSA), which provides a measure of the number and the strength of the neural networks that significantly contribute to brain activation. CSA, applied to fMRI data of anti-saccades, enabled us to verify whether the brain regions involved in the task are dominated by a single network or serve as key nodes for particular networks interaction. Moreover, when applying CSA to the atlas-defined regions-of-interest, results indicated that activity of the parieto-medial temporal network was suppressed by the eye field network and the default mode network. Thus, this effect of networks cancelation explains the absence of parieto-medial temporal activation within the GLM results. Together, those findings indicate that brain activations are a result of complex network interactions. Applying CSA appears to be a useful tool to reveal additional findings outside the scope of the "fixed-model" GLM and data-driven ICA approaches.

Rectangular loop-gap resonator with the light access to the sample.

A modified rectangular loop-gap resonator for X-band electron paramagnetic resonance (EPR) studies of aqueous samples, enabling the light access, is described. Changes introduced into rectangular resonator geometry, previously presented in Piasecki et al. (1998) [1], and redesigned coupling structure lead to the better thermal and mechanical stability. The modified structure makes provision for the controlled light access to the sample placed in a flat cell during an EPR experiment. The sensitivity of the resonator for aqueous samples as well as an experimentally tested microwave magnetic field homogeneity are presented. Results of simulations and experimental tests indicate that the presence of light access holes in the resonator's front side does not disturb the uniformity of microwave magnetic field distribution in the nodal plane. The optimal flat cell thickness for unsaturable and saturable aqueous samples has been calculated for this new structure. A modified rectangular geometry of the loop-gap resonator ensures a good performance for aqueous samples allowing its convenient and efficient light illumination during EPR signal recording .

Reorientation of cytochrome c2 upon interaction with oppositely charged macromolecules probed by SR EPR: implications for the role of dipole moment to facilitate collisions in proper configuration for electron transfer.

The reaction of water-soluble cytochrome c (c(2)) with its physiological redox partners is facilitated by electrostatic attractions between the two protein surfaces. Using spin-labeled cytochrome c(2) from Rhodobacter capsulatus and pulse electron paramagnetic resonance (EPR) measurements we compared spatial orientation of cytochrome c(2) upon its binding to surfaces of opposite charge. We observed that cytochrome c(2) can use its negatively charged "back" side when exposed to interact with positively charged surfaces (DEAE resin) which is the opposite to the use of its positively charged "front" side in physiological interaction with negatively charged binding domain of cytochrome bc(1). The later orientation is also adopted upon non-physiological binding of cytochrome c(2) to negatively charged carboxymethyl cellulose resin. These results directly demonstrate how the electric dipolar nature of cytochrome c(2) influences its orientation in interactions with charged surfaces, which may facilitate collisions with other redox proteins in a proper orientation to support physiologically-competent electron transfer. Saturation recovery EPR provides an attractive tool for monitoring spatial orientation of proteins in their interaction with surfaces in liquid phase. It is particularly valuable for metalloproteins engaged in redox reactions as a means to monitor the geometry and dynamics of formation of protein complexes in measurements that are independent of electron transfer processes.

W-band frequency-swept EPR.

This paper describes a novel experiment on nitroxide radical spin labels using a multiarm EPR W-band bridge with a loop-gap resonator (LGR). We demonstrate EPR spectroscopy of spin labels by linear sweep of the microwave frequency across the spectrum. The high bandwidth of the LGR, about 1 GHz between 3 dB points of the microwave resonance, makes this new experiment possible. A frequency-tunable yttrium iron garnet (YIG) oscillator provides sweep rates as high as 1.8x10(5) GHz/s, which corresponds to 6.3 kT/s in magnetic field-sweep units over a 44 MHz range. Two experimental domains were identified. In the first, linear frequency sweep rates were relatively slow, and pure absorption and pure dispersion spectra were obtained. This appears to be a practical mode of operation at the present level of technological development. The main advantage is the elimination of sinusoidal magnetic field modulation. In the second mode, the frequency is swept rapidly across a portion of the spectrum, and then the frequency sweep is stopped for a readout period; FID signals from a swept line oscillate at a frequency that is the difference between the spectral position of the line in frequency units and the readout position. If there is more than one line, oscillations are superimposed. The sweep rates using the YIG oscillator were too slow, and the portion of the spectrum too narrow to achieve the full EPR equivalent of Fourier transform (FT) NMR. The paper discusses technical advances required to reach this goal. The hypothesis that trapezoidal frequency sweep is an enabling technology for FT EPR is supported by this study.

Magnetic interactions sense changes in distance between heme b(L) and the iron-sulfur cluster in cytochrome bc(1).

During the operation of cytochrome bc(1), a key enzyme of biological energy conversion, the iron-sulfur head domain of one of the subunits of the catalytic core undergoes a large-scale movement from the catalytic quinone oxidation Q(o) site to cytochrome c(1). This changes a distance between the two iron-two sulfur (FeS) cluster and other cofactors of the redox chains. Although the role and the mechanism of this movement have been intensely studied, they both remain poorly understood, partly because the movement itself is not easily traceable experimentally. Here, we take advantage of magnetic interactions between the reduced FeS cluster and oxidized heme b(L) to use dipolar enhancement of phase relaxation of the FeS cluster as a spectroscopic parameter which with a unique clarity and specificity senses changes in the distance between those two cofactors. The dipolar relaxation curves measured by EPR at Q-band in a glass state of frozen solution (i.e., under the conditions trapping a dynamic distribution of FeS positions that existed in a liquid phase) of isolated cytochrome bc(1) were compared with the curves calculated for the FeS cluster occupying distinct positions in various crystals of cytochrome bc(1). This comparison revealed the existence of a broad distribution of the FeS positions in noninhibited cytochrome bc(1) and demonstrated that the average equilibrium position is modifiable by inhibitors or mutations. To explain the results, we assume that changes in the equilibrium distribution of the FeS positions are the result of modifications of the orienting potential gradient in which the diffusion of the FeS head domain takes place. The measured changes in the phase relaxation enhancement provide the first direct experimental description of changes in the strength of dipolar coupling between the FeS cluster and heme b(L).

Demonstration of short-lived complexes of cytochrome c with cytochrome bc1 by EPR spectroscopy: implications for the mechanism of interprotein electron transfer.

One of the steps of a common pathway for biological energy conversion involves electron transfer between cytochrome c and cytochrome bc1. To clarify the mechanism of this reaction, we examined the structural association of those two proteins using the electron transfer-independent electron paramagnetic resonance (EPR) techniques. Drawing on the differences in the continuous wave EPR spectra and saturation recoveries of spin-labeled bacterial and mitochondrial cytochromes c recorded in the absence and presence of bacterial cytochrome bc1, we have exposed a time scale of dynamic equilibrium between the bound and the free state of cytochrome c at various ionic strengths. Our data show a successive decrease of the bound cytochrome c fraction as the ionic strength increases, with a limit of approximately 120 mm NaCl above which essentially no bound cytochrome c can be detected by EPR. This limit does not apply to all of the interactions of cytochrome c with cytochrome bc1 because the cytochrome bc1 enzymatic activity remained high over a much wider range of ionic strengths. We concluded that EPR monitors just the tightly bound state of the association and that an averaged lifetime of this state decreases from over 100 micros at low ionic strength to less than 400 ns at an ionic strength above 120 mm. This suggests that at physiological ionic strength, the tightly bound complex on average lasts less than the time needed for a single electron exchange between hemes c and c1, indicating that productive electron transfer requires several collisions of the two molecules. This is consistent with an early idea of diffusion-coupled reactions that link the soluble electron carriers with the membranous complexes, which, we believe, provides a robust means of regulating electron flow through these complexes.

Saturation recovery EPR and ELDOR at W-band for spin labels.

A reference arm W-band (94 GHz) microwave bridge with two sample-irradiation arms for saturation recovery (SR) EPR and ELDOR experiments is described. Frequencies in each arm are derived from 2 GHz synthesizers that have a common time-base and are translated to 94 GHz in steps of 33 and 59 GHz. Intended applications are to nitroxide radical spin labels and spin probes in the liquid phase. An enabling technology is the use of a W-band loop-gap resonator (LGR) [J.W. Sidabras, R.R. Mett, W. Froncisz, T.G. Camenisch, J.R. Anderson, J.S. Hyde, Multipurpose EPR loop-gap resonator and cylindrical TE(011) cavity for aqueous samples at 94 GHz, Rev. Sci. Instrum. 78 (2007) 034701]. The high efficiency parameter (8.2 GW(-1/2) with sample) permits the saturating pump pulse level to be just 5 mW or less. Applications of SR EPR and ELDOR to the hydrophilic spin labels 3-carbamoyl-2,2,5,5-tetra-methyl-3-pyrroline-1-yloxyl (CTPO) and 2,2,6,6,-tetramethyl-4-piperidone-1-oxyl (TEMPONE) are described in detail. In the SR ELDOR experiment, nitrogen nuclear relaxation as well as Heisenberg exchange transfer saturation from pumped to observed hyperfine transitions. SR ELDOR was found to be an essential method for measurements of saturation transfer rates for small molecules such as TEMPONE. Free induction decay (FID) signals for small nitroxides at W-band are also reported. Results are compared with multifrequency measurements of T(1e) previously reported for these molecules in the range of 2-35 GHz [J.S. Hyde, J.-J. Yin, W.K. Subczynski, T.G. Camenisch, J.J. Ratke, W. Froncisz, Spin label EPR T(1) values using saturation recovery from 2 to 35 GHz. J. Phys. Chem. B 108 (2004) 9524-9529]. The values of T(1e) decrease at 94 GHz relative to values at 35 GHz.

Estimation of binding parameters for the protein-protein interaction using a site-directed spin labeling and EPR spectroscopy.

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c(2) interacting with cytochrome bc(1) complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein-protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.

Multipurpose EPR loop-gap resonator and cylindrical TE011 cavity for aqueous samples at 94 GHz.

A loop-gap resonator (LGR) and a cylindrical TE(011) cavity resonator for use at W band, 94 GHz, have been designed and characterized using the Ansoft (Pittsburgh, PA) high frequency structure simulator (HFSS; Version 10.0). Field modulation penetration was analyzed using Ansoft MAXWELL 3D (Version 11.0). Optimizing both resonators to the same sample sizes shows that EPR signal intensities of the LGR and TE(011) are similar. The 3 dB bandwidth of the LGR, on the order of 1 GHz, is a new advantage for high frequency experiments. Ultraprecision electric discharge machining (EDM) was used to fabricate the resonators from silver. The TE(011) cavity has slots that are cut into the body to allow penetration of 100 kHz field modulation. The resonator body is embedded in graphite, also cut by EDM techniques, for a combination of reasons that include (i) reduced microwave leakage and improved TE(011) mode purity, (ii) field modulation penetration, (iii) structural support for the cavity body, and (iv) machinability by EDM. Both resonators use a slotted iris. Variable coupling is provided by a three-stub tuning element. A collet system designed to hold sample tubes has been implemented, increasing repeatability of sample placement and reducing sample vibration noise. Initial results include multiquantum experiments up to 9Q using the LGR to examine 1 mM 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) in aqueous solution at room temperature and field modulation experiments using the TE(011) cavity to obtain an EPR spectrum of 1 microM TEMPO.

Microwave frequency modulation in CW EPR at W-band using a loop-gap resonator.

Loop-gap resonator (LGR) technology has been extended to W-band (94GHz). One output of a multiarm Q-band (35GHz) EPR bridge was translated to W-band for sample irradiation by mixing with 59 GHz; similarly, the EPR signal was translated back to Q-band for detection. A cavity resonant in the cylindrical TE011 mode suitable for use with 100 kHz field modulation has also been developed. Results using microwave frequency modulation (FM) at 50 kHz as an alternative to magnetic field modulation are described. FM was accomplished by modulating a varactor coupled to the 59 GHz oscillator. A spin-label study of sensitivity was performed under conditions of overmodulation and gamma2H1(2)T1T2<1. EPR spectra were obtained, both absorption and dispersion, by lock-in detection at the fundamental modulation frequency (50 kHz), and also at the second and third harmonics (100 and 150 kHz). Source noise was deleterious in first harmonic spectra, but was very low in second and third harmonic spectra. First harmonic microwave FM was transferred to microwave modulation at second and third harmonics by the spins, thus satisfying the "transfer of modulation" principle. The loaded Q-value of the LGR with sample was 90 (i.e., a bandwidth between 3 dB points of about 1 GHz), the resonator efficiency parameter was calculated to be 9.3 G at one W incident power, and the frequency deviation was 11.3 MHz p-p, which is equivalent to a field modulation amplitude of 4 G. W-band EPR using an LGR is a favorable configuration for microwave FM experiments.

Influence of the disulfide bond configuration on the dynamics of the spin label attached to cytochrome c.

A series of multi-nanosecond molecular dynamics (MD) simulations of wild-type cytochrome c and its spin-labeled variants with the methanethiosulfonate moiety attached at position C102 were performed (1) to elucidate the effect of the spin probe presence on the protein structure and (2) to describe the structure and dynamics of the spin-label moiety. Comparisons with the reference crystal structure of cytochrome c (PDB entry: 1YCC) indicate that the protein secondary structure is well preserved during simulations of the wild-type cytochrome c but slightly changed in simulations of the cytochrome c labeled at position C102. At the time scale covered in our simulations, the spin label exhibits highly dynamical behavior. The number of observed distinct conformations of the spin label moiety is between 3 and 13. The spin probe was found to form short-lived hydrogen bonds with the protein. Temporary hydrophobic interactions between the probe and the protein were also detected. The MD simulations directly show that the disulfide bond in the tether linking a spin probe with a protein strongly influence the behavior of the nitroxide group. The conformational flexibility and interaction with the protein are different for each of the two low energy conformations of the disulfide bond.

Accessibility of nitroxide side chains: absolute Heisenberg exchange rates from power saturation EPR.

In site-directed spin labeling, the relative solvent accessibility of spin-labeled side chains is taken to be proportional to the Heisenberg exchange rate (W(ex)) of the nitroxide with a paramagnetic reagent in solution. In turn, relative values of W(ex) are determined by continuous wave power saturation methods and expressed as a proportional and dimensionless parameter Pi. In the experiments presented here, NiEDDA is characterized as a paramagnetic reagent for solvent accessibility studies, and it is shown that absolute values of W(ex) can be determined from Pi, and that the proportionality constant relating them is independent of the paramagnetic reagent and mobility of the nitroxide. Based on absolute exchange rates, an accessibility factor is defined (0 < rho < 1) that serves as a quantitative measure of side-chain solvent accessibility. The accessibility factors for a nitroxide side chain at 14 different sites in T4 lysozyme are shown to correlate with a structure-based accessibility parameter derived from the crystal structure of the protein. These results provide a useful means for relating crystallographic and site-directed spin labeling data, and hence comparing crystal and solution structures.

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR.

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.

Determination of T1-spin-lattice relaxation time in a two-level system by continuous wave multiquantum electron paramagnetic resonance spectroscopy in a presence of tetrachromatic microwave irradiation.

Applicability of continuous wave multiquantum EPR methods to study relaxation times at X-band is examined. Multiquantum transitions excited in a two-level system by tetrachromatic irradiation are used for these studies. The Bloch equation model is applied to simulate lineshapes of the three quantum transitions as a function of frequency difference between exciting fields. The dependence of multiquantum transition signals on relaxation times and microwave amplitude is shown. On this basis a method of deducing relaxation times from these signals is formulated. The case of a homogeneously and inhomogeneously broadened resonance line is considered. Two experimental methods are used to verify the proposed hypothesis: the X-band continuous wave multiquantum EPR with four frequencies microwave field and saturation recovery EPR. The values of T(1) obtained from CW MQ EPR and SR EPR are compared.