Mechanism of interaction between electromagnetic fields and living organisms

Based on nonlinear phenomena of biophoton emission observed in the past, an interference model concerning with the mechanism of interaction between living organisms and electromagnetic fields was raised. Caused by biological nonlinearly polarizable double layer, destructive interference of incoming and reflected waves establishes in the outside. As a consequence, in the inside constructive interference takes place at the same time. The interference patterns may play an important role in biological self organization and in biological functions. We investigate the boundary conditions necessary for explaining these non-linear optical effects in terms of the phase conjugation. It turns out that there are solutions of the Maxwell equations which satisfy destructive interference of biophotons in agreement with the experimental results. Necessary provisions are nonlinearly polarizable optically active double layers of distances which are small compared to the wavelength of light. In addition, they have to be a     

A theoretical study of the principles regulating the specificity for Al(III) against Mg(II) in protein cavities

Several toxic effects arise from Al's presence in living systems, one of them being the alteration of the natural role of enzymes and non-enzyme proteins. Al(III) is capable of entering protein active sites that in normal conditions should be occupied by other metals. Even if Mg(II) is an ubiquitous metal in biological systems, the interference of aluminium in magnesium metabolism is not clear yet. In this work, a systematic study of the affinity of Al(III) for different protein binding sites is presented, with special attention on structural parameters, the role of the charge and the presence of different ligands in the protein cavity. The specificity of the binding site for Al(III) against Mg(II) has been studied, and also the thermodynamical propensity of a Mg(II)/Al(III) exchange. Quantum mechanical methods that proved to be reliable in previous works have been used, namely, the density functional theory (DFT) and polarizable continuum model (PCM).     

Bio-soliton model that predicts non-thermal electromagnetic frequency bands,that either stabilize or destabilize living cells

Solitons, as self-reinforcing solitary waves, interact with complex biological phenomena such as cellular self-organization. A soliton model is able to describe a spectrum of electromagnetism modalities that can be applied to understand the physical principles of biological effects in living cells, as caused by endogenous and exogenous electromagnetic fields and is compatible with quantum coherence. A bio-soliton model is proposed, that enables to predict which eigen-frequencies of non-thermal electromagnetic waves are life-sustaining and which are, in contrast, detrimental for living cells. The particular effects are exerted by a range of electromagnetic wave eigen-frequencies of one-tenth of a Hertz till Peta Hertz that show a pattern of 12 bands, and can be positioned on an acoustic reference frequency scale. The model was substantiated by a meta-analysis of 240 published articles of biological electromagnetic experiments, in which a spectrum of non-thermal electromagnetic waves were exposed to living cells and intact organisms. These data support the concept of coherent quantized electromagnetic states in living organisms and the theories of Fröhlich, Davydov and Pang. It is envisioned that a rational control of shape by soliton-waves and related to a morphogenetic field and parametric resonance provides positional information and cues to regulate organism-wide systems properties like anatomy, control of reproduction and repair.     

Transparent polymeric cell culture chip with integrated temperature control and uniform media perfusion

Modern microfabrication and microfluidic technologies offer new opportunities in the design and fabrication of miniaturized cell culture systems for online monitoring of living cells. We used laser micromachining and thermal bonding to fabricate an optically transparent, low-cost polymeric chip for long-term online cell culture observation under controlled conditions. The chip incorporated a microfluidic flow equalization system, assuring uniform perfusion of the cell culture media throughout the cell culture chamber. The integrated indium-tin-oxide heater and miniature temperature probe linked to an electronic feedback system created steady and spatially uniform thermal conditions with minimal interference to the optical transparency of the chip. The fluidic and thermal performance of the chip was verified by finite element modeling and by operation tests under fluctuating ambient temperature conditions. HeLa cells were cultured for up to 2 weeks within the cell culture chip and monitored using a time-lapse video recording microscopy setup. Cell attachment and spreading was observed during the first 10-20 h (lag phase). After approximately 20 h, cell growth gained exponential character with an estimated doubling time of about 32 h, which is identical to the observed doubling time of cells grown in standard cell culture flasks in a CO2 incubator.     

Nonlethal assay system of β-glucuronidase activity in transgenic tobacco roots

Previously, assay conditions for the GUS enzyme have required lethal and/or destructive conditions which do not allow for the continued observation of living plants. The replacement of sodium phosphate buffer with potassium phosphate buffer and the removal of potassium ferrocyanide and EDTA resulted in an assay for the GUS enzyme that is both nondestructive and nonlethal to tobacco plants and therefore allows observations of tobacco roots through time.     

Minitags for small molecules: detecting targets of reactive small molecules in living plant tissues using 'click chemistry'

Small molecules offer unprecedented opportunities for plant research since plants respond to, metabolize, and react with a diverse range of endogenous and exogenous small molecules. Many of these small molecules become covalently attached to proteins. To display these small molecule targets in plants, we introduce a two-step labelling method for minitagged small molecules. Minitags are small chemical moieties (azide or alkyne) that are inert under biological conditions and have little influence on the membrane permeability and specificity of the small molecule. After labelling, proteomes are extracted under denaturing conditions and minitagged proteins are coupled to reporter tags through a 'click chemistry' reaction. We introduce this two-step labelling procedure in plants by studying the well-characterized targets of E-64, a small molecule cysteine protease inhibitor. In contrast to biotinylated E-64, minitagged E-64 efficiently labels vacuolar proteases in vivo . We displayed, purified and identified targets of a minitagged inhibitor that targets the proteasome and cysteine proteases in living plant cells. Chemical interference assays with inhibitors showed that MG132, a frequently used proteasome inhibitor, preferentially inhibits cysteine proteases in vivo . The two-step labelling procedure can be applied on detached leaves, cell cultures, seedlings and other living plant tissues and, when combined with photoreactive groups, can be used to identify targets of herbicides, phytohormones and reactive small molecules selected from chemical genetic screens.     

Dynamic light scattering microscopy. A novel optical technique to image submicroscopic motions. I: theory

The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy.     

Reactive modifications of the autonomous time structure in the human organism

The spectrum of biological rhythms exhibits characteristic principles of biological time structure which also rule the functional behaviour. With increasing period lengths the rhythms become increasingly complex. In the long-wave section the rhythmic functions find their corresponding cycles in the environment, whereas the shorter waves represent only endogenous autonomous rhythms, which maintain an internal time order by means of frequency- and phase-coordination. Under resting conditions and in a state of complete adaptation only a few spontaneous rhythms dominate in the spectrum. However, under loading conditions as well as in pathological situations further periodicities come up. The spectrum of rhythms can be divided into certain blocks, with the period lengths predominating in each of these whole number frequency ratios forming a harmonic system. Frequency- and phase coordination establish a system of co-action which favours the functional economy of the organism. A tripartite organization of the autonomous rhythms involves different functional behaviours with regard to frequency, amplitude, and phase. Slower rhythms act upon the faster rhythms preferably by modulating their frequencies, while changes of the faster rhythms influence the slower ones by enhancing their amplitudes, multiplying their period lengths and shifting their phases. In principle the reactions of living systems are periodically structured. Reactive periodicity brings to appearance an endogenous time structure, which prefers whole number relationships with the spontaneous rhythms. The phase position of reactive periods depends on the stimulus. The amplitudes dampen down with increasing compensation. From the medical point of view so-called circaseptan (about 7 days) reactive periods are of predominant interest. This periodicity can be observed in numerous adaptive and compensating processes. It does not depend on the external week cycle and was already known to the antiquity.     

Kinetic dimensions of cells and crypts in irradiated intestinal epithelium of mice]

The average value of axial cryptal section area and cell section area on it were studied during 8 days after total X-ray irradiation of male mice (400 rad). A small reducing of cryptal area (20%) during destructive period (1-2 days) is followed by a big overshoot (60%) during regenerative time (3-7 days). The cryptal sizes in regenerative period deviate from a steady state more than during destructive time. There are two high waves of abnormal growth of cell sizes above the steady level: the first one during destructive time and the second one during regeneration. This level seems to be near to minimal sizes of cryptal proliferative cells which are necessary for proliferation. It means that normal intestinal epithelium is a very economical and stabilized system. It is possible to evaluate quantitatively the associated with proliferation flow of substance per crypt cell for normal and irradiated intestine by means of index Iv where I is mitotical index and v - the cell volume. Cell hypertrophy at the time of regeneration on the 4th-7th days and later after irradiation (130-160%), was revealed. The crypt cell hypertrophy is the factor of destabilization of irradiated intestinal epithelium.     

On peristaltic flow and its efficiency

The question of efficiency in performing biological functions is raised in the context of peristaltic fluid transport. To deal with this problem a complete solution for peristaltic flow in a pipe and in a channel, assuming a given time mean flow, is developed, by a double expansion in terms of the Reynolds number and the square of the wave number. This solution is valid for arbitrary waveshapes. We resolve a long-standing problem and show that quite generally the pressure rise per wave length is constant on a cross section. We also show that for a sinusoidal wave (and others) the interaction of Reynolds number and wave number is a third-order effect for this pressure rise. Plow-type waves, nipple-type waves and the sinusoidal wave are compared for maximum efficiency and for minimum energy usage. It is found that large plows are best from mechanical efficiency considerations, but large nipples use the least energy. The biological implications of these results are discussed.     

基于双工程菌的“锁扣”生物活材料构建及其体内肠道滞留应用

生物活材料的研究主要集中在利用单一细菌生产生物膜、水塑料等体外应用。由于菌株尺寸较小，当其应用于体内时，容易发生逃逸，导致滞留效果较差。为解决这一难题，本研究借助大肠杆菌(Escherichia coli)表面展示系统(Neae)，在两个菌株表面分别展示SpyTag和SpyCatcher，构建一种双菌“锁扣”型生物活材料生产系统。两菌株之间通过SpyTag和SpyCatcher的结合，发生原位交联，从而长时间滞留在肠道部位。体外实验表明两菌株混合几分钟后，会发生明显的沉降。此外，利用共聚焦成像和微流控平台进一步证明了该系统在流动状态下的粘附效果。最后，为了验证该系统在体内应用的可行性，小鼠连续3d口服A菌(p15A-Neae-SpyTag/sfGFP)和B菌(p15A-Neae-SpyCatcher/mCherry)，收集肠道组织进行冷冻切片染色。结果表明，相较于未结合菌株，该双菌系统能更多滞留在小鼠肠道，为生物活材料进一步的体内应用奠定了基础。     

Fluorescence correlation spectroscopy in living cells

Fluorescence correlation spectroscopy (FCS) is an ideal analytical tool for studying concentrations, propagation, interactions and internal dynamics of molecules at nanomolar concentrations in living cells. FCS analyzes minute fluorescence-intensity fluctuations about the equilibrium of a small ensemble (<10(3)) of molecules. These fluctuations act like a 'fingerprint' of a molecular species detected when entering and leaving a femtoliter-sized optically defined observation volume created by a focused laser beam. In FCS the fluorescence fluctuations are recorded as a function of time and then statistically analyzed by autocorrelation analysis. The resulting autocorrelation curve yields a measure of self-similarity of the system after a certain time delay, and its amplitude describes the normalized variance of the fluorescence fluctuations. By fitting the curves to an appropriate physical model, this method provides precise information about a multitude of measurement parameters, including diffusion coefficients, local concentration, states of aggregation and molecular interactions. FCS operates in real time with diffraction-limited spatial and sub-microsecond temporal resolution. Assessing diverse molecular dynamics within the living cell is a challenge well met by FCS because of its single-molecule sensitivity and high dynamic resolution. For these same reasons, however, intracellular FCS measurements also harbor the large risk of collecting artifacts and thus producing erroneous data. Here we provide a step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell. A discussion of advantages and disadvantages of one-photon versus two-photon excitation for FCS is available in Supplementary Methods online.     

Size Matters: Ryanodine Receptor Cluster Size Heterogeneity Potentiates Calcium Waves

Ryanodine receptors (RyRs) mediate calcium (Ca)-induced Ca release and intracellular Ca homeostasis. In a cardiac myocyte, RyRs group into clusters of variable size from a few to several hundred RyRs, creating a spatially nonuniform intracellular distribution. It is unclear how heterogeneity of RyR cluster size alters spontaneous sarcoplasmic reticulum (SR) Ca releases (Ca sparks) and arrhythmogenic Ca waves. Here, we tested the impact of heterogeneous RyR cluster size on the initiation of Ca waves. Experimentally, we measured RyR cluster sizes at Ca spark sites in rat ventricular myocytes and further tested functional impacts using a physiologically detailed computational model with spatial and stochastic intracellular Ca dynamics. We found that the spark frequency and amplitude increase nonlinearly with the size of RyR clusters. Larger RyR clusters have lower SR Ca release threshold for local Ca spark initiation and exhibit steeper SR Ca release versus SR Ca load relationship. However, larger RyR clusters tend to lower SR Ca load because of the higher Ca leak rate. Conversely, smaller clusters have a higher threshold and a lower leak, which tends to increase SR Ca load. At the myocyte level, homogeneously large or small RyR clusters limit Ca waves (because of low load for large clusters but low excitability for small clusters). Mixtures of large and small RyR clusters potentiates Ca waves because the enhanced SR Ca load driven by smaller clusters enables Ca wave initiation and propagation from larger RyR clusters. Our study suggests that a spatially heterogeneous distribution of RyR cluster size under pathological conditions may potentiate Ca waves and thus afterdepolarizations and triggered arrhythmias.     

Proton Dispersion Forces: Secondary-Structure Stabilizing Forces between the Hydrogen Bonds of the Polynucleotides

In the double helix formed by the semiprotonated polycytidylic acid (poly C), both strands are linked via NH⁺...N hydrogen bonds. It is a known fact that such symmetrical hydrogen bonds with a double minimum potential well are extremely polarizable. This polarizability causes interaction effects, in particular the proton dispersion forces between such hydrogen bonds. These forces result in a shift of the energy levels and a continuum is observed in the infrared (IR) spectra of solutions in which such hydrogen bonds are present. The continuum occurs in the IR spectrum of the semiprotonated poly C, when the former is present in coiled state. If the double helix forms, an extremely broad band of the NH stretching vibration is observed instead of the continuum, since in the double helix all hydrogen bonds are oriented equally to one another and polarize each other mutually to a strong degree. The proton dispersion forces between the hydrogen bonds balance a considerable part of the electrostatic repulsion of the protons and hence enable the double helix to form. It is conceivable that an unsymmetrical double minimum potential well is present in the NH...N bonds in the DNA and RNA. Such bonds may likewise be considerably more polarizable than electron systems and thus, in this case too, proton dispersion forces would contribute to helix stabilization.     

Efficient racemization of N‐phenylacetyl‐D‐glufosinate for L‐glufosinate production

Most amino acids contain chiral centres and exist as both D‐enantiomer and L‐enantiomer. The optically pure enantiomer is often more valuable than the racemate. Enzymatic resolution provides an effective strategy to obtain optically pure amino acids but often results in large amounts of unwanted isomer. In this study, optically pure L‐glufosinate (L‐PPT) was obtained by coupling amidase‐mediated hydrolysis of N‐phenylacetyl‐D,L‐glufosinate with racemization of N‐phenylacetyl‐D‐glufosinate (NPDG), which exclusively exhibits effective herbicidal properties compared with its D‐enantiomer. To improve the yield of L‐PPT, the racemization reaction conditions were optimized, and through single‐factor experiments, the optimal reaction temperature, reaction time, and mole ratio of phenylacetic acid to NPDG were determined to be 150°C, 30 minutes, and 1.5, respectively. The response surface methodology was applied to further optimize the racemization conditions, and the final yield of L‐PPT reached 96.13% with optimum reaction temperature of 154°C, reaction time of 23 minutes, and phenylacetic acid/NPDG mole ratio of 1.7, respectively. Moreover, adding a small amount of acetic anhydride further raised the yield of L‐PPT to 97.02%.     

Velocity-curvature relationship of colliding spherical calcium waves in rat cardiac myocytes.

Colliding spherical calcium waves in enzymatically isolated rat cardiac myocytes develop new wavefronts propagating perpendicular to the original direction. When investigated by confocal laser scanning microscopy (CLSM), using the fluorescent Ca2+ indicator fluo-3 AM, "cusp"-like structures become visible that are favorably approximated by double parabolae. The time-dependent position of the vertices is used to determine propagation velocity and negative curvature of the wavefront in the region of collision. It is evident that negatively curved waves propagate faster than positively curved, single waves. Considering two perfectly equal expanding circular waves, we demonstrated that the collision of calcium waves is due to an autocatalytic process (calcium-induced calcium release), and not to a simple phenomenon of interference. Following the spatiotemporal organization in simpler chemical systems maintained under conditions far from the thermodynamic equilibrium (Belousov-Zhabotinskii reaction), the dependence of the normal velocity on the curvature of the spreading wavefront is given by a linear relation. The so-called velocity-curvature relationship makes clear that the velocity is enhanced by curvature toward the direction of forward propagation and decreased by curvature away from the direction of forward propagation (with an influence of the diffusion coefficient). Experimentally obtained velocity data of both negatively and positively curved calcium waves were approximated by orthogonal weighted regression. The negative slope of the straight line resulted in an effective diffusion coefficient of 1.2 x 10(-4) mm2/s. From the so-called critical radius, which must be exceeded to initiate a traveling calcium wave, a critical volume (with enhanced [Ca2+]i) of approximately 12 microm3 was calculated. This is almost identical to the volume that is occupied by a single calcium spark.     

Engineering FKBP-Based Destabilizing Domains to Build Sophisticated Protein Regulation Systems

Targeting protein stability with small molecules has emerged as an effective tool to control protein abundance in a fast, scalable and reversible manner. The technique involves tagging a protein of interest (POI) with a destabilizing domain (DD) specifically controlled by a small molecule. The successful construction of such fusion proteins may, however, be limited by functional interference of the DD epitope with electrostatic interactions required for full biological function of proteins. Another drawback of this approach is the remaining endogenous protein. Here, we combined the Cre-LoxP system with an advanced DD and generated a protein regulation system in which the loss of an endogenous protein, in our case the tumor suppressor PTEN, can be coupled directly with a conditionally fine-tunable DD-PTEN. This new system will consolidate and extend the use of DD-technology to control protein function precisely in living cells and animal models.     

Molecular Dynamics Simulations with Interaction Potentials Including Polarization Development of a Noniterative Method and Application to Water

A general method is suggested for the implementation of polarization in molecular dynamics simulations of small molecules. Induced dipole moments are evaluated on selected polarizability centers and represented by separation of charges. The positive polarization charges reside on the selected atoms. The negative polarization charges are treated as additional particles. The positions of these polarization charges are determined from the electrical fields due to the permanent charges of the system. Thus the induction is treated explicitly, while the higher order contributions, the polarization due to induced dipoles, are taken into account in an average way by modification of potential parameters. The forces can be evaluated for the new charge distribution in the conventional way. As an illustration of this approach initial results are reported for the development of a polarizable water model. The higher order polarization is treated in an average way by slight increase of the permanent charges as compared to the values that would give the gas phase dipole moment. The increase in CPU time is comparable to the addition of one atom per polarizable center.     

Effect of initial stresses on the wave propagation in arteries

A theoretical analysis for the problem of wave propagation in arteries is presented. Blood is treated as a Newtonian, viscous incompressible fluid. On the basis of information derived from experimental investigations on the mechanical properties of wall tissues, the arterial wall is considered to be nonlinearly viscoelastic and orthotropic. The analysis is carried out for a cylindrical artery, under the purview of membrane theory, by taking account the effect of initial stresses. The motion of the wall and that of the fluid are assumed to be axisymmetric. Particular emphasis has been paid to the propagation of small amplitude harmonic waves whose wavelength is large compared to the radius of the vessel. By employing the equations of motion of the fluid and those for the wall, together with the equations of continuity, a frequency equation is derived by exploiting the conditions of continuity of the velocity of the arterial wall and that of blood on the endosteal surface of the wall. In order to illustrate the validity of the derived analytical expressions a quantitative analysis is made for the variations of the phase velocities as well as the transmission coefficient with frequency corresponding to different transmural pressures which relate to different initial stresses. Computational results indicate that phase velocities increase with the increase of transmural pressures.     

Kinetics of the light flash of the living firefly: a supramolecular process.

The light intensity vs. time curve of the light flash of the living firefly has been measured. Unlike the purified firefly enzyme system in aqueous solution, the living system does not show light decay conforming to a double exponential time curve, to simple first or second order decay, or to solid-state Elovich kinetics. Light decay of the living flash does show linearity in a probit vs. square root of time plot, which may indicate a reaction rate-limited by cooperative interactions of a biological phase transition. The observation that the kinetics of the firefly light system differ in the living cell from those in the purified system suggests that in the living system supramolecular factors control the rate of the reaction.