How electron interference can be used in probing the surface of minute bioobjects and in fast reading of polynucleotides

A method is proposed for examining the surface of small biological objects such as macromolecules and their complexes. Based on interference of low-energy electrons, it allows constructing analogs of optical holograms but with a resolution on the order of interatomic distances. A set of such holograms obtained at different electron energies can provide sufficient information for identifying the surface groups of the object. Thus the method can be used for fast reading of e.g. polynucleotide sequences.     

"Either-or" two-slit interference: stable coherent propagation of individual photons through separate slits

In quantum theory, nothing that is observable, be it physical, chemical, or biological, is separable from the observer. Furthermore, ". all possible knowledge concerning that object is given by its wave function" (Wigner, E. 1967. Symmetries and Reflections. Indiana University Press, Bloomington, IN), which can only describe probabilities of future events. In physical systems, quantum mechanical probabilistic events that are microscopic must, in turn, account for macroscopic events that are associated with a greater degree of certainty. In biological systems, probabilistic statistical mechanical events, such as secretion of microscopic synaptic vesicles, must account for macroscopic postsynaptic potentials; probabilistic single-channel events sum to produce a macroscopic ionic current across a cell membrane; and bleaching of rhodopsin molecules (responsible for quantal potential "bumps") produces a photoreceptor generator potential. Among physical systems, a paradigmatic example of how quantum theory applies to the observation of events concerns the interactions of particles (e.g., photons, electrons) with the two-slit apparatus to generate an interference pattern from a single common light source. For two-slit systems that use two independent laser sources with brief (<1 ms) intervals of mutual coherence (Paul, H. 1986. Rev. Modern Phys. 58:209-231), each photon has been considered to arise from both beams and has a probability amplitude to pass through each of the two slits. Here, a single laser source two-slit interference system was constructed so that each photon has a probability amplitude to pass through only one or the other, but not both slits. Furthermore, all photons passing through one slit could be distinguished from all photons passing through the other slit before their passage. This "either-or" system produced a stable interference pattern indistinguishable from the interference produced when both slits were accessible to each photon. Because this system excludes the interaction of one photon with both slits, phase correlation of photon movements derives from the "entanglement" of all photon wave functions due to their dependence on a common laser source. Because a laser source (as well as Young's original point source) will have stable time-averaged spatial coherence even at low intensities, the "either-or" two-slit interference can result from distinct individual photons passing one at a time through one or the other slit-rather than wave-like behavior of individual photons. In this manner, single, successive photons passing through separate slits will assemble over time in phase-correlated wave distributions that converge in regions of low and high probability.     

Quantum-like brain: "Interference of minds"

We present a contextualist statistical realistic model for quantum-like representations in physics, cognitive science, and psychology. We apply this model to describe cognitive experiments to check quantum-like structures of mental processes. The crucial role is played by interference of probabilities for mental observables. Recently one such experiment based on recognition of images was performed. This experiment confirmed our prediction on the quantum-like behavior of mind. In our approach "quantumness of mind" has no direct relation to the fact that the brain (as any physical body) is composed of quantum particles. We invented a new terminology "quantum-like (QL) mind." Cognitive QL-behavior is characterized by a nonzero coefficient of interference lambda. This coefficient can be found on the basis of statistical data. There are predicted not only cos theta-interference of probabilities, but also hyperbolic cosh theta-interference. This interference was never observed for physical systems, but we could not exclude this possibility for cognitive systems. We propose a model of brain functioning as a QL-computer (there is a discussion on the difference between quantum and QL computers).     

Changes in water electric conductivity upon heating by various heat sources including biological objects

The temperature dependence of conductivity of water heated by a biological object, as well as electrical, aqueous and other types of heaters was studied. Substantial differences in the conductivity parameters of water heated by bioobjects and physical sources of heat were found. The effect of "memory" of water in response to the biological influence and differences in the entropy of water upon biological treatment and upon heating by physical sources of heat were revealed.     

Electron microscopic-cytochemical study of the enzyme activity of energy metabolism in "dark" and "light" neurons of the rat cerebral cortex

The ultrastructural localization of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) activity in "dark" and "light" neurons of the intact rat's frontal brain cortex has been studied. The enzymes' activity was detected with using potassium ferricyanide as artificial acceptor of electrons. In the "light" cells SDH activity is localized in the mitochondria and plasma membranes. LDH activity is localized in the mitochondria, plasma membranes and hyaloplasm. SDH and LDH activity was not found in the "dark" cells.     

Dependence of magnetic suspectibility of various biological objects on their physiological conditions and viability

The magnetic susceptibility of seeds of cereals and worker honey bees was studied. A correlation between the magnetic susceptibility of bioobjects and their physiological state was established. The theory of magnetic orientation of migrating animals is discussed.     

Protein Equalizer Technology : the quest for a "democratic proteome"

No proteome can be considered "democratic", but rather "oligarchic", since a few proteins dominate the landscape and often obliterate the signal of the rare ones. This is the reason why most scientists lament that, in proteome analysis, the same set of abundant proteins is seen again and again. A host of pre-fractionation techniques have been described, but all of them, one way or another, are besieged by problems, in that they are based on a "depletion principle", i.e. getting rid of the unwanted species. Yet "democracy" calls not for killing the enemy, but for giving "equal rights" to all people. One way to achieve that would be the use of "Protein Equalizer Technology" for reducing protein concentration differences. This comprises a diverse library of combinatorial ligands coupled to spherical porous beads. When these beads come into contact with complex proteomes (e.g. human urine and serum, egg white, and any cell lysate, for that matter) of widely differing protein composition and relative abundances, they are able to "equalize" the protein population, by sharply reducing the concentration of the most abundant components, while simultaneously enhancing the concentration of the most dilute species. It is felt that this novel method could offer a strong step forward in bringing the "unseen proteome" (due to either low abundance and/or presence of interference) within the detection capabilities of current proteomics detection methods. Examples are given of equalization of human urine and serum samples, resulting in the discovery of a host of proteins never reported before. Additionally, these beads can be used to remove host cell proteins from purified recombinant proteins or protein purified from natural sources that are intended for human consumption. These proteins typically reach purities of the order of 98%: higher purities often become prohibitively expensive. Yet, if incubated with "equalizer beads", these last impurities can be effectively removed at a small cost and with minute losses of the main, valuable product.     

Glucose-sensitive holographic sensors

Holographic sensors for monitoring glucose were fabricated from hydrogel films containing chemical ligands based on phenylboronic acid. The films were transformed into reflection holograms using a diffusion method coupled with exposure to laser light. The diffraction wavelength of the holograms was used to monitor the swelling of the hydrogel film in the presence of glucose. Fully reversible changes in diffraction wavelength were demonstrated, highlighting the potential for using these holograms as glucose sensors.     

Individual filamentous phage imaged by electron holography

An in-line electron hologram of an individual f1.K phage was recorded with a purpose-built low energy electron point source (LEEPS) microscope. Cryo-microscopic methods were employed to prepare the specimen so that a single phage could be presented to the coherent low energy electrons: An aqueous phage suspension was applied to a thin carbon membrane with micro-machined slits. The membrane was rapidly cooled to freeze the remaining water as an amorphous ice sheet, which was then sublimated at low temperatures and pressures to leave individual free-standing phages suspended across slits. An image of a phage particle, depicted as the amplitude of the object wave, was reconstructed numerically from a digitized record of the hologram, obtained using 88 eV coherent electrons. The reconstructed image shows a single phage suspended across a slit in a supporting carbon membrane, magnified by a factor of 100,000. The width and shape in the reconstructed image compared well with a TEM image of the same filament. It is thus possible to record and reconstruct electron holograms of an individual phage. The challenge now is to improve the resolution of reconstructed images obtained by this method and to extend these structural studies to other biological molecules.     

On the "NPD ratio" as a test for crossover interference

The "NPD ratio," widely used by yeast geneticists, is of limited applicability and is prone to falsely indicate significant crossover interference in a chi-square test. A simple, better chi-square test for interference in two-factor crosses is described.     

Sensitive detection of viral antigens with a new method, "laser magnet immunoassay".

A new method, "laser magnet immunoassay" (LMIA), has been developed for sensitive detection of viral antigens. Target viruses captured on microbeads were made to react with antibodies labeled with magnetite particles. In a magnetic field, magnetically labeled antigens dispersed in water were attracted to and concentrated at one point on the surface, resulting in the lifting up of a small surface area. A laser beam which was incident on the point reflected, making an interference fringe. The intensity of the fringe indicates the amount of the magnetite conjugated with antigen. A very low concentration of antigens, such as 5 particles of influenza virus and 0.1 pg/ml of human immunodeficiency virus (HIV) p24 antigen in human serum, could be detected by this method. Application of this method to diagnoses of viral diseases in early stages is discussed.     

Collective movement of ions in the cytoplasm

A theoretical model of propagation in the cytoplasm of self-consistent electromagnetic waves of the millimeter-infrared range has been developed, cytoplasmic ions surrounded by water ??coats?? being the main carriers of these waves. It has been discovered that not only own long-wavelength transverse oscillations, but also longitudinal waves that cannot leave the cytoplasm can exist in tissues of living organisms. Frequencies and logarithmic decrements of such perturbations have been found, and it is shown that these frequencies are close to those of ion oscillations inside the ??coats.?? Passage of laser radiation in bioobjects at the indicated frequencies has been analyzed, revealing bands of body impenetrability for waves. A new mechanism of swinging of cytoplasm own oscillations is proposed, based on the existence of an extreme border of the ion movement area. It has been shown that with this mechanism, the electric field amplitude for the longitudinal waves is six to seven orders of magnitude greater than the Planck fluctuation level.     

Xanthine oxidase activity: simultaneous HPLC evaluation of the "D" and "O" forms

A new HPLC method was set up for the simultaneous evaluation of the amount of uric acid and NADH produced by incubation of tissue fractions containing xanthine oxidase, from which the activity of both type "O" (oxidase) and type "D" (dehydrogenase) xanthine oxidase can be calculated. After incubation of the enzyme fraction and ethanol extraction, HPLC analysis is directly carried out. Sensitivity of the method is high enough for the evaluation of xanthine oxidase activity at the lowest reported tissue values. The reliability of the method was tested measuring the enzyme activity in rat heart and kidney extracts.     

Towards the real-time monitoring of glucose in tear fluid: holographic glucose sensors with reduced interference from lactate and pH

Glucose-selective holographic sensors were fabricated from unique tetrahedral 2-acrylamidophenylboronic acid (2-APB) incorporated with co-monomers poly(ethylene glycol) acrylate (PEG), (3-acrylamidopropyl)trimethylammonium chloride (ATMA) and [2-(acryloyloxy)ethyl]-trimethylammonium chloride (AETA) into thin hydrogel films which were transformed into volume holograms using a diffusion method coupled with holographic recording using a frequency-doubled Nd:YAG laser (532 nm). The results showed that the 2-APB-based holographic sensors contracted upon addition of glucose due to the formation of a 2:1 complex between the tetrahedral 2-APB and glucose. More significantly, the 2-APB-based holographic sensors had greatly reduced lactate dependence and a hugely reduced pH effect over the physiological range of pH. These features are vital for development of contact lens-based glucose sensor, where the pH variability is greater (pH 5.8-7.8) and the lactate concentration is substantially higher than in blood. Furthermore, the 2-APB-based holographic sensors also displayed fast response to glucose. The successful union of holograms and the tetrahedral 2-APB receptor for glucose detection in artificial tear fluid is also demonstrated. This new type of holographic sensors responding to glucose with features of minor pH effect and negligible interference from lactate is applicable to the detection of glucose concentrations in tear fluid for the management of diabetes.     

Hydrogenases in the "active" state: determination of g-matrix axes and electron spin distribution at the active site by 1H ENDOR spectroscopy

Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix ( g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the beta-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g1- g2 plane is nearly coplanar (18.3 degrees) with the plane defined by nickel and three sulfur atoms, and the g2 axis deviates by 22.9 degrees from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.     

Oxidation and reduction of exogenous cytochrome c by the activity of the respiratory chain.

Oxidation of exogenous NADH by isolated rat liver mitochondria is generally accepted to be mediated by endogenous cytochrome c which shuttles electrons from the outer to the inner mitochondrial membrane. More recently it has been suggested that, in the presence of added cytochrome c, NADH oxidation is carried out exclusively by the cytochrome oxidase of broken or damaged mitochondria. Here we show that electrons can be transferred in and out of intact mitochondria. It is proposed that at the contact sites between the inner and the outer membrane, a "bi-trans-membrane" electron transport chain is present. The pathway, consisting of Complex III, NADH-b5 reductase, exogenous cytochrome c and cytochrome oxidase, can channel electrons from the external face of the outer membrane to the matrix face of the inner membrane and viceversa. The activity of the pathway is strictly dependent on both the activity of the respiratory chain and mitochondrion integrity.     

Calculation of the proximity function of electrons

A new semianalytical method to calculate the proximity function for electrons is proposed. An integral equation for the proximity function that can be solved by using information on the spatial dose distributions is obtained. The proximity function for electrons in the energy range from 10 eV to 10 keV is calculated by solving the equation numerically, using a set of electron collision cross sections for water vapor. The results are in good agreement with those obtained using the Monte Carlo method. The proposed method can be used for electrons of high energies much more efficiently than the Monte Carlo method.     

Interference with bottom-up feature detection by higher-level object recognition

Drawing portraits upside down is a trick that allows novice artists to reproduce lower-level image features, e.g., contours, while reducing interference from higher-level face cognition. Limiting the available processing time to suffice for lower- but not higher-level operations is a more general way of reducing interference. We elucidate this interference in a novel visual-search task to find a target among distractors. The target had a unique lower-level orientation feature but was identical to distractors in its higher-level object shape. Through bottom-up processes, the unique feature attracted gaze to the target. Subsequently, recognizing the attended object as identically shaped as the distractors, viewpoint invariant object recognition interfered. Consequently, gaze often abandoned the target to search elsewhere. If the search stimulus was extinguished at time T after the gaze arrived at the target, reports of target location were more accurate for shorter (T<500 ms) presentations. This object-to-feature interference, though perhaps unexpected, could underlie common phenomena such as the visual-search asymmetry that finding a familiar letter N among its mirror images is more difficult than the converse. Our results should enable additional examination of known phenomena and interactions between different levels of visual processes.     

UPGMA树的不惟一性问题及其解决方法

对于一组给定的DNA或蛋白质序列,UPGMA算法构建的二叉进化树可能是不惟一的,其具体拓扑结构与序列输入顺序相关,这一现象通常被称为"tied trees"。提出了UPGMA的一种改进算法——不加权算术平均组群方法(UMGMA),用以解决UPGMA树的不惟一问题。在UPGMA树惟一时,该方法产生的进化树与UPGMA树相同;而在UPGMA树不惟一时,该方法可以产生一棵惟一的、与序列输入顺序无关的多叉进化树,而且该算法还具有一个可调的容差参数,来控制生成进化树的主要分枝结构,这对于突出大规模进化树的总体脉络具有重要意义。     

A versatile graphene-based fluorescence "on/off" switch for multiplex detection of various targets

We have designed a versatile molecular beacon (MB)-like probe for the multiplex sensing of targets such as sequence-specific DNA, protein, metal ions and small molecule compounds based on the self-assembled ssDNA-graphene oxide (ssDNA-GO) architecture. The probe employs fluorescence "on/off" switching strategy in a single step in homogeneous solution. Compared to traditional molecular beacons, the proposed design is simple to prepare and manipulate and has little background interference, but still gives superior sensitivity and rapid response. More importantly, this ssDNA-GO architecture can serve as a universal beacon platform by simply changing the types of ssDNA sequences for the different targets. In this work, the ssDNA-GO architecture probe has been successfully applied in the multiplex detection of sequence-specific DNA, thrombin, Ag(+), Hg(2+) and cysteine, and the limit of detection was 1 nM, 5 nM, 20 nM, 5.7 nM and 60 nM, respectively. The results demonstrate that the ssDNA-GO architecture can be an excellent and versatile platform for sensing multiplex analytes, easily replacing the universal molecular beacon.