Modeling of Brain Tissue Heating Caused by Direct Cortical Stimulation for Assessing the Risk of Thermal Damage.

This paper aims to employ the numerical simulations to assess the risk of cellular damage during the application of a novel paradigm of electrical stimulation mapping (ESM) used in neurosurgery. The core principle of the paradigm is the use of short, high-intensity and high-frequency stimulation pulses. We developed a complex numerical model and performed coupled electro-thermal transient simulations. The model was optimized by incorporating ESM electrodes' resistance obtained during multiple intraoperative measurements and validated by comparing them with the results of temperature distribution measurement acquired by thermal imaging. The risk of heat-induced cellular damage was assessed by applying the Arrhenius equation integral on the computed time-dependent spatial distribution of temperature in the brain tissue. Our results suggest that the impact of the temperature increase during our novel ESM paradigm is thermally non-destructive. The presented simulation results match the previously published thermographic measurement and histopathological examination of the stimulated brain tissue and confirm the safety of the novel ESM.

Mitochondrial metabolism - neglected link of cancer transformation and treatment.

Physical processes in living cells were not taken into consideration among the essentials of biological activity, regardless of the fact that they establish a state far from thermodynamic equilibrium. In biological system chemical energy is transformed into the work of physical forces for various biological functions. The energy transformation pathway is very likely connected with generation of the endogenous electrodynamic field as suggested by experimentally proved electrodynamic activity of biological systems connected with mitochondrial and microtubule functions. Besides production of ATP and GTP (adenosine and guanosine triphosphate) mitochondria form a proton space charge layer, strong static electric field, and water ordering around them in cytosol - that are necessary conditions for generation of coherent electrodynamic field by microtubules. Electrodynamic forces are of a long-range nature in comparison with bond and cohesive forces. Mitochondrial dysfunction leads to disturbances of the electromagnetic field; its power and coherence may be diminished, and frequency spectrum altered. Consequently, defective electrodynamic interaction forces between cancer and healthy cells may result in local invasion of cancer cells. Further deformation of interaction forces connected with experimentally disclosed spatial disarrangement of the cytoskeleton and disordered electrodynamic field condition metastatic process. Cancer therapeutic strategy targeting mitochondria may restore normal physiological functions of mitochondria and open the apoptotic pathway. Apoptosis of too much damaged cancer cells was observed. Considerable experience with DCA (dichloroacetate) cancer treatment in humans was accumulated. Clinical trials should assess DCA therapeutic potential and collect data for development of novel more effective drugs for mitochondrial restoration of various cancers.

Removal of interference from fetal MEG by frequency dependent subtraction.

Fetal magnetoencephalography (fMEG) recordings are contaminated by maternal and fetal magnetocardiography (MCG) signals and by other biological and environmental interference. Currently, all methods for the attenuation of these signals are based on a time-domain approach. We have developed and tested a frequency dependent procedure for removal of MCG and other interference from the fMEG recordings. The method uses a set of reference channels and performs subtraction of interference in the frequency domain (SUBTR). The interference-free frequency domain signals are converted back to the time domain. We compare the performance of the frequency dependent approach with our present approach for MCG attenuation based on orthogonal projection (OP). SUBTR has an advantage over OP and similar template approaches because it removes not only the MCG but also other small amplitude biological interference, avoids the difficulties with inaccurate determination of the OP operator, provides more consistent and stable fMEG results, does not cause signal redistribution, and if references are selected judiciously, it does not reduce fMEG signal amplitude. SUBTR was found to perform well in simulations and on real fMEG recordings, and has a potential to improve the detection of fetal brain signals. The SUBTR removes interference without the need for a model of the individual interference sources. The method may be of interest for any sensor array noise reduction application where signal-free reference channels are available.

High-frequency electric field and radiation characteristics of cellular microtubule network.

Microtubules are important structures in the cytoskeleton, which organizes the cell. Since microtubules are electrically polar, certain microtubule normal vibration modes efficiently generate oscillating electric field. This oscillating field may be important for the intracellular organization and intercellular interaction. There are experiments which indicate electrodynamic activity of variety of cells in the frequency region from kHz to GHz, expecting the microtubules to be the source of this activity. In this paper, results from the calculation of intensity of electric field and of radiated electromagnetic power from the whole cellular microtubule network are presented. The subunits of microtubule (tubulin heterodimers) are approximated by elementary electric dipoles. Mechanical oscillation of microtubule is represented by the spatial function which modulates the dipole moment of subunits. The field around oscillating microtubules is calculated as a vector superposition of contributions from all modulated elementary electric dipoles which comprise the cellular microtubule network. The electromagnetic radiation and field characteristics of the whole cellular microtubule network have not been theoretically analyzed before. For the perspective experimental studies, the results indicate that macroscopic detection system (antenna) is not suitable for measurement of cellular electrodynamic activity in the radiofrequency region since the radiation rate from single cells is very low (lower than 10⁻²° W). Low noise nanoscopic detection methods with high spatial resolution which enable measurement in the cell vicinity are desirable in order to measure cellular electrodynamic activity reliably.

Neutrophilic differentiation modulates the apoptotic response of HL-60 cells to sodium butyrate and sodium valproate.

Differentiation of myeloid leukemic cells may result in less sensitivity to various apoptotic stimuli. We examined whether human leukemia HL-60 cells differentiating by all-trans retinoic acid (ATRA) acquired resistance to the apoptogenic activity of two histone deacetylase (HDAC) inhibitors, butyrate and valproate. In undifferentiated cells, the cytotoxicity of both butyrate and valproate was associated with activation of the intrinsic apoptotic pathway since we observed dissipation of mitochondrial membrane potential, induction of caspase-9 and caspase-3 activities, appearance of sub-G1 DNA and loss of plasma membrane asymmetry and/or integrity. Both HDAC inhibitors were also able to induce accumulation of undifferentiated cells in the G0/G1 phase of the cell cycle. ATRA was found to enhance the apoptotic effect of both butyrate and valproate in undifferentiated cells. This aside, ATRA appeared to synergize with butyrate in the induction of the G0/G1 cell cycle arrest. In cells pretreated for 72 h with ATRA, butyrate and valproate in combination with ATRA induced lower dissipation of mitochondrial membrane potential and weaker apoptotic and/or necrotic changes in plasma membrane, whereas DNA fragmentation was not diminished compared to undifferentiated cells. Similar results were also obtained when butyrate or valproate were combined with another neutrophilic differentiation inducer, dimethyl sulfoxide. We conclude that neutrophilic differentiation modulates but does not abrogate the apoptotic response of HL-60 cells to butyrate and valproate, and nuclei are preferentially affected during apoptosis in differentiated cells.

Fetal MEG evoked response latency from beamformer with random field theory.

Analysis of fetal magnetoencephalographic brain recordings is restricted by low signal to noise ratio (SNR) and non-stationarity of the sources. Beamformer techniques have been applied to improve SNR of fetal evoked responses. However, until now the effect of non-stationarity was not taken into account in detail, because the detection of evoked responses is in most cases determined by averaging a large number of trials. We applied a windowing technique to improve the stationarity of the data by using short time segments recorded during a flash-evoked study. In addition, we implemented a random field theory approach for more stringent control of false-positives in the statistical parametric map of the search volume for the beamformer. The search volume was based on detailed individual fetal/maternal biometrics from ultrasound scans and fetal heart localization. Average power over a sliding window within the averaged evoked response against a randomized average background power was used as the test z-statistic. The significance threshold was set at 10% over all members of a contiguous cluster of voxels. There was at least one significant response for 62% of fetal and 95% of newborn recordings with gestational age (GA) between 28 and 45 weeks from 29 subjects. We found that the latency was either substantially unchanged or decreased with increasing GA for most subjects, with a nominal rate of about -11 ms/week. These findings support the anticipated neurophysiological development, provide validation for the beamformer model search as a methodology, and may lead to a clinical test for fetal cognitive development.

Understanding dynamics of the system using Hilbert phases: an application to study neonatal and fetal brain signals.

The Hilbert phase phi(t) of a signal x(t) exhibits slips when the magnitude of their successive phase difference |phi(t(i+1))-phi(t(i))| exceeds pi. By applying this approach to periodic, uncorrelated, and long-range correlated data, we show that the standard deviation of the time difference between the successive phase slips Deltatau normalized by the percentage of slips in the data is characteristic of the correlation in the data. We consider a 50x50 square lattice and model each lattice point by a second-order autoregressive (AR2) process. Further, we model a subregion of the lattice using a different set of AR2 parameters compared to the rest. By applying the proposed approach to the lattice model, we show that the two distinct parameter regions introduced in the lattice are clearly distinguishable. Finally, we demonstrate the application of this approach to spatiotemporal neonatal and fetal magnetoencephalography signals recorded using 151 superconducting quantum interference device sensors to identify the sensors containing the neonatal and fetal brain signals and discuss the improved performance of this approach over the traditionally used spectral approach.

Conventional protein kinase C isoenzymes undergo dephosphorylation in neutrophil-like HL-60 cells treated by chelerythrine or sanguinarine.

The quaternary benzo[c]phenanthridine alkaloid chelerythrine is widely used as an inhibitor of protein kinase C (PKC). However, in biological systems chelerythrine interacts with an array of proteins. In this study, we examined the effects of chelerythrine and sanguinarine on conventional PKCs (cPKCs) and PKC upstream kinase, phosphoinositide-dependent protein kinase 1 (PDK1), under complete inhibition conditions of PKC-dependent oxidative burst. In neutrophil-like HL-60 cells, sanguinarine and chelerythrine inhibited N-formyl-Met-Leu-Phe, phorbol 12-myristate 13-acetate (PMA)-, and A23187-induced oxidative burst with IC(50) values not exceeding 4.6 micromol/L, but the inhibition of PMA-stimulated cPKC activity in intact cells required at least fivefold higher alkaloid concentrations. At concentrations below 10 micromol/L, sanguinarine and chelerythrine prevented phosphorylation of approximately 80 kDa protein and sequestered approximately 60 kDa phosphoprotein in cytosol. Moreover, neither sanguinarine nor chelerythrine impaired PMA-stimulated translocation of autophosphorylated PKCalpha/betaII isoenzymes, but both alkaloids induced dephosphorylation of the turn motif in PKCalpha/betaII. The dephosphorylation did not occur in unstimulated cells and it was not accompanied by PKC degradation. Furthermore, cell treatment with sanguinarine or chelerythrine resulted in phosphorylation of approximately 70 kDa protein by PDK1. We conclude that PKC-dependent cellular events are affected by chelerythrine primarily by multiple protein interactions rather than by inhibition of PKC activity.

Bootstrap significance of low SNR evoked response.

In order to obtain adequate signal to noise ratio (SNR), stimulus-evoked brain signals are averaged over a large number of trials. However, in certain applications, e.g. fetal magnetoencephalography (MEG), this approach fails due to underlying conditions (inherently small signals, non-stationary/poorly characterized signals, or limited number of trials). The resulting low SNR makes it difficult to reliably identify a response by visual examination of the averaged time course, even after pre-processing to attenuate interference. The purpose of this work was to devise an intuitive statistical significance test for low SNR situations, based on non-parametric bootstrap resampling. We compared a two-parameter measure of p-value and statistical power with a bootstrap equal means test and a traditional rank test using fetal MEG data collected with a light flash stimulus. We found that the two-parameter measure generally agreed with established measures, while p-value alone was overly optimistic. In an extension of our approach, we compared methods to estimate the background noise. A method based on surrogate averages resulted in the most robust estimate. In summary we have developed a flexible and intuitively satisfying bootstrap-based significance measure incorporating appropriate noise estimation.

Evaluation of novel microtubules interfering agents myoseverin, tubulyzine and E2GG in primary cultures of rat hepatocytes.

We investigated the effects of novel microtubules interfering agents (MIAs) in primary cultures of rat hepatocytes. Cells were treated for 24 h with a known compound colchicine and newly synthesized derivatives myoseverin, tubulyzine, and E2GG. We examined the effects of MIAs on microtubules network integrity and on the polymerization capability of isolated tubulin. All tested MIAs inhibited microtubules assembly with the following IC(50) values: tubulyzine (4.4 + or - 0.9 micromol/l), myoseverin (7.0 + or - 0.8 micromol/l), E2GG (16 + or - 2 micromol/l), colchicine (2.0 + or - 0.4 micromol/l). The potency of MIAs to perturb microtubular network integrity (monitored by immune-histochemistry) increased in the order tubulyzine < myoseverin < E2GG < colchicine. We described recently deleterious effects of MIAs on the expression of drug metabolizing enzymes, including CYP1A1. Here we observed inhibitory effects of novel MIAs on dioxin-inducible expression of CYP1A1 mRNA in rat hepatocytes. We conclude that novel MIAs exert analogical biological response as classical MIAs such as colchicine or nocodazole. This further supports the hypothesis that tubulin is the primordial target of MIAs within the cell and that perturbation of microtubules dynamics and/or integrity triggers the biological effects described here.

Validation of the flash-evoked response from fetal MEG.

Flash-evoked responses can be recorded from the fetus in utero. However, a standard analysis approach based on orthogonal projection (OP) to attenuate maternal and fetal cardiac signals leads to a spatial redistribution of the signal. This effect prevents the correlation of source location with a known fetal head location in some cases and the signal-to-noise ratio (SNR) is sometimes limited such that the response latency is difficult to determine. We used a modified beamformer model search analysis to avoid the redistribution shortcoming and to improve the SNR. We included a statistical test for residual interference in the average and quantified significance of the evoked response with a bootstrap method. Selected source locations compared favorably to fetal head locations estimated from ultrasound exams. The evoked response time course was found to have a significant post-trigger peak with a latency between about 180 and 770 ms in more than 90% of the subject measurements. These results confirm that the combined application of a beamformer model search and bootstrap significance test provides a validation of the flash-evoked response observed in OP processed fetal MEG channels.

Searching for the best model: ambiguity of inverse solutions and application to fetal magnetoencephalography.

Fetal brain signals produce weak magnetic fields at the maternal abdominal surface. In the presence of much stronger interference these weak fetal fields are often nearly indistinguishable from noise. Our initial objective was to validate these weak fetal brain fields by demonstrating that they agree with the electromagnetic model of the fetal brain. The fetal brain model is often not known and we have attempted to fit the data to not only the brain source position, orientation and magnitude, but also to the brain model position. Simulation tests of this extended model search on fetal MEG recordings using dipole fit and beamformers revealed a region of ambiguity. The region of ambiguity consists of a family of models which are not distinguishable in the presence of noise, and which exhibit large and comparable SNR when beamformers are used. Unlike the uncertainty of a dipole fit with known model plus noise, this extended ambiguity region yields nearly identical forward solutions, and is only weakly dependent on noise. The ambiguity region is located in a plane defined by the source position, orientation, and the true model centre, and will have a diameter approximately 0.67 of the modelled fetal head diameter. Existence of the ambiguity region allows us to only state that the fetal brain fields do not contradict the electromagnetic model; we can associate them with a family of models belonging to the ambiguity region, but not with any specific model. In addition to providing a level of confidence in the fetal brain signals, the ambiguity region knowledge in combination with beamformers allows detection of undistorted temporal waveforms with improved signal-to-noise ratio, even though the source position cannot be uniquely determined.

Optimal reduction of MCG in fetal MEG recordings.

Recording fetal magnetoencephalographic (fMEG) signals in-utero is a demanding task due to biological interference, especially maternal and fetal magnetocardiographic (MCG) signals. A method based on orthogonal projection of MCG signal space vectors (OP) was evaluated and compared with independent component analysis (ICA). The evaluation was based on MCG amplitude reduction and signal-to-noise ratio of fetal brain signals using exemplary datasets recorded during ongoing studies related to auditory evoked fields. The results indicate that the OP method is the preferable approach for attenuation of MCG and for preserving the fetal brain signals in fMEG recordings.

Fluorescence labelling of phosphatase activity in digestive glands of carnivorous plants.

A new ELF (enzyme labelled fluorescence) assay was applied to detect phosphatase activity in glandular structures of 47 carnivorous plant species, especially Lentibulariaceae, in order to understand their digestive activities. We address the following questions: (1) Are phosphatases produced by the plants and/or by inhabitants of the traps? (2) Which type of hairs/glands is involved in the production of phosphatases? (3) Is this phosphatase production a common feature among carnivorous plants or is it restricted to evolutionarily advanced species? Our results showed activity of the phosphatases in glandular structures of the majority of the plants tested, both from the greenhouse and from sterile culture. In addition, extracellular phosphatases can also be produced by trap inhabitants. In Utricularia, activity of phosphatase was detected in internal glands of 27 species from both primitive and advanced sections and different ecological groups. Further positive reactions were found in Genlisea, Pinguicula, Aldrovanda, Dionaea, Drosera, Drosophyllum, Nepenthes, and Cephalotus. In Utricularia and Genlisea, enzymatic secretion was independent of stimulation by prey. Byblis and Roridula are usually considered as "proto-carnivores", lacking digestive enzymes. However, we found high activity of phosphatases in both species. Thus, they should be classified as true carnivores. We suggest that the inflorescence of Byblis and some Pinguicula species might also be an additional "carnivorous organ", which can trap a prey, digest it, and finally absorb available nutrients.

Human fetal brain imaging by magnetoencephalography: verification of fetal brain signals by comparison with fetal brain models.

Fetal magnetoencephalogram (fMEG) is measured in the presence of a large interference from maternal and fetal magnetocardiograms (mMCG and fMCG). This cardiac interference can be successfully removed by orthogonal projection of the corresponding spatial vectors. However, orthogonal projection redistributes the fMEG signal among channels. Such redistribution can be readily accounted for in the forward solution, and the signal topography can also be corrected. To assure that the correction has been done properly, and also to verify that the measured signal originates from within the fetal head, we have modeled the observed fMEG by two extreme models where the fetal head is assumed to be either electrically transparent or isolated from the abdominal tissue. Based on the measured spontaneous, sharp wave, and flash-evoked fMEG signals, we have concluded that the model of the electrically isolated fetal head is more appropriate for fMEG analysis. We show with the help of this model that the redistribution due to projection was properly corrected, and also, that the measured fMEG is consistent with the known position of the fetal head. The modeling provides additional confidence that the measured signals indeed originate from within the fetal head.

How many channels are needed for MEG?

Channel count in modern MEG systems has been steadily increasing, but are more channels necessary? Assuming that the spatial sampling considerations are satisfied, this question can be answered by examining the MEG system's ability to localize and resolve brain sources. For the simple situation where only uncorrelated sensor noise is present, dipole localization accuracy monotonically increases with increasing number of channels, while for spatially correlated brain noise the accuracy increases only until the number of channels reaches 100 to 200. Beyond this limit the inter-channel separation is comparable to the brain noise correlation distance and increasing the channel count does not help. Contrary to the above dipole result, we show by simulations with up to several thousand channels, that if the data is analyzed by beamformers even in the presence of correlated brain noise, the two-source resolvability and single-source localization accuracy monotonically improve with increasing number of channels. We demonstrate such behavior for a 275 channels system, where we have inserted an artificial dipole into real measured brain noise and resampled the number of channels to 138. Beamformer analysis of the data shows markedly improved localization accuracy when the number of channels is increased from 138 to 275. This finding also signifies that the beamformer performance is not limited by system imperfections when the number of channels is as large as 275. To clarify these results, we illustrate analytically the mechanism of beamformer resolution dependence on the number of channels, using an example of a simple system containing two dipole sources, and uncorrelated sensor noise.

Forward solution ambiguity for fMEG model.

Accurate knowledge of the electromagnetic model of the fetal brain is required for quantitative fetal MEG (fMEG) signal analysis. The model is defined by the fetal head position, assuming that volume currents are confined to it. Head position can be located by 3D ultrasound. Since the ultrasound is presently not integrated with fMEG, there is a potential for fetal head motion when the mother moves between the two instruments. To interpret the fMEG data without accurate information about the fetal head position, a model search must be undertaken to localize not only the fMEG source within the fetal head, but also the fetal head within the maternal abdomen. Such model search results in multiple solutions with different source amplitudes, but with roughly the same fit errors [Vrba, 2004]. We show that this behaviour is caused by the forward solution ambiguity. For a given measurement, there is a large region within the maternal abdomen within which all fetal head centers produce reasonably good explanation of the observed signal. The source positions within these different fetal heads are different, but the resulting signals are indistinguishable in the presence of even a small noise. Similar ambiguity also exists in adult MEG, where it usually does not pose a problem because the head position is well known.

Cleaning fetal MEG using a beamformer search for the optimal forward model.

We present a new method for improving the signal-to-noise ratio (SNR) of event-related fetal MEG signals based upon the SAM minimum-variance beamformer. SAM could separate the evoked response source activity from the remaining fMEG signal and interference if the evoked response source coordinates and forward model were know. However, this requires knowledge of both the coordinate of the evoked response source and its forward model. In late gestation, the vernix caseosa effectively insulates the fetus from the amniotic fluid. Hence, the forward model could be approximated by an equivalent current dipole in a homogeneously conducting sphere with its origin at the center of the fetal head. In the absence of accurate anatomical data, a beamformer could be used to evaluate all feasible source-origin combinations--selecting the combination giving the best SNR for the evoked response. Application of this approach to measured fMEG data reveals that the optimal model sphere location is described not by a single local sphere origin, but rather by all origins lying within an extended region. This result is explained by model predictions showing the same region of ambiguity [Vrba, 2004]. Although the model search does not localize the sources of the fetal evoked response, it does significantly improve SNR. This was demonstrated by analysis of fetal auditory evoked response data.

Advanced electronics for the CTF MEG system.

Development of the CTF MEG system has been advanced with the introduction of a computer processing cluster between the data acquisition electronics and the host computer. The advent of fast processors, memory, and network interfaces has made this innovation feasible for large data streams at high sampling rates. We have implemented tasks including anti-alias filter, sample rate decimation, higher gradient balancing, crosstalk correction, and optional filters with a cluster consisting of 4 dual Intel Xeon processors operating on up to 275 channel MEG systems at 12 kHz sample rate. The architecture is expandable with additional processors to implement advanced processing tasks which may include e.g., continuous head localization/motion correction, optional display filters, coherence calculations, or real time synthetic channels (via beamformer). We also describe an electronics configuration upgrade to provide operator console access to the peripheral interface features such as analog signal and trigger I/O. This allows remote location of the acoustically noisy electronics cabinet and fitting of the cabinet with doors for improved EMI shielding. Finally, we present the latest performance results available for the CTF 275 channel MEG system including an unshielded SEF (median nerve electrical stimulation) measurement enhanced by application of an adaptive beamformer technique (SAM) which allows recognition of the nominal 20-ms response in the unaveraged signal.

Impact of ionic aluminium on extracellular phosphatases in acidified lakes.

We studied direct inhibiting effects of aluminium (Al) on extracellular phosphatases produced by the plankton of acidified lakes in the Bohemian Forest. In laboratory experiments we tested the effect of different Al concentrations (0-1000 microg l(-1)) on kinetic parameters of acid phosphatases (pH optimum approximately 5.0) at pH between 4.5 and 5.2. We observed a significant reduction of an apparent substrate affinity at Al concentrations between 300 and 1000 microg l(-1) at pH 4.5 and 4.8 (but not at 5.2). In contrast, maximum acid phosphatase activity (AcPA) remained unchanged. Such behaviour of saturation kinetics is compatible with the assumption that ionic Al acts as a competitive inhibitor of acid phosphatases. To decide whether the observed Al effects could be explained alternatively by complexation of Al with substrate, we tested statistically the best fits of data with both possible models (competitive versus complexation). Experimental results supported the competitive hypothesis rather than the complexation model suggested originally by some authors. Furthermore, we tested the Al effect within a wide range of pH from 4.0 to 6.0. For pH values < 5.2, the results of an Al-pH matrix experiment gave a more detailed picture: the higher the Al concentration, the wider the pH range in which Al could negatively affect AcPA. The ecological ramifications of this effect were evaluated in the context of field AcPA data on three strongly acidified lakes.